TW201827070A - Synthetic polypeptide, composition comprising the same, antibody produced thereby, and uses thereof - Google Patents

Abstract

Disclosed herein are polypeptides overexpressed on the surface of cancer cells. According to one embodiment, the polypeptide comprises the amino acid sequence of SEQ ID NO: 1, 2 or 3. The present polypeptides are useful in the treatment and/or prophylaxis of a cancer in a subject in need thereof. Accordingly, also disclosed herein are compositions comprising the polypeptides, antibodies that bind to the polypeptides, and their applications in the prophylaxis and/or treatment of cancer.

Description

Synthetic polypeptide, composition containing the same, antibody produced therefrom and use thereof

This disclosure is about the field of treating cancer. More specifically, the present disclosure relates to a polypeptide associated with cancer, a vaccine composition comprising the polypeptide, an antibody that binds to the polypeptide, and the use thereof for preventing and/or treating cancer.

The cancer department has a disease with abnormal cell proliferation that has the potential to invade or spread to other parts of the body. More than 100 different types of cancer have been identified in humans. Among them, cancer that occurs in men includes lung cancer, prostate cancer and colorectal cancer; breast cancer, lung cancer and colorectal cancer are mainly caused by women.

Most of the cancer comes from environmental factors, including alcohol, smoking, obesity, infection, radiation exposure, stress, lack of exercise and environmental pollution. In addition, there are reports that heredity plays an important role in the cause of cancer. Hereditary cancers are mainly caused by genetic defects (such as genetic mutations). For example, related studies have confirmed that mutations in the gene BRCA1 or BRCA2 are associated with cancer production in breast, ovarian or pancreatic cancer.

Prophylactic cancer vaccines protect individuals from cancer by triggering an immune response against a tumor-associated antigen (TAA). However, different types of cancer usually show different TAAs (for example, hepatocellular carcinoma mainly exhibits alpha-fetoprotein (AFP), ovarian cancer mainly exhibits CA-125, and prostate cancer mainly exhibits prostate specificity. Prostate-specific antigen (PSA); In this case, it is not easy to predict the risk of different individuals suffering from a particular cancer, and to design an immunization strategy for those cancers. Given that most preventive cancer vaccines are designed for the target TAA that will be manifested in a particular type of cancer, these vaccines are not suitable for human therapy. Accordingly, there is a need in the art for a TAA that is widely manifested in different cancers; and in turn, a cancer vaccine that is effective in protecting humans from cancer.

Current major cancer therapies include surgery, radiation therapy, chemotherapy, hormonal therapy, and targeted therapy. In general, treatment varies with the type of cancer, location and severity, and the patient's health and preferences. However, due to limited specificity, insufficient efficacy, and/or adverse side effects, most of these treatments do not produce satisfactory results for cancer patients.

Immunotherapy is an alternative treatment that has been exploited in recent years to exploit part of the immune system of an individual to attack cancer cells. Immunotherapy mainly includes therapeutic cancer vaccines, immune checkpoint inhibitors, monoclonal antibodies and non-specific immunotherapy. Therapeutic cancer vaccines typically contain one or more specific antigens that stimulate immune cells (such as dendritic cells and T cells) to recognize and attack cancer cells that express the antigen. Unfortunately, such cancer therapies on average only prolong the life of the patient for months and cannot cure the cancer. In addition, they can also cause undesirable side effects and/or are associated with over-activated/non-specific immune responses such as fever, chills, burnout, pain, nausea and headache. For immunological checkpoints and their inhibitors, PD-1 and CTLA-4 are two known immunological checkpoint proteins that are expressed by ligands on cancer cells (ie PD-L1 and CD80/CD86). Binding, while inhibiting T cell function and / or reducing immune response. Thus, immunological checkpoint inhibitors provide a therapeutic method for treating cancer cells by stimulating an immune response. However, it is worth noting that in addition to cancer cells, normal cells also exhibit these ligands (ie, PD-L1 and CD80/CD86); therefore, such treatments may cause serious or even life-threatening side effects in individuals. . Monoclonal antibodies can recognize cancer cells that exhibit antigen-specific antigens (eg, TAA), thereby inhibiting cancer cell proliferation and/or causing cancer cell death (eg, apoptosis or cell necrosis) Necrosis)) to achieve the efficacy of treating cancer. In addition, once bound to an antigen, monoclonal antibodies also activate a complement system that is resistant to cancer cells. In terms of efficacy, monoclonal antibodies still have some drawbacks that need to be improved, including poor stability, insufficient affinity, and lack of specificity. As for non-specific immunotherapy, such as interleukins or interferons, the immune system is stimulated. A limitation of this type of treatment is that it is not specifically labeled for cancer cells and may trigger a cytokine storm in the individual.

In view of the above, there is a need in the related art for a cancer-related polypeptide that is widely expressed in different cancer cells to prepare cancer vaccines and antibodies that are effective for preventing and/or treating different cancers in an individual in need thereof.

BRIEF DESCRIPTION OF THE DRAWINGS A brief summary of the invention is presented below to provide a basic understanding of the invention. This Summary is not an extensive overview of the invention, nor is it a critical/critical element of the disclosure, or the scope of the disclosure. Its sole purpose is to present some concepts of the invention in a simplified

A first aspect of the present disclosure relates to a synthetic polypeptide for preventing and/or treating cancer in an individual in need thereof. According to an embodiment of the present disclosure, the synthetic polypeptide comprises a first polypeptide having an amino acid sequence having a sequence similarity to sequence number: 1, 2 or 3 of at least 85%. According to one embodiment of the present disclosure, the first polypeptide has an amino acid sequence of SEQ ID NO: 1, 2 or 3.

According to an optional embodiment, the N-terminus of the synthetic polypeptide has an acetylated, glycosylated or formylated. Alternatively or additionally, the C-terminus of the synthetic polypeptide has an amidated or glycosylated.

Optionally, the synthetic polypeptide may further comprise a second polypeptide located at the N-terminus or C-terminus of the first polypeptide, wherein the second polypeptide is selected from the group consisting of ovalbumin (OVA), Bovine serum albumin (BSA), keyhole limpet haemocyanin (KLH), β-galactosidase, thyroglobulin (TGB) and combinations thereof The group consisting of.

A second aspect of the present disclosure relates to a vaccine composition for preventing and/or treating a cancer of a body, wherein the vaccine composition comprises a synthetic polypeptide of the present invention, and a pharmaceutically acceptable adjuvant. In general, the pharmaceutically acceptable adjuvant is selected from the group consisting of fatty alcohol polyoxyethylene ether-D (Emulsigen-D), aluminum hydroxide, incomplete Fruend's adjuvant (IFA). Complete Fruend's adjuvant (CFA), endotoxin based adjuvant, mineral oil, mineral oil and surfactant, Ribi adjuvant (Ribi adjuvant), gold adjuvant (Titer-max), Syntax adjuvant formulation, aluminum salt adjuvant, nitrocellulose adsorbed antigen, immunostimulating complex ( Immune stimulating complex), Gebru adjuvant, super carrier, copolymer of ethylene and vinyl acetate (Elvax 40w), L-tyrosine, oil-based emulsion (montanide) Adju prime, squalene, sodium phthalyl lipopolysaccharide Ide, SPLPS), calcium phosphate, saponin, and muramyl dipeptide (MDP).

In certain embodiments, the synthetic polypeptide of the vaccine composition of the present invention may further comprise a second polypeptide at the N-terminus or C-terminus of the first polypeptide, wherein the second polypeptide is selected from OVA, A group consisting of BSA, KLH, β-galactosidase, TGB, and combinations thereof.

A third aspect of the present disclosure relates to a method for preventing or treating cancer in an individual in need thereof. The method comprises administering to the individual an effective amount of a vaccine composition of the invention.

According to certain embodiments, administration of the vaccine composition produces from about 0.8 micrograms to 80 milligrams of the synthetic polypeptide of the invention per kilogram of body weight (i.e., from 0.8 micrograms to 80 milligrams per kilogram per dose); preferably, per dose per dose. The body weight is from about 8 micrograms to about 8 milligrams; more preferably, from about 80 micrograms to about 800 micrograms per kilogram of body weight. According to some embodiments, the vaccine composition is administered to the individual at least twice in the course of vaccination. According to a preferred embodiment, the vaccine composition is administered to the individual 6 to 10 times during the vaccination session.

The vaccine composition of the invention may be administered by any effective route, such as transmucosal, intranasal, subcutaneous, intradermal, intramuscular, intravenous or intraperitoneal. (intraperitoneal). As can be appreciated, the vaccine compositions of the present invention can be administered to an individual in combination with other therapies selected from the group consisting of surgery, radiation therapy, chemotherapy, hormonal therapy, antiangiogenic therapy, and immunotherapy. A group of its combination.

A fourth aspect of the disclosure provides an antibody or fragment thereof having binding affinity for a polypeptide of the invention. According to an embodiment of the present disclosure, the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the sequence number: 6, the sequence number : 7 and the amino acid sequence of SEQ ID NO: 8, and the VL comprises the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11.

According to certain embodiments, the disclosure provides a monoclonal antibody or binding fragment thereof having binding affinity for a polypeptide of the invention, wherein the antibody or binding fragment thereof comprises a VH sequence and a VL sequence, wherein the VH sequence and sequence Number: 12 has a sequence similarity of at least 85%, and the VL sequence has a sequence similarity to sequence number: 13 of at least 85%. In some embodiments, the sequence similarity between the VH sequence and SEQ ID NO: 12, and the sequence similarity between the VL sequence and SEQ ID NO: 13 are at least 85%, 86%, 87%, 88%, 89, respectively. %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%. According to a particular embodiment, the VH has the amino acid sequence of SEQ ID NO: 12 and the VL has the amino acid sequence of SEQ ID NO: 13.

Optionally, the antibody of the invention can be conjugated to a reporter molecule, a contrast agent or an anti-cancer drug.

The invention also discloses a pharmaceutical composition for treating cancer in an individual in need thereof. The pharmaceutical compositions of the present disclosure comprise a monoclonal antibody of the invention or a binding fragment thereof, and a pharmaceutically acceptable carrier.

Accordingly, a fifth aspect of the present disclosure provides a method of treating cancer in a subject in need thereof using the antibody of the present invention or the pharmaceutical composition of the present invention. In particular, the method comprises administering to the individual an effective amount of an antibody of the invention or a pharmaceutical composition of the invention.

According to certain embodiments, the subject is administered from about 0.8 micrograms to 80 milligrams of the antibody of the invention per kilogram of body weight per dose; preferably, from about 8 micrograms to about 8 milligrams per kilogram of body weight per dose; more preferably, per dose per dose The weight of the kilogram is about 80 micrograms to 800 micrograms. In these embodiments, at least the antibody of the invention is administered to the individual twice during the vaccination session.

Alternatively, the pharmaceutical composition of the present invention is administered to the individual, wherein the pharmaceutical composition produces from about 0.8 micrograms to 80 milligrams of the antibody of the invention per kilogram of body weight per dose; preferably, about 8 micrograms per kilogram of body weight per dose. 8 mg; more preferably, about 80 micrograms to 800 micrograms per kilogram of body weight per dose.

In general, the vaccine composition, antibody and pharmaceutical composition of the invention can be administered to the individual by any effective route, including transmucosal, intranasal, subcutaneous, intratumoral, intradermal, intramuscular, intravenous. Or abdominal cavity. Optionally, the vaccine compositions, antibodies, and pharmaceutical compositions of the present invention can be administered to an individual in combination with other therapies selected from the group consisting of surgery, radiation therapy, chemotherapy, hormonal therapy, anti-angiogenesis therapy, and A group of immunotherapy.

Exemplary cancers that are acceptable for treatment with vaccine compositions, antibodies, and/or pharmaceutical compositions of the invention include, but are not limited to, gastric cancer, lung cancer, bladder cancer, breast cancer ), pancreatic cancer, renal cancer, colorectal cancer, cervical cancer, ovarian cancer, brain tumor, prostate cancer Prostate cancer), hepatocellular carcinoma, melanoma, esophageal carcinoma, multiple myeloma, and head and neck squamous cell carcinoma.

In certain embodiments, the individual is a vertebrate. In certain embodiments, the vertebrate is a mammal, wherein the mammal includes, but is not limited to, agricultural animals (eg, cattle), sports animals, pets (eg, cats, dogs, and horses), primates, Mouse and rat. Preferably, the individual is a human.

The accompanying features and advantages of the present disclosure are readily apparent from the following detailed description and claims.

The detailed description and the accompanying drawings are intended to be in the This description sets forth the functions of the embodiments and the sequence of steps used to construct and operate the embodiments. However, other embodiments may be utilized to achieve the same or equivalent functions and sequence of steps.

1. Definition

For the sake of convenience, some of the proper nouns used in the specification, examples, and appended claims are hereby incorporated. The scientific and technical terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention pertains, unless otherwise defined herein. In the absence of conflict with context, the singular noun used in this specification covers the plural of the noun, and the plural noun used also covers the singular of the noun. In the specification and claims, the singular forms "a", "," In addition, in the scope of the present specification and the patent application, "at least one" has the same meaning as "one or more", and both represent one, two, three or more. Unless otherwise defined by the present specification, the present disclosure is practiced by conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are conventional in the art to which the present invention pertains. Such techniques have been explained in detail in the open literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 1989); DNA Cloning, Volumes I and II (DN Glover ed., 1985); Culture Of Animal Cells (RI Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); Methods In Enzymology (Academic Press, Inc., NY); Gene Transfer Vectors For Mammalian Cells (JH Miller and MP Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Antibodies: A Laboratory Manual, by Harlow and Lane (Cold Spring Harbor Laboratory Press, 1988); and Handbook Of Experimental Immunology, Volumes I-IV (DM Weir And CC Blackwell, eds., 1986).

Although numerical ranges and parameters are used to define a broad range of values for the present invention, the relevant values in the specific embodiments have been presented as precisely as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. As used herein, the term "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a particular value or range. Alternatively, the term "about" means that the actual value falls within the acceptable standard error of the average, depending on the considerations of those of ordinary skill in the art to which the invention pertains. Except for the experimental examples, or unless otherwise explicitly stated, all ranges, quantities, values, and percentages used herein are understood (eg, to describe the amount of material used, the length of time, the temperature, the operating conditions, the quantity ratio, and the like. Are all modified by "about". Therefore, unless otherwise indicated to the contrary, the numerical parameters disclosed in the specification and the appended claims are intended to be At a minimum, these numerical parameters should be understood as the number of significant digits indicated and the values obtained by applying the general carry method.

The term "polypeptide" refers to a polymer of an amino acid, regardless of the length of the polymer; thus, the terms "peptides", "oligopeptides", and "proteins" They are all included within the definition of a polypeptide and can be used interchangeably here. These terms do not specify or exclude chemical or post-expression modifications of the polypeptides of the invention, although chemical or post-expression modifications of the polypeptide may be included or excluded in certain embodiments. Thus, for example, modifications of the polypeptide include glycosyl groups, acetyl groups, phosphate groups, lipid groups, and covalent attachments. All of them are explicitly covered within the scope of the term polypeptide. More specifically, polypeptides having such modifications can be designated as individual species and included in or excluded from the present invention.

The term "synthetic polypeptide" as used in the present invention means that the polypeptide does not comprise the entire naturally occurring protein molecule. The polypeptide is "synthetic", which means that human intervention can be performed by techniques such as chemical synthesis, recombinant genetic techniques, or ragmentation of whole antigen or the like. Prepared.

As contemplated by the present invention, minor variations in the amino acid sequence of the polypeptide are contemplated to be encompassed within the scope of the present disclosure and claimed inventive concepts, provided that the amino acid sequence changes are maintained at least 85%. More than the sequence similarity, such as at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% sequence similarity. The polypeptide of the present invention can be specifically modified to alter the characteristics of a polypeptide that is not related to its physiological activity. For example, in this study, certain amino acids can be altered and/or deleted without affecting the physiological activity of the polypeptide (ie, its immune response that triggers tumor specificity). In particular, substitution of a conservative amino acid can be expected. Conservative substitutions refer to substitutions within a family of related amino acids in their side chains. The amino acid encoded by the gene is roughly divided into several families: (1) acidity = aspartate, glutamate; (2) alkali = lysine, arginine (arginine), histidine; (3) non-polar = alanine, valine, leucine, isoleucine, proline (proline) ), phenylalanine, methionine, tryptophan; and (4) uncharged polarity = glycine, asparagine, glutamine Glutamine, cysteine, serine, threonine, tyrosine. More preferably, the family is: alanine and threonine are aliphatic-hydroxy families; aspartame and glutamine are a family containing a guanamine bond; alanine, proline, Leucine and isoleucine are aliphatic families; and phenylalanine and tryptophan tyrosine are aromatic families. For example, it may be reasonably expected to replace leucine with isoleucine or valine, replace aspartic acid with glutamic acid, replace sulphonic acid with serine, or replace a structurally related amino acid. A similar substitution of an amino acid does not affect the binding force or properties of the resulting molecule, especially if the substitution is not related to the amino acid at the framework position. Whether or not the amino acid change produces a functional peptide can be easily determined by analyzing the specific activity of the peptide derivative. Fragments or analogs of the protein/peptide of the present invention can be readily prepared by those of ordinary skill in the art to which the present invention pertains. The preferred amine and carboxy terminal positions of the fragment or analog occur at the near boundary of the functional domain. In one embodiment, a monobasic acid residue (eg, proline) of the synthetic peptide of the present invention is conservatively substituted (eg, substituted with leucine). In other embodiments, the diamino acid residue of the synthetic peptide of the present invention is conservatively substituted into other suitable amino acid residues, for example, proline (V) and arginine (R). Replacement with a pair of amino acids including, but not limited to, methionine (M) and lysine (K), lysine (K) and valine (P), tryptophan ( W) and isoleucine (I), isoleucine (I) and proline (P), aspartame (N) and proline (V), and glutamine (G) and Amino acid (K).

The term "Percentage (%)") means the percentage of the amino acid residue of the first peptide sequence that is identical to the amino acid residue of the second peptide sequence; In the case of alignment, the sequence is aligned and a gap is introduced as needed to achieve a maximum percent identity, and no conservative substitution is considered as part of the sequence similarity. The alignment can be performed using methods well known to those skilled in the relevant art and determining the same percentage of the two sequences; for example, alignment can be performed using computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) analysis. One skilled in the relevant art can use appropriate parameters (including various algorithms) to measure the alignment to produce the largest aligned sequence in the full length sequence of the amino acid. In the present disclosure, sequence alignment between diamino acid sequences is performed using the Blastp (protein-protein BLAST) computer software provided online by the National Center for Biotechnology Information (NCBI). Specifically, the amino acid sequence between a specific amino acid sequence A and a specific amino acid sequence B is the same percentage (may also be expressed as a specific amino acid sequence A and a specific amino acid sequence B has a certain The percentage of amino acid sequence identity is calculated using the following formula: Where X is the number of amino acid residues that match each other between the A and B sequences after comparison with the BLAST, and Y is the total number of amino acid residues in the A or B sequence (whichever is shorter).

The term "vaccine" as used in this disclosure refers to a composition that, when inoculated, can elicit an immune response in the animal, which can completely or partially protect the animal against a disease (eg, Cancer) or the symptoms of the disease. The term "vaccine" covers prophylactic or therapeutic vaccines. The term "combination vaccine" refers to a conjugate vaccine that combines two or more vaccines.

The term "pharmaceutically acceptable" means a "generally regarded as safe" molecular entity and composition, for example, such molecular entities and compositions are physiologically tolerable, And when administered to the human body, there is usually no allergic or similar adverse reaction, such as symptoms of stomach discomfort, dizziness or the like. Preferably, "pharmaceutically acceptable" in this disclosure means that the molecular entity and composition have been certified by the competent authority of the federal government or have been certified by the competent authority of the state government, or listed in the US Pharmacopoeia or other generally recognized The Pharmacopoeia is intended to be used in animals, especially for the human body.

The term "adjuvant", unless defined otherwise in the present disclosure, is used herein to mean any of enhances, increments, upwardly modulates, diversifies or others. A substance or mixture of substances used to modulate an immune response (eg, humoral or cellular immune response) against an antigen.

The term "antigen" or "antigenic agent", unless otherwise defined in the present disclosure, means that when introduced into an immune-sound human or animal body, it can elicit humoral immunity or Any medium for cellular immune responses. The antigen can be a pure substance, a mixture, a specific material (including cells, cell debris or cell-derived fragments) or a living (usually attenuated) organism or virus. Exemplary suitable antigens include, but are not limited to, proteins, glycoproteins, lipoproteins, polypeptides, peptides, carbohydrates/polysaccharides. ), lipopolysaccharide, toxin, virus, bacterium, fungus, and parasite.

The term "immunogenicity" as used in this disclosure refers to the ability of an immunogen, antigen or vaccine to elicit an immune response.

The term "vector" as used in this disclosure means a nucleic acid molecule that can transfer another nucleic acid molecule to which it is linked. One type of vector is "plasmid", which refers to a circular double-stranded DNA that can bind an additional DNA fragment. Another type of vector is a phage vector. Another type of vector is a viral vector in which an additional DNA fragment can be ligated into the viral genome. Certain vectors can be autonomously replicated in the host cell into which they are introduced (e.g., a bacterial vector having a bacterial origin of replication and a mammalian vector of an additional genetic unit (episomal)). Other vectors (e.g., mammalian vectors that are not additional genetic units) can be integrated into the host cell's genome when introduced into the host cell, and thus can be replicated along with the host gene. In addition, certain vectors may dominate the expression of the gene on its operably linked genes. These vectors are referred to herein as "recombinant expression vectors" (or simply "recombinant vectors"). In general, expression vectors for use in recombinant DNA techniques are typically in the form of plasmids. In the present disclosure, "plasmid" and "vector" are used interchangeably, since the plasmid is the most commonly used form of vector.

"Polynucleotide" and "nucleic acid" are interchangeable terms in the present disclosure and refer to a polymer of nucleotides of any length, including DNA and RNA. The nucleotide may be a deoxyribonucleotide, a ribonucleotide, a modified nucleotide or base, and/or an analog thereof, or any of which may be by DNA or RNA polymerase or a synthetic reaction. A matrix incorporated into the polymer.

The term "antibody" as used in this disclosure refers to an immunoglobulin molecule that specifically binds to an epitope on an antigen. The antibody can be an intact immunoglobulin of natural origin or from recombinant technology and can be an immunoreactive portion of the intact immunoglobulin. Antibodies are typical tetramers of immunoglobulin molecules. Antibodies may exist in various forms in the disclosure, including, for example, polyclonal antibodies, monoclonal antibodies (eg, full length or intact monoclonal antibodies), monovalent antibodies, multivalent antibodies (multivalent) Antibodies, multispecific antibodies (eg, bispecific antibodies, as long as the antibody exhibits a particular biological activity), may also include certain antibody fragments, including variant fragments (Fv), antigen-binding fragments (Fab) and F(ab)2 fragments (having two Fab portions joined to each other by a disulfide bond), and single-chain variant fragment (scFv) antibodies. The antibody can be a chimeric, humanized antibody, human antibody, and/or affinity matured antibody.

The term "isolated" refers to an antibody that is identified and isolated, and/or a component that is recovered from the native native environment. Contaminant components recovered from the natural native environment are those that interfere with the research, diagnostic or therapeutic use of the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In one embodiment, the antibody is purified: (1) an antibody greater than 95% molecular weight is purified using an antibody quantification method, such as the Lowry's method (Folin-phenol reagent method), and in certain embodiments, greater than 99 % molecular weight, (2) using, for example, a spinning cup sequenator to obtain at least the extent of 15 amino acid residues from the N-terminus or within the amino acid internal sequence, or (3) The homologues in SDS-PAGE in the reduced or non-reduced state are purified by, for example, Coomassie blue staining or silver staining. An isolated antibody includes an antibody in situ in a recombinant cell, since at least one component of the native native environment of the antibody is absent. In general, however, at least one purification step is required to prepare the isolated antibody.

The term "complementarity determining region" (CDR) in the present disclosure refers to a hypervariable region of an antibody molecule that forms a surface complementary to the three-dimensional surface of a sessile antigen. Advancing from the N-terminus to the C-segment, the heavy and light chains of each antibody comprise three CDRs (CDR 1, CDR 2 and CDR3). Thus, an antigen binding site comprises a total of six CDRs comprising three CDRs from the heavy chain variant region and three CDRs from the light chain variant region. The amino acid residues of the CDRs are in intimate contact with the immobilizing antigen, with the most intense antigenic contact typically associated with the heavy chain CDR3.

The term "substantially similar" or "equivalent" in the present disclosure means that there is a substance between two values (for example, one relating to the molecule of the invention and the other to the reference/comparative molecule). A high degree of similarity such that the skill in the art to which the present invention pertains will be considered to be the difference between two values within the context of the biological properties measured by the values (e.g., half-life, Kd value, antiviral effect, etc.). Has little or no biological and/or statistical significance. As a function of the numerical value of the reference/compare molecule, the difference between the two values is, for example, less than about 50%, less than about 40%, less than about 30%, less than about 20%, and less than about 10%.

The term "binding affinity" generally refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise indicated, "binding affinity" in this disclosure refers to the intrinsic binding affinity that reflects a 1:1 interaction between a member of a binding pair (eg, an antibody and an antigen). The affinity of the molecule X for its partner Y can generally be expressed by the dissociation constant (Kd). Affinity can be measured by conventional methods well known in the art, including those described in this disclosure. Low-affinity antibodies typically bind antigen slowly and tend to dissociate easily, while high-affinity antibodies generally bind antigen more rapidly and tend to maintain longer binding. A variety of methods for measuring binding affinity are well known in the art, any of which can be used for the purposes of this disclosure.

The term "variable region" or "variable domain" of an antibody refers to the amino terminal domain of an antibody heavy or light chain. These domains are generally the most variable part of the antibody and comprise an antigen binding site.

The term "variable" refers to the fact that certain portions of the variant region differ widely in sequence between antibodies and are used for the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the entire domain of the antibody. It focuses on three segments called complementarity determining regions (CDRs) or hypervariable regions (HVRs) in the light and heavy chain variant domains. The more highly conserved part of the variability domain is called the framework region (FR). The variant domains of the native heavy and light chains each comprise four FRs, most of which adopt a beta-sheet configuration, joined by three CDRs that form a circular junction and in some cases form part of a beta-sheet structure. The CDRs in each chain are held together very closely by the FR and together with the CDRs of the other chain contribute to the formation of the antigen binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domain is not directly involved in the binding of the antibody to the antigen, but exhibits multiple effector functions, such as the involvement of antibodies in antibody-dependent cellular toxicity.

Digestion of antibodies with papain produces two identical antigen-binding fragments, called "Fab" fragments, each with an antigen-binding site and a remaining "Fc" fragment whose name reflects its ability to crystallize readily. . Pepsin treatment produces an F(ab')2 fragment that has two antigen binding sites and is still capable of cross-linking antigen.

The term "Fv" is the smallest antibody fragment that contains the entire antigen recognition and binding site. In the double-stranded Fv species, this region consists of a dense, non-covalently bound one heavy chain variant domain and one light chain variant domain dimer. In the single-chain Fv (scFv) species, one heavy chain variant domain and one light chain variant domain can be covalently linked by a flexible peptide linker such that the light and heavy chains resemble a double strand. The "dimer" structure of the Fv species is combined. It is in this configuration that the three CDRs of each variant interact to define an antigen binding site on the surface of the VH-VL dimer. The six CDRs together confer antigen specificity to the antibody. However, even a single variant domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind antigen, but with a lower affinity than the entire binding site.

The Fab fragment also comprises a constant domain of the light chain and a first constant domain of the heavy chain. The Fab' fragment differs from the Fab fragment in that the carboxy terminus of the first constant domain of the heavy chain is increased by a few residues, including one or more cysteine acids from the antibody hinge region. Fab'-SH is the designation of Fab' in which the constant domain cysteine residues carry a free thiol group in the disclosure. The F(ab')2 antibody fragment was originally formed as a pair of Fab' fragments having hinged cysteine between the Fab' fragments. Other chemical ligation methods for antibody fragments are also well known.

According to the amino acid sequence of its constant domain, the "light chains" of antibodies (immunoglobulins) from any vertebrate species can be classified into one of two distinct types, called kappa. , κ) and lambda (λ).

Antibodies (immunoglobulins) can be assigned to different classes based on their heavy chain constant domain amino acid sequences. Immunoglobulins are divided into five major types: IgA, IgD, IgE, IgG, and IgM, and some of them can be further divided into subtypes (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different types of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively. Subunit structures and three-dimensional structural lines of different types of immunoglobulins are well known and are generally described in reference to Abbas et al. Cellular and Mol. Immunology, 4th ed. (2000). An antibody can be part of a larger fusion molecule formed by covalent or non-covalent association of an antibody with one or more other proteins or peptides.

The term "antibody fragments" encompasses only a portion of an intact antibody, wherein the portion retains at least one, up to most, or all of the functions normally associated with the portion when present in the intact antibody. In one embodiment, an antibody fragment comprises an antigen binding site of an intact antibody, thereby retaining the ability to bind antigen. In another embodiment, an antibody fragment, eg, an antibody fragment comprising an Fc region, retains at least one biological function typically associated with the Fc region, such as FcRn binding, antibody half-life regulation, when the Fc region is present in an intact antibody , ADCC function and complement combination. In one embodiment, the antibody fragment is a monovalent antibody having an in vivo half-life substantially similar to the intact antibody. For example, such an antibody fragment can comprise an antigen binding arm and is linked to an Fc sequence capable of conferring stability to the fragment in vivo.

The term "monoclonal antibody" as used in this disclosure refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except that there may be minimal amounts of naturally occurring mutations. Thus, the modifier "monoclonal" indicates that the antibody is not a feature of a discrete mixture of antibodies. Such monoclonal antibodies typically comprise an antibody comprising a polypeptide sequence that binds to the subject, wherein the target binding polypeptide sequence is obtained by a process comprising selecting a single target binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be to select a unique colony from a collection of numerous strains, such as fusion tumor strains, phage colonies, or recombinant DNA strains. It will be appreciated that the selected target binding sequence may be further altered, for example, to increase affinity for the subject, humanize the target binding sequence, increase its yield in cell culture, and reduce its immunogenicity in vivo. An antibody that comprises a multi-specific antibody, and the like, and which comprises the altered binding sequence is also a monoclonal antibody of the present invention. Unlike typical antibody preparations that contain different antibodies directed against different epitopes, each monoclonal antibody of a monoclonal antibody preparation is directed against a single epitope on the antigen. In addition to their specificity, monoclonal antibody preparations have the advantage that they are generally not contaminated by other immunoglobulins. The term "monoclonal" as used herein to indicate that a antibody is obtained from a substantially homogeneous population of antibodies should not be construed as requiring that the antibody be prepared by any particular method. For example, monoclonal antibodies used in accordance with the present disclosure can be prepared by a variety of techniques, including, for example, fusion tumor methods (e.g., Kohler et al., Nature, 256: 495 (1975); Harlow et al., Antibodies : A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA methods (see for example U.S. Patent 4,816,567, phage display technology (see, for example, Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Sidhu et al J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004)), and for having partial or entire Techniques for preparing human or human-like antibodies in an animal of an immunoglobulin locus or a gene encoding a human immunoglobulin sequence (see, for example, WO 98/24893; WO 96/3409 6; WO 96/33735; WO 91/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993); Bruggemann et al. , Year in Immunol. 7:33 (1993); US Patent 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016; Marks et al., Bio. Technology 10: 779-783 (1992); Lonberg et al., Nature 368 : 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al., Nature Biotechnol. 14: 845-851 (1996); Neuberger, Nature Biotechnol. 14: 826 (1996) and Lonberg And Huszar, Intern. Rev. Immunol. 13: 65-93 (1995)).

Monoclonal antibodies specifically include in the present disclosure "chimeric" antibodies in which a portion of the heavy and/or light chain is identical to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody type or subtype or Homologous, and the remainder of the strand is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody type or subtype, and fragments of such antibodies, as long as they exhibit a particular biological activity ( U.S. Patent 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

A "humanized" form of a non-human (e.g., murine) antibody refers to a chimeric antibody that minimally comprises a sequence derived from a non-human immunoglobulin. In one embodiment, the humanized anti-system refers to a highly variable region residue in a human immunoglobulin (receptor antibody) using a non-human species (donor antibody) having a specific specificity, affinity and/or ability, such as small Immunoglobulins replaced by highly variable region residues of murine, rat, rabbit or non-human primates. In certain instances, the framework region (FR) residues of human immunoglobulin are replaced with corresponding non-human residues. Furthermore, humanized antibodies may comprise residues not found in the recipient antibody or in the donor antibody. These modifications can be made to further improve the performance of the antibody. In general, a humanized antibody will comprise at least one, usually two substantially entire, variant domains, wherein all or substantially all of the hypervariable loops correspond to a variant loop of a non-human immunoglobulin, and all or substantially All FRs are FRs of human immunoglobulin sequences. The humanized antibody will also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically a constant region of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); Presta, Curr. Op. Struct. Biol. 2:593-596 ( 1992). See also the following review and references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23: I 035-I 038 (1995) Hur Ie and Gross, Curr. Op. Biotech. 5: 428-433 (1994).

The term "hypervariable region", "HVR" or "HV", in this disclosure, refers to a region of antibody variant domain that is highly variable in sequence and/or forms a structurally defined loop. In general, an antibody comprises six highly variable regions; three in VH (HI, H2, H3) and three in VL (LI, L2, L3). There are many descriptions of highly variable regions that are in use, and are covered by this disclosure. The Kabat complementarity determining region (CDR) is based on sequence variability and is most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. ( 1991)). Chothia refers to the position of the structural loop of substitution (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The AbM hypervariable region is a compromise between the Kabat CDR and Chothia structural loops and is used by the Oxford molecular AbM antibody modeling software. The "contact" highly variable region is based on available composite crystal structure analysis. Each of the residues from these highly variable regions are listed below.

Ring Kabat AbM Chothia Contact

L1 L24-L34 L24-L34 L26-L32 L30-L36

L2 L50-L56 L50-L56 L50-L52 L46-L55

L3 L89-L97 L89-L97 L91-L96 L89-L96

H1 H31-H35B H26-H35B H26-H32 H30-H35B

(Kabat number)

H1 H31-H35 H26-H35 H26-H32 H30-H35

(Chothia number)

H2 H50-H65 H50-H58 H53-H55 H47-H58

H3 H95-H102 H95-H102 H96-H101 H93-H101

The highly variable region may comprise the following "extended hypervariable regions": 24-36 or 24-34 (L1), 46-56 or 49-56 or 50-56 or 52-56 in the VL. (L2) and 89-97 (L3), and 26-35 (Hl), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3) in VH. According to Kabat et al., supra, each of the variant domain residues in these definitions is numbered.

A "framework" or "FR" residue is one of its variant domain residues other than the highly variable region residues as defined by the present disclosure.

The term "Kabat variant residue number" or "Kabat amino acid position number" and the like refers to the numbering system used in the antibody compilation for the heavy chain variant domain or the light chain variant domain, which is compiled as Kabat. Et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to the FR or HVR of the truncated or inserted variant domain. For example, a heavy chain variation domain can include insertion of a single amino acid (residue 52a according to Kabat) after residue 52 of H2, and insertion of a residue after position 82 of the heavy chain FR residue (according to Kabat, such as residues 82a, 82b, and 82c, etc.). For a particular antibody, the Kabat numbering of the residues can be determined by alignment of the "standard" Kabat numbered sequences against the homologous regions of the antibody sequences.

A "single-chain Fv" or "scFv" antibody fragment comprises the VH and VL domains of an antibody, wherein such domains are present on a single polypeptide chain. In general, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains such that the scFv is capable of forming a specific structure that binds to the antigen. For a review of scFv, see Pluckthun, in: The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

The term "diabodies" refers to a small antibody fragment having two antigen-binding sites that contain linked heavy chain variant domains (VH) and light chain variants in the same polypeptide chain (VH-VL). Domain (VL). By using a short linker, the two domains on the same chain cannot be paired, forcing these domains to pair with the complementary domains of the other chain, resulting in two antigen binding sites. Diabodies are more fully described, for example, in EP 404,097; WO 93/1161; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).

The term "human antibody" refers to an amino acid sequence having an amino acid sequence corresponding to an antibody produced by a human and/or an antibody prepared using any of the techniques disclosed in the disclosure for the preparation of a human antibody. . This definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen binding residues.

The term "affinity matured" refers to one or more alterations in one or more HVRs of an antibody, resulting in an affinity of the antibody for the antigen compared to the parent antibody without such alteration. Improved antibodies. In one embodiment, the affinity matured antibody has an affinity for a target antigen on the order of nanomolar or even picomoles. Affinity matured antibodies can be prepared by procedures well known in the art. Affinity maturation by VH and VL domain shuffling is described by Marks et al. Bio/Technology 10:779-783 (1992). Random mutagenesis of CDR and/or framework residues is described in the following literature: Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al., Gene 169:147-155 (1995) Yelton et al., J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol. 226:889-896 (1992).

The term "blocking" antibody or "antagonist" antibody refers to an antibody that inhibits or reduces the biological activity of the antigen to which it binds. Certain blocking antibodies or sputum resistant antibodies can substantially or completely inhibit the biological activity of the antigen.

The term "agonist antibody" as used in this disclosure refers to an antibody that mimics the functional activity of at least one particular polypeptide.

"effective amount" - as used herein, refers to an amount of a component sufficient to produce a desired therapeutic response. In other words, the term "effective amount" refers to an effective necessary dose to achieve the desired therapeutic or prophylactic effect. For therapeutic purposes, an effective amount also refers to a compound or composition whose therapeutic benefit effects exceed the toxic or detrimental effects of the ingredient. The specific effective amount depends on various factors such as the particular condition being treated, the physiological condition of the patient (eg, patient weight, age or sex), the mammal or animal species being treated, the duration of treatment, current therapy (if any) The nature of the words, the particular dosage form employed, and the structure of the compound or its derivatives. For example, an effective amount can be expressed in grams, milligrams or micrograms or a few milligrams per kilogram of body weight (mg/kg, mg/kg). Alternatively, an effective amount can be expressed as the concentration of the active ingredient (e.g., synthetic polypeptide or antibody in the disclosure), such as molar concentration, mass concentration, volume concentration, weight molarity. Molarity, mole fraction, mass fraction, and mixing ratio. In particular, there is a "therapeutically effective amount" of a synthetic polypeptide or an antibody in the present disclosure, which in the present disclosure means sufficient to alleviate or ameliorate an individual and cancer. Synthetic polypeptides of related symptoms or antibody doses in the present disclosure. The art to which the present invention pertains has a human equivalent dose (HED) that can be converted into a drug, such as a synthetic polypeptide or antibody of the present disclosure, based on the dose obtained by a person skilled in the art. For example, those skilled in the art to which the present invention pertains can generally estimate the maximum safe starting dose of an adult clinical volunteer in an initial clinical treatment according to the US Food and Drug Administration (FDA). (Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers) to estimate the highest safe dose for human use.

A "therapeutically effective amount" of a polypeptide or antibody, unless otherwise indicated, refers to an amount sufficient to provide a therapeutic benefit in the treatment or management of the disease or condition, or in delaying or minimizing one or more symptoms associated with the disease or condition. A therapeutically effective amount of a polypeptide or antibody is indicative of the amount of a therapeutic agent, alone or in combination with other therapies, to provide a therapeutic benefit in the treatment or management of the disease or condition. A "therapeutically effective amount" can encompass a quantity that improves overall efficacy, reduces or avoids the symptoms or causes of the disease or condition, or enhances the therapeutic efficacy of another therapeutic agent.

The term "prophylactically effective amount" of a polypeptide or antibody, unless otherwise indicated, means that the amount is sufficient to prevent, or prevent, one or more symptoms associated with, or associated with, the disease or condition. A prophylactically effective amount of a polypeptide or antibody refers to the amount of a therapeutic agent, alone or in combination with other agents, which provides a prophylactic benefit in the prevention of disease. The term "prophylactically effective amount" can encompass an amount that improves overall prevention or enhances the preventive effect of another therapeutic agent. Typically, but not necessarily, the prophylactically effective amount will be less than the therapeutically effective amount because the prophylactic dose used in the individual is earlier or in the early stages of the disease.

The term "subject" or "patient" refers to a vertebrate. In certain embodiments, the vertebrate is a mammal. Mammals include, but are not limited to, agricultural animals (e.g., cattle), sports animals, pets (e.g., cats, dogs, and horses), primates, mice, and rats. In certain embodiments, the mammal refers to a human that can be treated by the methods of the invention of the present disclosure. Unless specifically stated as one of the genders, the term "individual" or "patient" means that the gender is male or female.

For the purposes of treatment, the term "mammal" ( mammal) means any animal classified as a mammal, including humans, households and agricultural animals, as well as zoos, sports or pet animals such as dogs, horses, cats, Cow, etc. In certain embodiments, the mammal is a human.

2. Description of the preferred embodiment

At least a portion of the present disclosure is based on three polypeptides that the inventors have found to overexpress on the surface of cancer cells as compared to normal cells. Thus, the polypeptides can be utilized as antigens to develop antibodies and vaccine compositions for the prevention and/or treatment of cancer.

(i) Polypeptides and their uses

The first aspect of the present disclosure relates to polypeptides that are overexpressed on the surface of different types of cancer cells, including gastric cancer, lung cancer, bladder cancer, breast cancer, pancreatic cancer, kidney cancer, colorectal cancer, cervical cancer, ovarian cancer. , brain tumor, prostate cancer, hepatocellular carcinoma, melanoma, esophageal cancer, multiple myeloma and head and neck squamous cell carcinoma.

According to some embodiments of the present disclosure, the polypeptide comprises an amino acid sequence having at least 85% sequence similarity to any of SEQ ID NO: 1 to 3; in other words, the amino group comprised by the polypeptide of the present invention Acid sequence and sequence number: 1, 2 or 3 may have 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 Sequence similarity of %, 98%, 99% or 100%. In one embodiment, the polypeptide having the amino acid sequence of SEQ ID NO: 1 is designated "polypeptide CH-1" (CH-1 polypeptide). In another embodiment, the polypeptide having the amino acid sequence of SEQ ID NO: 2 is designated "polypeptide CH-2" (CH-2 polypeptide). In still another embodiment, the polypeptide having the amino acid sequence of SEQ ID NO: 3 is designated "CH-3 polypeptide".

Depending on the particular purpose, one of ordinary skill in the art to which the present invention pertains can replace one or more of the amino acid residues of the polypeptides CH-1, CH-2 or CH-3 of the present invention with a conservative amino acid (such as to brighten The amino acid is replaced by isoleucine or valine or alanine is replaced by proline to achieve the same/similar stimulatory efficacy.

Preferably, the N-terminus of the polypeptide of the invention has an acetamidine modification, a saccharification modification or a methylation modification. Alternatively or additionally, the C-terminus of the polypeptide of the invention is modified by amidoximation or saccharification.

Optionally, the N-terminus or C-terminus of the polypeptide of the invention is conjugated to a carrier molecule to increase the immunogenicity of the polypeptide. Exemplary common carrier molecules include, but are not limited to, OVA, BSA, KLH, TGB, and combinations thereof.

The polypeptide of the present invention as described above can be synthesized by a conventional method such as a FMOC protection method such as t-BOC or an α-amine group. Both of these methods are related to the stepwise synthesis method, that is, starting from the C-terminus of the polypeptide, an amino acid is added in each step. Alternatively, the polypeptides of the invention can be synthesized using the common solid phase peptide synthesis methods. Alternatively, host cells (e.g., HEK293 cells) transfected with a nucleic acid encoding the polypeptide can be used to prepare a synthetic polypeptide of the invention.

Based on the above-mentioned polypeptides, which are excessively expressed on the surface of cancer cells, the polypeptides can be used as tumor antigens to stimulate an individual to produce an immune response specific to the antigen, thereby providing protection to the individual. More specifically, once a polypeptide is administered to an individual via an appropriate route (eg, via mucosa, subcutaneous, intratumoral, intradermal, intramuscular, intravenous, or intraperitoneal), the polypeptide can elicit a polypeptide-associated immune response in the individual ( For example, a T cell that produces specificity for a polypeptide is produced, and the immune effect produced by the challenge locks and attacks cancer cells that express the polypeptide.

Accordingly, another aspect of the present disclosure is directed to a method for preventing and/or treating cancer in an individual in need thereof; for example, the individual is at risk of developing cancer, or the subject is suffering from or suspected of having cancer . The methods of the invention comprise administering to the individual an effective amount (e.g., a prophylactically effective amount or a therapeutically effective amount) of a polypeptide of the invention.

The polypeptide of the invention may be administered to the individual via a route selected for a particular purpose, wherein the pathway is selected from the group consisting of mucosa, subcutaneous, intratumoral, intradermal, intramuscular, venous, and intraperitoneal.

It will be appreciated that the methods of the invention may be administered to an individual, or co-administered to an individual in combination with other therapies known to increase the immune response of the individual and/or reduce the immunosuppressive effects. For example, the polypeptide of the present invention can be administered to an individual together with interleukin-12 (IL-12, a cytokine known to enhance T cell response); alternatively, the polypeptide of the present invention can be combined with a polypeptide For immunosuppressive cells (eg, regulatory T cells (Treg), myeloid derived suppressor cells (MDSC) and type 2 macrophages) and/or cytokines (such as interleukin- 10 (IL-10) and transforming growth factor-β (TGF-β)) The production and/or function of the inhibitory effect of the agent is co-administered into the individual. The subject invention can be administered to the subject prior to, concurrently with, or after administration of the therapy, depending on the particular therapeutic purpose.

(ii) a vaccine composition comprising the polypeptide of the invention and use thereof

Another aspect of the disclosure relates to vaccine compositions developed using the polypeptides of the invention. In particular, the vaccine compositions of the invention comprise a polypeptide of the invention and a pharmaceutically acceptable adjuvant.

As is well known to those of ordinary skill in the art to which the present invention pertains, an adjuvant is a substance that increases the immune response of an antigen, such as a polypeptide of the invention. Exemplary adjuvants useful for enhancing the polypeptides of the invention include, but are not limited to, fatty alcohol polyoxyethylene ether-D, aluminum hydroxide, Freund's incomplete adjuvant, Freund's complete adjuvant, and endotoxin-related Agent, mineral oil, mineral oil and surfactant, Ribi adjuvant, gold adjuvant, Syntax adjuvant preparation, aluminum salt adjuvant, nitrocellulose adsorption antigen, immunostimulating complex, Gebru adjuvant, overload agent, ethylene and Copolymer of vinyl acetate, L-tyrosine acid, oil-based emulsion, Adju prime adjuvant, squalene, sodium phthalocyanine, calcium phosphate, saponin and cell wall dipeptide. According to an operational embodiment, the adjuvant is a fatty alcohol polyoxyethylene ether-D.

The present disclosure also provides a method for preventing and/or treating a cancer in an individual in need thereof (eg, the individual has a risk of developing cancer, or the individual is suffering from or suspected of having cancer). The methods of the invention comprise administering to the individual an effective amount (e.g., a prophylactically effective amount or a therapeutically effective amount) of a vaccine composition of the invention, whereby the individual is vaccinated to produce an immune response against cancer.

In general, the effective amount of the vaccine composition or active ingredient (i.e., the polypeptide of the invention) contained therein may depend on a number of factors, such as the physiological condition of the patient (e.g., the body mass, age or sex of the patient), to be treated The type of mammal or animal, the duration of the treatment, and the nature of the concurrent treatment, if any.

According to one embodiment, the individual is a mouse. To produce an vaccination effect in mice, the subject is administered about 0.01 to 1,000 mg of the polypeptide of the invention per kilogram of body weight per dose; for example, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06 per kilogram of body weight per dose, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 5 00, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990 or 1000 mg of the polypeptide of the invention. Preferably, each dose is from 0.1 to 100 mg per kg of body weight. More preferably, each dose is from 1 to 20 mg per kilogram of body weight. According to an operative embodiment, administration of 4 to 5 mg of the polypeptide of the invention per kilogram of body weight per individual is sufficient to elicit an immune response (e.g., production of antibodies) in the individual.

The art to which the present invention pertains has a human equivalent dose (HED) that can be converted into any of the polypeptides of the present invention by a dose that can be obtained by a person of ordinary skill based on an experimental animal model. Thus, for humans, the effective HED of the polypeptides of the invention is from about 0.8 micrograms to about 80 milligrams per kilogram of body weight; in other words, for humans, the effective HED of the polypeptides of the invention can be 0.8 per kilogram of body weight per dose. 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900 or 950 micrograms or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 per kilogram of body weight 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, Any of 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 mg; preferably, for each The agent is from 8 micrograms to 8 milligrams per kilogram of body weight; more preferably, from 80 micrograms to 800 micrograms per kilogram of body weight per dose. In a preferred embodiment, the effective HED is from 300 micrograms to 400 micrograms per kilogram of body weight per dose.

According to an embodiment of the present disclosure, the vaccine composition is administered to the individual at least twice during the vaccination session. For example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more are administered. The vaccination regimen may depend on a variety of factors including, but not limited to, the patient's physiological condition (eg, the patient's physical quality, age, or gender), the individual class to be vaccinated, and the antigen to be immunized/ The essence of the vaccine composition. Basically, the vaccine composition can be administered to the individual 6 to 10 times, from several days to several years.

For example, in one embodiment of the present disclosure, the individual is a mouse, and the vaccine composition of the present invention is administered to the individual every week for 8 weeks, thereby inducing specificity to the polypeptide in the individual. Sexual immune response.

The vaccine composition of the invention can be administered to the individual via any suitable route, such as via mucosa, subcutaneous, intradermal, intramuscular, intravenous or intraperitoneal. According to a particular embodiment, the subject is administered a vaccine composition of the invention via subcutaneous injection.

In accordance with certain embodiments of the present disclosure, the methods of the invention facilitate eliciting an anti-tumor response (e.g., producing an anti-tumor antibody). In one embodiment, the serum antibody (i.e., the mouse serum following immunization of the mouse with an antigen, comprising an antibody specific for the antigen) elicited by the method of the present invention has a cytotoxic effect on the tumor cells. In another embodiment, the serum antibodies elicited by the methods of the invention inhibit tumor growth in an individual.

According to one embodiment, the antibody elicited by the polypeptide CH-1, CH-2 or CH-3 compared to the antibody produced by the polypeptides CH-4 (SEQ ID NO: 4) and CH-5 (SEQ ID NO: 5), respectively It can trigger a higher amount of cancer cells to apoptosis.

It will be appreciated that the methods of the invention may be administered to an individual, either alone or in combination with other methods of treatment, including surgery, radiation therapy, chemotherapy, hormonal therapy, anti-angiogenic therapy, and immunotherapy. For example, the subject can be administered a composition of the invention, followed by administration of any of the immunotherapies to enhance the immune response elicited by the vaccine compositions of the invention. The subject invention can be administered to the subject prior to, concurrently with, or after administration of the therapy, depending on the particular therapeutic purpose.

Essentially, the individual to be treated by the methods of the invention is a mammal, for example, human, mouse, rat, hamster, guinea pig, rabbit, dog, cat, cow, goat, sheep, monkey, and horse. Preferably, the individual is a human.

(iii) antibodies produced by the polypeptides and/or vaccine compositions of the invention and uses thereof

Another aspect of the present disclosure relates to an antibody produced by an immunological reaction specific for a polypeptide as described in Section (ii) of the present disclosure.

Exemplary antibody preparation method

Exemplary antibodies that bind to the polypeptides of the present disclosure can be made using any of the methods well known in the art. See, for example, Harlow and Lane, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.

Host animal immunization and fusion tumor technology

The polypeptide of the invention described in Section (i) or the vaccine composition described in Section (ii) can be immunized into a host animal, such as a mouse, rat or rabbit, to prepare an exemplary antagonist against the polypeptide of the present disclosure. Multiple antibodies. The antigen can be immunized into any mammal to produce a particular antibody. In general, animals such as rodents, rabbits or primates can be used. Rodents include, for example, mice, rats, and hamsters. Rabbits include, for example, rabbits. Primates include, for example, Catarrhini monkeys (Taiwan monkeys) such as Macaca fascicularis, rhesus monkeys, baboons, and chimpanzees. Immunization can be carried out according to conventional procedures. There is no special restriction on the negotiating time schedule. The time course for performing immunization may be from several days to several weeks, preferably from 2 to 10 times per week until a specific antibody titer is reached.

According to certain embodiments, the vaccine compositions of the present invention are vaccinated against the host animal by subcutaneous (sc) or intraperitoneal (ip) (iso) routes for 8 consecutive weeks on a weekly basis. In certain embodiments, it may be helpful to conjugate a polypeptide of the present disclosure to a protein that is immunogenic in the species to be immunized, such as keyhole limpet hemocyanin, serum albumin. Fetal calf thyroglobulin or the use of bifunctional or derivatized agents (eg, maleic iminobenzamide sulfo amber ylide (joined by cysteine residues), N-hydroxy amber Soybean trypsin inhibitor of imine (joined by an amine acid residue), glutaraldehyde, succinic anhydride, sulfoxide, etc.).

After the final immunization, the spleen cells and regional lymph nodes of the experimental animals were removed. Further, blood samples of the experimental animals are periodically taken after the start of immunization, and the serum is separated by centrifugation. The antibody titer in the obtained serum is measured by an appropriate detection method including, but not limited to, enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA). Or radio immunoassay (RIA). In a preferred embodiment, antibody titers are detected by ELISA assay. Next, the final immunization of the experimental animals which produced high antibody titers to the polypeptide of the present invention was carried out. An antibody-producing cell is prepared from spleen cells and regional lymph nodes or the like of the above immunized animal. Tissue fragments and red blood cells are removed as much as possible during the manufacture of antibody-producing cells. A commercially available erythrocyte remover product can be utilized for this purpose. Alternatively, ammonium chloride and Tris buffer can be prepared and used. The antibody-producing cells prepared above are immediately fused with immortal cells (such as myeloma cells) to prepare a hybridoma cell, which can be semi-eternally sustained and proliferated simultaneously. antibody. A common cell strain derived from an animal such as a mouse can be used for this purpose. Cell lines useful in the present invention should not survive in HAT selective cultures (components include hypoxanthine, thymidine, and aminopterin), but when the cells are produced After the cells of the antibody are fused, they should be grown in the culture solution. Exemplary myeloma cells include, but are not limited to, mouse myeloma cell lines (such as myeloma FO cells, sourced from the Salk Institute Cell Distribution Center, San Diego, Calif. USA). The cells of the mouse tumors MOPC-21 and MPC-11, as well as the SP-2 cells supplied by the American Type Culture Collection, Rockville, Md. USA). The published literature has documented human myeloma cell lines and mouse-human heteromyeloma cell lines, including Karpas 707H and others described in Kozbor, J. Immunol. , 133:3001 (1984); and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987).

Immunocytes (i.e., antibody producing cells) and myeloma cells can be fused by known methods, for example, by Milstein et al. (Galfre et al., Methods Enzymol. 73:3-46, 1981). Cell fusion is usually a cell fusion promoter (such as polyethylene glycol (PEG), which has an average molecular weight of 200 to 20,000 Daltons or its analogs (Goding, Monoclonal Antibodies: Principles and Practice, In the presence of pp. 59-103 (Academic Press, 1986)), spleen cells or lymph node cells are mixed with myeloma cells. Alternatively, cell fusion can be performed in a cell fusion apparatus employing current stimulation (e.g., electroporation). After cell fusion, the resulting cells are diluted and cultured in HAT medium to screen for fusion-fused fusion tumor cells.

Specific fusion tumor cells are selected from the above fused cells. Fusion cells that are able to survive in HAT medium will form colonies. The supernatant of each well was collected and tested for the presence or absence of antibody titers against the polypeptide of the invention. Preferably, immunoprecipitation or other in vitro binding assays such as ELISA, EIA or RIA and/or immunofluorescence assays can be used to detect individual plants produced by fusion tumor cells. The specificity of the binding of antibodies. Once the culture well positive for the antibody titer is detected, the cells contained in the culture well are cultured in an HT culture solution (without ammonia sputum). After a period of incubation, the antibody titer of the supernatant was reconfirmed. The selected cells are selected by limiting dilution procedures, and the cells are cultured by a conventional method to obtain a single cell population that secretes a single specific antibody, such as a monoclonal antibody (Goding, Monoclonal). Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). A strain that secretes a highly specific antibody to the polypeptide of the present invention is screened and propagated to a certain extent to establish a fusion tumor.

It can be suitably used from culture medium, ascites or serum by conventional immunoglobulin purification methods such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis. (gel electrophoresis), dialysis or affinity chromatography, isolation of monoclonal antibodies secreted by the strain.

In addition, hybridoma cells can be grown in vivo as an ascites tumor in an animal. For example, the resulting hybridoma can be implanted into the abdominal cavity of a mouse and ascites can be collected.

This can be achieved, for example, by ammonium sulfate precipitation, protein A or protein G column, DEAE ion exchange chromatography, or a polypeptide having the present disclosure. The resulting individual antibody was purified by affinity column. The antibodies of the present disclosure are useful not only for the purification and detection of proteins of the present disclosure, but also as agonists and antagonists of the proteins of the present disclosure. Furthermore, the antibodies can be administered to antibody therapies for diseases associated with the proteins of the present disclosure.

Recombination technology

The resulting monoclonal antibodies can also be recombinantly produced using genetic engineering techniques (see, for example, Borrebaeck C. A. K. and Larrick J. W. Therapeutic Monoclonal Antibodies, published in the United Kingdom by MacMillan Publishers LTD, 1990). The DNA encoding the antibody can be selected from an immune cell (such as a hybridoma or a lymphocyte which produces an antibody after immunization), inserted into an appropriate vector, and introduced into a host cell to prepare a recombinant antibody. The disclosure also provides recombinant antibodies prepared as described above.

When the resulting antibody is administered to a human (antibody treatment), a human antibody or a humanized antibody is a preferred choice for reducing immunogenicity. For example, an antigen selected from a polypeptide, a cell expressing a polypeptide, or a lysates thereof can be immunized with a transgenic animal carrying a human antibody gene. The antibody-producing cells are then collected in an automated object and fused with myeloma cells to obtain hybridomas from which human antibodies against the protein can be prepared. Alternatively, immune cells (such as immunized lymphocytes) from which an oncogene will produce antibodies can be immortalized and used to prepare monoclonal antibodies.

DNA encoding a monoclonal antibody can be readily isolated and sequenced using conventional methods (e.g., using oligonucleotide probes that specifically bind to the murine antibody heavy and light chain genes). Hybridoma cells can be a preferred source of such DNA. Once isolated, the DNA can be placed in a performance vector, and the expression vector can be subsequently transfected into a host cell, such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO). Cells, or myeloma cells that do not originally produce immunoglobulin, thereby synthesizing monoclonal antibodies in recombinant host cells. Retrospective literature on the expression of antibody-encoding DNA in bacteria, including Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) and Pluckthun, Immunol. Rev., 130:151-188 ( 1992). DNA encoding an antibody produced by the above-described fusion tumor cells can be genetically modified by conventional techniques to prepare genetically engineered antibodies. Genetically engineered antibodies (such as humanized antibodies, chimeric antibodies, single chain antibodies, and bispecific antibodies) can be prepared, for example, by conventional recombinant techniques. The DNA can then be modified, for example, by replacing the human heavy and light chain constant domain coding sequences to replace the homologous murine sequence (Morrison et al., (1984) Proc. Nat. Acad. Sci. 81:6851), Or by covalently linking all or a portion of the non-immunoglobulin polypeptide coding sequence to an immunoglobulin coding sequence. In this manner, a genetically engineered antibody having a binding specificity of a target antigen, such as a "chimeric" or "fusion" antibody, can be prepared.

Techniques for preparing "chimeric antibodies" are well known in the art. See, for example, orrison et al. (1984) Proc. Natl. Acad. Sci. USA 81, 6851; Neuberger et al. (1984) Nature 312, 604; and Takeda et al. (1984) Nature 314:452.

Typically, such non-immunoglobulin polypeptides are replaced by antibody constant domains or substituted into variant domains of an antigen binding site of an antibody to create a chimeric bivalent antibody comprising An antigen binding site that is specific for an antigen and another antigen binding site that is specific for a different antigen.

Chimeric or fusion antibodies can also be prepared in vitro using known methods in synthetic protein chemistry, including those involving cross-linkers. For example, an immunotoxin can be constructed using a disulfide exchange reaction or by forming a thioether bond. Exemplary suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.

Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues from a non-human source. Such non-human amino acid residues are often referred to as "input" residues, which are typically taken from the "input" variant domain. Humanization can be based essentially on the method of Winter and colleagues (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)), replacing the corresponding sequence of a human antibody with a rodent CDR or CDR sequence. Thus, such "humanized" antibodies are chimeric antibodies (U.S. Patent 4,816,567) in which the corresponding sequence substitutions from non-human species are substantially less than the intact human variant domain. In practice, a humanized antibody is a human antibody that typically replaces certain CDR residues and possibly certain FR residues with residues at analogous sites of a rodent antibody.

The selection of human variant regions for the preparation of humanized antibodies, including both light and heavy chains, is important to reduce antigenicity. The entire library of known human variant region sequences was screened for the rodent antibody variant region sequence according to the so-called "best-fit" method. The human sequence closest to the rodent is then selected as the human framework region (FR) of the humanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol ., 196:901 (1987)). Another approach is to use specific framework regions derived from the consensus sequence of all human antibodies of a particular light or heavy chain subpopulation. Several different humanized antibodies can be used in the same framework region (Carter et al., Proc. Natl. Acad Sci. USA, 89: 4285 (1992); Pretaetal., J. Immnol., 151: 2623 (1993)).

More importantly, antibodies retain high affinity for antigens and other beneficial biological properties after humanization. To accomplish this, humanized antibodies are prepared according to a preferred method using a three-dimensional model of the parental and humanized sequences to analyze the parental sequences and various conceptual humanized products. Three-dimensional immunoglobulin models are commonly available and are well known in the art. A computer program can be obtained that graphically illustrates and displays the possible three-dimensional conformational structure of the selected candidate immunoglobulin sequences. Examination of such display images enables analysis of the possible role of residues in the functioning of candidate immunoglobulin sequences, i.e., analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this manner, FR residues can be selected from the receptor and input sequences and combined to achieve desired antibody characteristics, such as increased affinity for the target antigen. In general, CDR residues are directly and most substantively involved in the effect on antigen binding.

Alternatively, it has now been possible to prepare transgenic animals (e.g., mice) that produce a human antibody repertoire after immunization in the absence of endogenous immunoglobulin production. For example, the published literature has documented that deletion of a homozygous zygote of an antibody heavy chain joining region (JH) gene in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. Transferring a human germline immunoglobulin gene array in such germline mutant mice will result in the production of human antibodies following antigen challenge. See, for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immuno., 7:33 (1993). Human antibodies can also be derived from phage display libraries (Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581-597 (1991)). .

Any of the nucleic acids encoding the anti-polypeptide antibodies (including heavy chains, light chains or both) of the invention, vectors comprising one or more of the nucleic acids, such as expression vectors, and host cells comprising one or more of the vectors are also Within the scope of this disclosure. In certain embodiments, the vector comprises a nucleic acid encoding a nucleotide sequence of a heavy chain variant region or a light chain variant region of an anti-polypeptide antibody of the invention. In other embodiments, the vector comprises a nucleotide sequence encoding a heavy chain variant region and a light chain variant region of an anti-polypeptide antibody, and its expression can be controlled by a single promoter or two separate promoters. Also provided herein are methods for the preparation of any of the anti-polypeptide antibodies of the disclosure, for example, by recombinant techniques as described in this section.

Other techniques for preparing antibodies

In other embodiments, fully human antibodies can be obtained using commercially available mice that have been engineered to exhibit specific human immunoglobulins. Humanized or human antibodies can also be prepared using transgenic animal designed to produce a more desirable (e.g., fully human antibody) or more robust immune response. Exemplary such techniques are Xenomouse RTM from Amgen, Inc. (Fremont, Calif.) and HuMAb-MouseRTM and TC MouseTM from Medarex, Inc. (Princeton, N.J.). In another alternative, the antibody can be produced recombinantly by phage display technology. See, for example, U.S. Patent Nos. 5,565,332; 5,580,717; 5,733,743; 6,265,150 and Winter et al., (1994) Annu. Rev. Immunol. 12:433-455. Alternatively, human antibodies and antibody fragments can be prepared in vitro using phage display technology (McCafferty et al., (1990) Nature 348: 552-553) using immunoglobulin variant (V) domain gene lineage from unimmunized donors. .

The antigen-binding fragment of the intact antibody (full length antibody) can be prepared by a conventional method. For example, an F(ab')2 fragment can be prepared by pepsin digestion of an antibody molecule, and a Fab fragment can be prepared by reducing a disulfide bridge of the F(ab')2 fragment.

Alternatively, self-utilizing McCafferty et al., Nature, 348:552-554 (1990); Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol Biol., 222: An antibody phage library (e.g., a single chain antibody phage library) prepared by the technique described in 581-597 (1991) is isolated from the anti-polypeptide antibody of the present invention. Subsequent publications describe chain shuffling (Marks et al., Bio/Technology, 10:779-783 (1992)), as well as combinatorial infection and recombination in vivo as a strategy for constructing a large phage library (Waterhouse) Et al., Nuc. Acids. Res., 21:2265-2266 (1993)), to prepare human antibodies with high affinity (in nanomolar range per liter). Thus, these techniques are viable alternatives to traditional monoclonal antibody fusion tumor technology for isolated antibodies to individual antibodies.

The antibody obtained according to the invention can be purified to homogeneity. For example, isolation and purification of antibodies can be carried out according to the separation and purification methods used in general proteins. For example, column chromatography (such as affinity chromatography), filtration, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing can be used by appropriate selection and combination. And others (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988), but are not limited thereto, to isolate and isolate antibodies. The antibody concentration obtained above can be determined by absorbance measurement and enzyme-linked immunosorbent assay (ELISA). Exemplary chromatographic methods other than affinity chromatography, including, for example, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography, and the like (Strategies for Protein) Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). The chromatography procedure can be performed by liquid chromatography (such as HPLC, FPLC).

Antibodies can be analyzed using methods of the art well known in the art. For example, one method is to identify antigenic epitopes to which an antigen binds, or "epitope mapping." A number of methods are known in the art for mapping and characterizing epitopes on proteins, including crystal structure of antibody-antigen complexes, competition assays, gene fragment expression assays, and assays based on synthetic peptides. As described, for example, in Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999, Chapter 11. In another example, epitope mapping can be utilized to determine the sequence to which an antibody binds. The epitope may be a linear epitope (ie, contained in a single-segment amino acid), or a conformational epitope, which is optionally included in a single-segment (primary structure linear sequence) amine The three-dimensional interaction of the base acid is formed. The peptides of different lengths (e.g., at least 4 to 6 amino acid lengths) can be isolated or synthesized (e.g., recombinantly) and used for binding assays of antibodies. In another example, an overlapping peptide derived from a target antigen sequence can be utilized in a systemic screen, and binding is determined by an antibody to determine the epitope to which the antibody binds. According to the gene fragment expression analysis, the open reading frame encoding the target antigen can be fragmented randomly or according to a specific genetic structure, and the reactivity of the fragment represented by the antigen can be determined by the antibody to be tested. A gene fragment can be prepared, for example, by PCR, followed by transcription in vitro and translation into a protein in the presence of a radioactive amino acid. The binding of the antibody to the radiolabeled antigenic fragment is determined by immunoprecipitation and gel electrophoresis. Certain epitopes can also be identified using a large library of random peptide sequences displayed on the surface of phage particles (phage libraries). Alternatively, the binding library of the overlapping peptide fragments to the antibody to be tested can be tested in a simple binding assay.

In another example, mutagenesis of the antigen binding domain, domain exchange assays, and alanine scanning mutagenesis can be performed to identify the requisite and/or necessary residues required for epitope binding. For example, domain exchange assays can be performed using target antigen mutants in which sequences from closely related, but antigenically different proteins, such as another member of the neurotrophin family, have been substituted (exchanged) for binding antibodies to the candidate antibody. Determine the different residues in the position. The importance of specific antigenic fragments for antibody binding can be assessed by assessing the binding of the antibody to the target protein of the mutation.

Alternatively, competition assays can be performed using other antibodies known to bind to the same antigen to determine if one antibody binds to the same epitope with other antibodies. The art to which the present invention pertains is well known to those skilled in the art.

Other aspects of an exemplary antibody preparation method

The present compositions can be combined with other active agents, carriers, vehicles, excipients that can be identified by reading the present disclosure by those of ordinary skill in the art to which the present invention pertains. Excipients) or adjuvant agents are included together in a pharmaceutical composition.

The pharmaceutical composition preferably comprises at least one pharmaceutically acceptable carrier. In such pharmaceutical compositions, the compositions of the present disclosure form an "active compound", also known as an "active agent". The term "pharmaceutically acceptable carrier" as used in the present disclosure includes solvents compatible with administration, dispersion media, coatings, antibacterial agents, and Antifungal agents, isotonic agents and absorption delaying agents and the like. Supplementary active compounds can also be incorporated into the compositions. The pharmaceutical composition can be formulated to be compatible with its intended route of administration. Exemplary routes of administration include non-oral routes such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal or subcutaneous administration may include the following ingredients: sterile diluents, such as water for injection, saline solution, fixed oils, Polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants, For example, ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid; a buffer such as acetates, citrates or phosphates And phosphates, such as sodium chloride or dextrose. The pH can be adjusted with an acid or a base such as hydrochloric acid or sodium hydroxide. The parenteral formulation can be sealed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Human antibody

A human anti-polypeptide antibody of the invention can be constructed by combining an Fv strain variant domain sequence selected from a human-derived phage display library with known human constant domain sequences as described above. Alternatively, a human monoclonal anti-polypeptide antibody of the invention can be prepared by a fusion tumor method. Publications such as Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al J. Immunol., 147: 86 (1991) has described human myeloma and mouse-human heteromyeloma cell lines for the preparation of human monoclonal antibodies.

It has now been possible to prepare transgenic animals (e.g., mice) capable of producing a human antibody repertoire after immunization in the absence of endogenous immunoglobulin production. For example, the published literature has documented that deletion of a homozygous zygote of an antibody heavy chain joining region (JH) gene in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. Transferring a human germline immunoglobulin gene array in such germline mutant mice will result in the production of human antibodies following antigen challenge. See, for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immuno., 7:33 (1993).

Human antibodies can also be derived from non-human (eg, rodent) antibodies using gene shuffling, and the human antibodies have similar affinities and specificities to the original non-human antibodies. According to this method (also referred to as "epitope imprinting"), the heavy or light chain variant region of the non-human antibody fragment obtained by the phage display technology as described above is replaced with the human V domain gene. Lineage, a population that forms a non-human chain/human chain scFv or Fab chimera. Selection with an antigen can result in the isolation of a non-human chain/human chain chimeric scFv or Fab, wherein the human chain restores the antigen binding site that is damaged when the corresponding non-human chain in the primary phage display line is removed. Point, which is the choice of the antigenic determinant (imprint) for human chain mate. When the method is repeated to replace the remaining non-human chain, a human antibody can be obtained (refer to PCT WO 93/06213 published on April 1, 1993). Unlike traditional humanization by CDR grafting of non-human antibodies, this technique provides fully human antibodies that do not have FR or CDR residues of non-human origin.

According to a preferred embodiment of the present disclosure, a fusion tumor 1C4 exhibiting binding affinity for the polypeptide CH-3 can be selected. The resulting monoclonal antibodies can be isolated or prepared by any of the known methods. For example, an antibody can be prepared by culturing a culture supernatant obtained by culturing a fusion tumor in a culture medium having a low serum concentration. Alternatively, the fusion tumor can be injected into the abdominal cavity of the experimental animal, and ascites produced by the animal can be collected to prepare an antibody. The antibody may be purified or isolated by affinity column chromatography, gel filtration chromatography, ion exchange chromatography or the like. Any of these known methods can be suitably selected or used in combination with other methods.

According to some embodiments of the present disclosure, the monoclonal antibody 1C4 comprises a heavy chain variant region (VH) and a light chain variant region (VL), wherein the VH comprises three complementarity determining regions (CDRs, ie, CDR-H1, CDRs) -H2 and CDR-H3) have an amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively, and the VL comprises three CDRs (ie, CDR-L1, CDR-L2 and CDR-L3) ) an amino acid sequence having SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11. More specifically, the VH comprises CDR-H1 (SEQ ID NO: 6), CDR-H2 (SEQ ID NO: 7), and CDR-H3 (SEQ ID NO: 8) from the N-terminus to the C-terminus; The N-terminus to the C-terminus comprise CDR-L1 (SEQ ID NO: 9), CDR-L2 (SEQ ID NO: 10) and CDR-L3 (SEQ ID NO: 11).

According to some embodiments, the heavy chain variant region (VH) has at least 85% with sequence number: 12 (eg, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93) %, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence similarity amino acid sequence; and the light chain variant region (VL) has a sequence number: 13 at least 85 % (eg 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) The sequence similarity of the amino acid sequence. In a specific embodiment, the VH has the amino acid sequence of SEQ ID NO: 12 and the VL has the amino acid sequence of SEQ ID NO: 13.

According to certain embodiments of the present disclosure, the antibody 1C4 is specifically labeled to cancer cells and inhibits their growth.

Optionally, the antibody of the invention can be conjugated to a reporter molecule (such as biotin or luciferin) or a developer such that upon detection of the reporter molecule or developer once injected into the individual, the target of the polypeptide of the invention can be monitored. Cancer cells. Alternatively, the antibody of the present invention can be conjugated to an anticancer drug to enhance the tumoricidal efficacy of the antibody of the present invention, thereby achieving a better therapeutic effect in the individual.

use

The antibodies of the invention can be used, for example, in vitro, ex vivo, and in vivo therapeutic methods. The antibody of the present invention can be used as an antagonist to partially or completely block specific antigen activity in vitro, in vivo, and/or in vivo. Furthermore, at least some of the antibodies of the invention neutralize antigenic activity from other species. Thus, antibodies of the invention can be used to inhibit specific antigenic activity, such as in cell cultures containing antigens, in human subjects, or in other mammalian individuals (e.g., orangutans, baboons, baboons) that are antigens that cross-react with antibodies of the invention. In monkeys, macaques and rhesus monkeys, pigs or mice). In one embodiment, an antibody of the invention can be used to inhibit antigenic activity, even if the antibody contacts the antigen to inhibit antigenic activity. In one embodiment, the antigen is a human protein molecule.

In one embodiment, a method of inhibiting antigenic activity in an individual having a disorder, such as cancer, can be used with an antibody of the invention, wherein the antigenic activity is deleterious, comprising administering to the individual an antibody of the invention Thereby, the antigen activity in the individual is inhibited. In one embodiment, the antigen is a human protein molecule and the individual is a human individual. Alternatively, the individual can be a mammal that exhibits an antigen to which the antibody of the invention binds. Alternatively, the individual can be a mammal into which an antigen has been introduced (e.g., by administering an antigen or by expressing an antigen transgene). The antibodies of the invention can be administered to a human subject for therapeutic purposes. Furthermore, the antibodies of the invention can be administered to non-human mammals (e.g., primates, pigs or mice) that exhibit antigens that cross-react with antibodies for veterinary purposes, or animal models of human disease. In relation to the latter, such animal models are useful for assessing the therapeutic efficacy of the antibodies of the invention (e.g., testing the dosage and time course of administration). The antibody of the present invention can be used to treat, inhibit, delay its course of disease, prevent/delay its recurrence, ameliorate, or prevent a disease, disorder, or condition associated with abnormal expression and/or antigenic activity of a polypeptide comprising the present disclosure, including, but Not limited to, cancer, muscle disorders, ubiquitin pathway-associated genetic disorders, immune/inflammatory disorders, neurological disorders, and other ubiquitin pathway-related disorders.

In one aspect, a blocking antibody of the invention is specific for an antigen comprising a polypeptide of the disclosure.

In certain embodiments, an immunoconjugate comprising an antibody of the invention conjugated to a cytotoxic agent is administered to a patient. In certain embodiments, the cell surface exhibits one or more internalized immunoconjugates of a protein associated with an antigen comprising a polypeptide of the disclosure and/or antigens thereof, thereby increasing immune conjugate killing The therapeutic efficacy of the combined target cells. In one embodiment, the cytotoxic agent is labeled to or interfere with nucleic acids in the target cell. Such exemplary cytotoxic agents include any of the chemotherapeutic agents noted in the present disclosure (such as maytansinoid (N2'-desethyl-N2'-(4-mercapto-4-methyl-1) -Oxopentyl)-6-methyl maytansine or calicheamicin), radioisotope or ribonuclease or DNA endonuclease.

The antibodies of the invention may be used alone or in combination with other compositions during the course of treatment. For example, an antibody of the invention can be administered with another antibody and/or an adjuvant/therapeutic agent (eg, a steroid). For example, an antibody of the invention may be combined with an anti-inflammatory and/or bactericidal agent in a therapeutic regimen, for example, for the treatment of any of the diseases of the present disclosure, including cancer, muscle disorders, ubiquitin pathway-associated genetic disorders, immunity/ Inflammatory conditions, neurological disorders, and other ubiquitin pathway-related disorders. Such combination therapies noted above include combination administration (incorporation of two or more agents into the same or separate dosage forms), and separate administration, in which case, prior to the administration of the adjuvant therapy and/or The antibody of the invention is then administered.

The antibodies (and adjuvant therapeutic agents) of the invention may be administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and if administered topically, are administered intralesionally. Non-oral infusions include intramuscular, intravenous, arterial, intraperitoneal, or subcutaneous administration. In addition, antibodies are administered by pulse infusion, as appropriate, especially in doses. Administration can be by any suitable route (e.g., by injection, such as intravenous or subcutaneous injection), depending in part on whether the administration is short-lived or chronic.

The position of the binding target of the antibody of the present invention can be considered in the preparation and administration of the antibody. When the binding targets are intracellular molecules, certain embodiments of the invention provide antibodies or antigen-binding fragments thereof that will be introduced into the cell in which the binding target is located. In one embodiment, the antibody of the invention can be expressed intracellularly as an intrabody. The term "intrabody" as used in the present disclosure refers to an antibody or antigen-binding portion thereof which is intracellularly expressed and capable of selectively binding to a target molecule as described in the following literature: Marasco, Gene Therapy 4:11-15 (1997); Kontermann, Methods 34: 163-170 (2004); U.S. Patent Nos. 6,004,940 and 6,329,173; U.S. Patent Application Publication No. 2003/0104402, and PCT Publication No. WO 2003/077945. Intracellular expression of intracellular antibodies is achieved by introducing a nucleic acid encoding a desired antibody or antigen binding portion thereof (lack of a wild-type leader sequence and a secretion signal normally associated with a gene encoding an antibody or antigen-binding fragment) into a target cell. Any of a variety of methods for introducing nucleic acids into cells can be used, including, but not limited to, microinjection, ballistic injection, electroporation, calcium phosphate precipitation, liposomes, and retroviruses carrying specific nucleic acids, adenoviruses, glands Related virus and vaccinia vector transfection. One or more nucleic acids encoding all or part of an anti-polypeptide antibody of the invention can be delivered to a subject cell, thereby expressing an antigen capable of binding intracellularly to a polypeptide comprising a polypeptide of the disclosure and modulating one or more of said antigen-associated cells One or more intracellular antibodies of the pathway.

In another embodiment, an internalization antibody is provided. The antibody may have certain properties that enhance the delivery of the antibody into the cell, or may be modified to have such characteristics. Techniques for achieving this are well known in the art. For example, cationization of antibodies is known to facilitate their uptake into cells (see, e.g., U.S. Patent 6,703,019). Liposomes or liposomes can also be used to deliver antibodies into cells. When antibody fragments are used, it is generally advantageous to bind minimally to the binding domain of the binding domain of the target protein. For example, based on the sequence of the antibody-variant region, a peptide molecule that retains the ability to bind to the target protein sequence can be designed. These peptides can be synthesized chemically and/or by recombinant DNA techniques. See, for example, Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993).

The modulating (cell penetrating or cell introgressing) polypeptide can be enhanced into the target cells by methods well known in the art. For example, certain sequences, such as those derived from HIV Tat or an antennal homeodomain protein, are capable of directing heterologous proteins to efficiently absorb across the cell membrane. See, for example, Chen et al., Proc. Natl. Acad. Sci. USA (1999), 96: 4325-4329.

When the binding targets are located in the brain, certain embodiments of the invention provide antibodies or antigen-binding fragments thereof that can cross the blood-brain barrier. Certain neurodegenerative diseases are associated with increased permeability of the blood-brain barrier such that antibodies or antigen-binding fragments are readily introduced into the brain. When the blood-brain barrier remains intact, molecular delivery across the blood-brain barrier can be accomplished by several methods well known in the art including, but not limited to, physical methods, lipid-based methods, and receptor- and channel-based methods.

Physical methods of delivering antibodies or antigen-binding fragments across the blood-brain barrier include, but are not limited to, completely evading the blood-brain barrier or forming open pores in the blood-brain barrier. Methods of circumvention include, but are not limited to, direct injection into the brain (see, for example, Papanastassiou et al., Gene Therapy 9: 398-406 (2002)), interstitial infusion/convection enhanced delivery (see, for example, Bobo et al., Proc) Natl. Acad. Sci. USA 91: 2076-2080 (1994)), and implanting a delivery device into the brain (see, for example, Gill et al., Nature Med. 9: 589-595 (2003); and Gliadel WafersTM) , Guildford Pharmaceutical). Methods of forming openings in the barrier include, but are not limited to, ultrasound (see, e.g., U.S. Patent Publication No. 2002/0038086), osmotic pressure (e.g., administration of high-tension mannitol (Neuwelt, EA, Implication of the Blood-Brain) Barrier and its Manipulation, Vols 1 & 2, Plenum Press, NY (1989))), by, for example, penetration of bradykinin or permeabilizer A-7 (see, e.g., U.S. Patent 5,112,596, 5,268,164, 5,506,206 and 5,686,416), and transfecting neurons across the blood-brain barrier with a vector containing a gene encoding an antibody or antigen-binding fragment (see, e.g., U.S. Patent Publication No. 2003/0083299).

Lipid-based methods of delivering antibodies or antigen-binding fragments across the blood-brain barrier include, but are not limited to, encapsulation of antibodies or antigen-binding fragments into liposomes, wherein the liposomes bind to receptors on the vascular endothelium of the blood-brain barrier The antibody-binding fragment is coupled (see, for example, U.S. Patent Application Publication No. 20040225313), and coated with low-density lipoprotein particles (see, for example, U.S. Patent Application Publication No. 20040204354) or Delipoprotein E (see, e.g., U.S. Patent Application Publication No. 20040131692). (coating) an antibody or antigen-binding fragment.

Receptor- and channel-based methods of delivering antibodies or antigen-binding fragments across the blood-brain barrier include, but are not limited to, the use of glucocorticoid blockers to increase the permeability of the blood-brain barrier (see, for example, U.S. Patent Application Publication No. 2002/0065259 , 2003/0162695 and 2005/0124533); activating potassium channels (see, for example, US Patent Application Publication No. 2005/0089473), inhibiting ABC drug transporters (see, for example, US Patent Application Publication No. 2003/0073713); coating antibodies with transferrin And modulating the activity of one or more transferrin receptors (see, e.g., U.S. Patent Application Publication No. 2003/0129186), and cationized antibodies (see, e.g., U.S. Patent No. 5,004,697).

The antibody compositions of the invention are formulated, administered and administered in a manner consistent with good medical practice. Factors considered in this context include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the condition, the delivery site of the agent, the method of administration, the time course of administration, and the medical practitioner known Other factors. The antibody may be, but not necessarily, formulated with one or more agents currently used to prevent or treat the condition. The effective amount of such other agents will depend on the amount of the antibody of the invention, the type of condition or treatment being present in the dosage form, and other factors discussed above. These agents are typically administered in the same dosages and in the administration routes described in the present disclosure, or from about 1% to 99% of the dosages described in the present disclosure, or in any one dose and in any one of the empirical/ It is considered clinically appropriate to use.

For the prevention or treatment of a disease, the appropriate dose of the antibody of the invention (when used alone or in combination with other agents such as chemotherapeutic agents) depends on the type of disease to be treated, the type of antibody, the severity of the disease and the course of the disease, for prophylactic or therapeutic purposes. The administration of antibodies, prior therapies, the patient's clinical history and response to antibodies, and the judgment of the attending physician. The antibody is administered to the patient appropriately or in a series of courses.

Depending on the type and severity of the disease, an antibody of from about 1 microgram to 15 milligrams per kilogram (e.g., from 0.1 mg to 10 mg per kilogram) may be the initial candidate dose for administration to a patient, whether for example by one or more times. Dispensing separately, or by continuous infusion. A typical daily dose may range from about 1 microgram to 100 milligrams per kilogram or more, depending on the factors mentioned above. For repeated administration over several days or longer, depending on the condition, it is usually sustainable until the disease symptoms are specifically inhibited.

An exemplary dosage of antibody ranges from about 0.05 mg per kilogram to about 10 mg per kilogram. Thus, one or more doses (or any combination thereof) of about 0.5 mg per kilogram, about 2.0 mg per kilogram, about 4.0 milligrams per kilogram, or about 10 milligrams per kilogram can be administered to a patient. However, such doses may be administered intermittently, such as weekly or every three weeks (e.g., such that the patient receives from about two to about twenty or, for example, about six doses of antibody). A higher starting dose can be administered followed by one or more lower doses. An exemplary dosing regimen comprises administering an initial dose of about 4 mg of antibody per kg followed by administration of about 2 mg of a weekly maintenance dose of antibody per kg. According to one embodiment, the dosage of the antibody of the invention to the individual is from about 0.8 micrograms to 80 milligrams per kilogram of body weight per dose; preferably, from about 8 micrograms to about 8 milligrams per kilogram of body weight per dose; more preferably, Each dose is from about 80 micrograms to 800 micrograms per kilogram of body weight. In these embodiments, at least the antibody is administered to the individual twice during the vaccination session. However, other dosage regimens can be useful. The progress of this therapy can be easily monitored by conventional techniques and analysis.

In accordance with certain embodiments of the present disclosure, the antibodies of the invention are useful for inhibiting the growth of cancers that overexpress a polypeptide on the cell surface. Exemplary cancers to be treated by the methods of the invention include, but are not limited to, gastric cancer, lung cancer, bladder cancer, breast cancer, pancreatic cancer, kidney cancer, colorectal cancer, cervical cancer, ovarian cancer, brain tumor, prostate cancer , hepatocellular carcinoma, melanoma, esophageal cancer, multiple myeloma and head and neck squamous cell carcinoma.

Using the antibodies of the invention, the disclosure further provides a method of treating cancer in an individual in need thereof (e.g., the individual has or is suspected of having cancer). The method comprises administering to the individual an effective amount (e.g., a prophylactically effective amount or a therapeutically effective amount) of an antibody of the invention, wherein the antibody can be a monoclonal antibody (e.g., a monoclonal antibody 1C4 of the invention) or a plurality of antibodies ( For example, serum antibodies that are isolated from an individual after immunization).

According to one embodiment, the individual is a mouse, wherein the subject is administered from about 0.01 to 1,000 mg of the antibody of the invention per kilogram per dose; for example, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06 per kilogram of body weight per dose , 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 , 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 , 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88 , 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230 , 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480 4 90, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990 or 1000 mg. Preferably, each dose is from 0.1 to 100 mg per kg of body weight. More preferably, each dose is from 1 to 20 mg per kilogram of body weight. According to one embodiment, 4 to 5 mg of the antibody of the invention per kg of body weight per dose is sufficient to inhibit tumor growth in the individual.

The art to which the present invention pertains has a dose equivalent to a human equivalent dose (HED) of any of the antibodies of the present invention, which can be converted by a person of ordinary skill based on the experimental animal model. Thus, for the human body, an effective amount of the antibody of the invention is from about 0.8 micrograms to 80 milligrams per kilogram of body weight per dose; for example, 0.8, 0.9, 1, 2, 3, 4, 5 per kilogram of body weight per dose. , 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650 , 700, 750, 800, 850, 900 or 950 micrograms or dose per kilogram of body weight 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 mg. In a preferred embodiment, the effective amount of the antibody of the invention is from about 300 micrograms to about 400 micrograms per kilogram of body weight per dose.

According to one embodiment, the subject is administered the antibody of the invention once every three days during the course of treatment. Preferably, at least the antibody of the invention is administered to the individual twice during the course of treatment; for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more times. The course of treatment may depend on a variety of factors including, but not limited to, the patient's physiological condition (eg, the patient's physical mass, age, or gender) and the severity of the disease (eg, cancer type and grade). In accordance with an embodiment of the present disclosure, the individual can be administered the antibody once every three days for 51 days.

The subject can be administered an antibody of the invention via any suitable route, such as transmucosal, subcutaneous, intradermal, intramuscular, intravenous, intratumoral or intraperitoneal. According to a specific embodiment, the subject is administered an antibody of the invention via intravenous injection.

As can be appreciated, the methods of the invention can be used alone or in combination with other additional therapies, including surgery, radiation therapy, chemotherapy, hormonal therapy, anti-angiogenic therapy, and immunotherapy. For example, after surgical removal of a tumor, the antibodies of the invention facilitate specific landmarks to eliminate and eliminate residual cancer cells in tissues or blood surrounding the tumor, and/or prevent tumor recurrence. Depending on the particular therapeutic purpose, the method of the invention may be applied to the individual prior to, during or after administration of the additional additional therapy.

Essentially, the individual treated by the methods described above and the embodiments of the present disclosure are mammals, for example, humans, mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, Cows, goats, sheep, monkeys and horses. Preferably, the individual is a human.

Various embodiments are set forth below to illustrate certain aspects of the present disclosure in order to facilitate the practice of the invention. These examples should not be construed as limiting the scope of the invention. It is believed that the present invention may be utilized and practiced without departing from the scope of the invention. All publications cited herein are hereby incorporated by reference in their entirety.

Example

Materials and Methods

Cell culture

Colorectal cancer cell lines (including SW480, HT29, Colo205, and HCT116), gastric cancer cell line AGS, lung cancer cell lines (including A549 and H1299), breast cancer cell lines (including MCF-7, SKBR3, and HS578T), cervical cancer cells Hela, hepatocellular carcinoma cell line HepG2, ovarian cancer cell line SKOV-3, and pancreatic cancer cell lines (including BxPC-3, Panc-1, and Patu8988T) were cultured in Du's modified Eagle culture medium containing 10% fetal bovine serum (Dulbecco's) Modified Eagle's medium, DMEM) or RPMI-1640 medium. In this experiment, human monocyte THP-1, human umbilical vein endothelial cells (HUVEC), human embryonic kidney 293 (HEK293) and human lung epithelial cells BEAS-2B, etc. As a normal control group. THP-1 cells were cultured in RPMI-1640 medium, human umbilical vein endothelial cells were cultured in culture medium 200 (GIBCO), BEAS-2B cell lines were cultured in BEBM medium, and human embryonic kidney cells were cultured. The culture was carried out in Minimum Essential Media (MEM) containing 10% fetal bovine serum. All cells were cultured in a 5% CO ₂ , 37 ° C incubator.

Protein detection

To detect the expression of the polypeptide HSP27, 2×10 ⁵ cells were specifically identified with different HSP27 fragments (including rabbit-derived anti-HSP27 antibodies, which recognize 10, 40, 80 to 150, 124 to 136 of HSP27, respectively. The 1 to 155 and 175 to 205 amino acid residues, as well as the serum antibody of the present invention, were reacted at 4 ° C for 1 hour. After the completion of the action, the cells were washed 3 times with phosphate-buffered saline (PBS), and labeled with 1 μg of goat-derived anti-IgG FITC antibody (GeneTex) and reacted for 30 minutes. The cells were analyzed by flow cytometry.

Immune mouse

100 μg of polypeptide CH-1 (comprising 36-65 amino acid residues of HSP27; SEQ ID NO: 1), CH-2 (containing 46-75 amino acid residues of HSP27; SEQ ID NO: 2) , CH-3 (comprising the 56th to 85th amino acid residues of HSP27; SEQ ID NO: 3), CH-4 (containing the 6th to 35th amino acid residues of HSP27; SEQ ID NO: 4), and CH5 ( The 86th to 115th amino acid residues comprising HSP27; SEQ ID NO: 5) are respectively mixed with an adjuvant fatty alcohol polyoxyethylene ether-D. At the first week, the mixture was administered to the mice by subcutaneous injection. The initially primed mice were re-immunized weekly for the same period of 7 weeks. Blood samples were collected weekly from the orbital sinus of immunized mice. Serum was collected and stored at -20 °C for subsequent analysis.

Serum antibody titer

Antibody titers were determined by indirect ELISA. Briefly, an ELISA is applied to a 100 ng antigen (ie, polypeptide CH-1, CH-2 or CH-3) or a 100 ng goat-derived anti-mouse kappa chain antibody. Hole plate. The wells were washed with PBS containing 0.05% Tween-20 (PBS-T) and filled with 100 ml of 1% skim milk in PBS to fill the excess space of the ELISA chassis. Serum samples were serially diluted with PBS-T and added to an ELISA plate for 1 hour at room temperature. Subsequently, the wells were washed with PBS-T and goat-derived anti-mouse IgG HRP antibody (diluted in PBS-T at a dilution ratio of 1:8,000) was added and allowed to act at room temperature for 1 hour. After washing, 100 μl of 3,3',5,5'-Tetramethylbenzidine (TMB) was added to each well and allowed to act at room temperature for 20 minutes. The reaction was stopped with 50 equivalents of 1 equivalent of HCL per liter and the absorbance (OD450) at a wavelength of 450 nm was read using an ELISA reader.

Purified monoclonal antibody

Monoclonal antibody 1C4 was purified by protein L agarose (protein L agorose, GenScript) and purified according to the protocol. Briefly, protein L agarose (1.5 ml) was prepared in 20% ethanol and injected into a disposable column. The protein L agarose resin was washed with 10 ml of washing buffer (20 mmol of NaHPO ₄ per liter, 0.15 mol of NaCl per liter, pH 8). The conditioned medium (30 ml) of the fusion tumor cell 1C4 was diluted with 30 ml of binding buffer. The mixed sample solution is loaded into the column and inverted to uniformly mix. Next, the column was washed twice with 30 ml of washing buffer until the OD280 was close to zero. The antibody was gently lysed by adding 5 ml of a dissolution buffer (0.1 mol of glycine per liter, pH 2.5), and the antibody was collected in a collection tube containing 500 μl of neutralizing buffer. The eluted antibody was concentrated, and the buffer was exchanged to a neutralization buffer with Sephadex G-25 resin (GE). Finally, the antibody concentration was quantified by OD280 absorbance.

Cloning and sequencing of the Ig variant domain (V) gene

Using TRIzol ^® reagent ^{(TRIzol ® Reagent, Thermo) 2} × 10 6 th RNA 1C4 hybridoma cell extracts, using SuperScript ^® III reverse transcriptase kit ^{(SuperScript ® III Reverse Transcriptase (RT} ) kit, Invitrogen) to RNA trans Transcribed into cDNA. Thereafter, the Ig-Primer Sets (Novagen) and GoTaq ^® G2 Green Fluorescent Standard Mixing Reagent (GoTaq ^® G2 Green Master Mix, Promega) were used to amplify the Ig heavy and light chain genes. The PCR product was cloned by the TOPO ^® TA Cloning ^® kit (TOPO ^® TA Cloning ^® Kit, Invitrogen). Finally, DNA sequencing was performed by Mission Biotech.

The analysis data indicates that the heavy chain variant region comprises the nucleotide sequence of SEQ ID NO: 14 which can be used to encode the amino acid sequence of SEQ ID NO: 12, and the light chain variant region comprises the nucleotide sequence of SEQ ID NO: 15. It can be used to encode the amino acid sequence of SEQ ID NO: 13.

Combining affinity

The binding affinity of monoclonal antibody 1C4 to polypeptide CH-3 was determined by ELISA. First, the culture well of the ELISA plate is coated with an antigen of 500 ng (for example, polypeptide CH-3), and then placed until the next day. 100 ng of antibody 1C4 was mixed with polypeptide CH-3 at various concentrations (including 0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 μg per ml), followed by reaction at room temperature for 1 hour. Next, the mixture was added to an ELISA plate and allowed to act at room temperature for 20 minutes. The culture wells were washed with PBS-T, and goat-derived anti-mouse IgG HRP antibody (diluted 1:10,000 with PBS-T) was added and allowed to stand at room temperature for 1 hour. After washing, 100 μl of TMB was added to each well and incubated at room temperature for 20 minutes. The reaction was stopped with 50 equivalents of 1 equivalent mole of HCL per liter, and the absorbance of OD450 was read with an ELISA reader.

Cell viability analysis

1 × 10 ⁴ cells were seeded in a 96-well culture dish and cultured in a constant temperature incubator at 37 ° C, 5% CO ₂ for 24 hours. Thereafter, the cells are combined with monoclonal antibody 1C4, or anti-CH-1, anti-CH-2 or anti-CH-3 serum antibodies (ie, polypeptides CH-1, CH-2, and CH-3, respectively). After immunization of the mice, serum was isolated from the mice) and reacted for 72 hours. Thereafter, 100 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (3-(4,5-cimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide, MTT) was added to each well and allowed to act at 37 ° C for 4 hours. The resulting triphenylmethyl ester crystals were dissolved in DMSO. Finally, the absorbance of OD595 was measured using an ELISA reader.

To test the effect of commercially available anti-HSP27 antibody (code number: 102-17173, RayBiotech) on cancer cells, 3×10 ³ HT29 cells were seeded in each well of a 96-well culture plate and placed at 37 ° C. Incubate in a constant temperature incubator with 5% CO ₂ . After 24 hours, 0.5 μl of anti-HSP27 antibody was added and cultured for 48 hours. Cell viability was assessed by MTT assay.

Apoptosis analysis

1 × 10 ⁵ HT29 cells were seeded in a 24-well culture dish and cultured in a 37 ° C, 5% CO ₂ incubator. After 24 hours, the cells were treated with anti-CH-1, anti-CH-2 or anti-CH-3 serum antibodies for 48 hours, respectively. HT29 cells were washed twice with pre-cooled PBS and suspended using 500 microliters of cell fixing solution (Cytofix solution). Then, 5 μl of Caspase-3 PE antibody (PE Active Caspase 3 Apoptosis Kit, BD) was added to the suspension, and Protect from light for 30 minutes at room temperature. The cells were analyzed by flow cytometry.

Animal experiment

1 × 10 ⁷ HT29 cells were implanted subcutaneously in nude mice. Mice bearing tumors were administered 100 micrograms of serum antibody every three days by intravenous injection. Regularly detect tumor size.

Example 1 Expression of HSP27 on the surface of cancer cells

An example is the use of flow cytometry to analyze the amount of HSP27 and its fragments. As shown in Figure 1, the amount of HSP27 on the surface of cancer cells was higher than that of normal cells (i.e., HUVEC cells, HEK293 cells, and BEAS-2B cells).

In order to investigate the fragment of HSP27 which is expressed extracellularly (ie, extended to the extracellular region), amino acid residues 10 to 40, 80 to 150, 124 to 136, 1 to 155 and 175 to 205 of HSP27 will be identified, respectively. The antibodies interacted with intact HT29 cells (i.e., the HT29 cells were not treated by a permeabilization step such that antibodies could not enter) and were analyzed by flow cytometry. Fluorescent signals were detected only on the surface of the cells cooperating with antibodies recognizing the amino acid residues 1 to 155 of HSP27 (results not shown). In other words, of the five antibodies tested, only antibodies recognizing the 1st to 155 amino acid residues of HSP27 bind to the surface of HT29 cells. Therefore, it is speculated that the 1st to 10th and 40th to 80th amino acid residues of HSP27 will be expressed on the surface of cancer cells.

Example 2 Analysis of serum antibodies against anti-CH-1, anti-CH-2 or anti-CH-3

Based on the findings of Example 1, three polypeptides were synthesized, wherein the polypeptide CH-1 was composed of the 36th to 65th amino acid residues of HSP27, and the polypeptide CH-2 was composed of the 46th to 75th amino acid residues of HSP27. Composition, and polypeptide CH-3 is composed of the 56th to 85th amino acid residues of HSP27.

The synthetic polypeptides CH-1, CH-2 and CH-3 are separately mixed with an appropriate adjuvant, and then the mice are immunized according to the procedure described in "Materials and Methods". Serum antibodies against anti-CH-1, anti-CH-2 or anti-CH-3 were isolated from immunized mice and detected by ELISA assay. The experimental data indicates that the three polypeptides can stimulate the production of IgG antibodies in mice (Fig. 2A), and the prepared serum antibodies can be specifically bound to their respective corresponding polypeptides (Fig. 2B).

The binding affinity of the serum antibody to cancer cells was then analyzed by flow cytometry. As shown in the results in Figure 3, all three serum antibodies can bind to HT29 cells (indicated by dashed lines), while the addition of peptides CH-1, CH-2 or CH-3 blocks the binding between the two (in dotted lines). Mark). This result indicates that serum antibodies against anti-CH-1, anti-CH-2 or anti-CH-3 can be specifically labeled to cancer cells. MTT and flow cytometry analysis data further indicated that anti-CH-1, anti-CH-2 or anti-CH-3 serum antibodies bind to cancer cells and inhibit cell proliferation (Fig. 4 to a to c), and will cause an apoptotic reaction of cancer cells by producing apoptotic protease-3 protein.

Serum antibodies stimulated by the polypeptide CH-3 elicit a higher amount of apoptosis in HT29 cells compared to serum antibodies stimulated by EGFP or polypeptides CH-4, CH-5 (Fig. 6). The results in Figure 6 indicate that anti-CH-4 and anti-CH-5 serum antibodies did not significantly induce cell death.

Next, animal models were used to evaluate the anti-tumor efficacy of anti-CH-1 and anti-CH-3 serum antibodies. Anti-CH-1 and anti-CH-3 serum antibodies significantly inhibited tumor growth compared to the control group (control serum) (Fig. 7).

Example 3 Analysis of the characteristics of monoclonal antibody 1C4

The monoclonal antibody 1C4 was purified from the fusion tumor according to the procedure described in "Materials and Methods", and its biological activity and inhibitory effect were examined. Figures 8 and 9 depict the results of these experiments, respectively.

As shown in the results of Fig. 8, the monoclonal antibody 1C4 has a binding affinity to the polypeptide CH-3, and its dissociation constant is about 1.78 × 10 ^-7 . In the cell-specific assay, it was found that antibody 1C4 can be specifically identified and labeled to all tested cancer cells, including HepG2, A549, AGS, SW480, HCT116, HT29 and Colo205 cells, but not to normal cells THP-1. (Fig. 8B).

The results in Figure 9 further demonstrate that antibody 1C4 inhibits tumor growth compared to the control group. It is worth noting that the commercially available antibody HSPB1 does not affect (e.g., reduce) the survival rate of cancer cells compared to the control group (i.e., simple culture) (Fig. 10).

Summarizing the above results, this study found that the polypeptides CH-1, CH-2 and CH-3 were overexpressed on the surface of various types of cancer cells. Based on this finding, three vaccine compositions comprising the polypeptides CH-1, CH-2 and CH-3, respectively, can be used to stimulate a specific immune response to cancer cells (eg, antibodies specific for cancer cells), thereby protecting And / or treat cancer. In accordance with an embodiment of the present disclosure, the antibody 1C4 produced can be specifically labeled to cancer cells and inhibit their growth. The polypeptides, vaccine compositions and antibodies described herein provide potential therapeutic modalities for the prevention and/or treatment of various types of cancer.

While the embodiments of the present invention have been disclosed in the foregoing embodiments, those of ordinary skill in the art of the present invention may be variously modified and modified. The above description, examples and experimental data are a complete description of the structure and the manner of use of the present disclosure as exemplary embodiments. Although various embodiments of the present disclosure have been described above with a certain degree of characteristics, or with reference to one or more specific embodiments, those of ordinary skill in the art to which the present invention pertains can still deviate from the disclosure. Numerous modifications to the disclosed embodiments are made in the context of the spirit and scope.

no

The content of the present invention can be more clearly understood from the following detailed description and the accompanying drawings.

1 is a bar graph depicting the amount of expression of polypeptide HSP27 on the surface of a particular cell line in accordance with an embodiment of the present disclosure.

2A is a bar graph illustrating an embodiment of the present disclosure, which illustrates polypeptide CH-1 (SEQ ID NO: 1), CH-2 (SEQ ID NO: 2), or CH-3, respectively. No.: 3) After immunizing the mice, the total serum IgG antibody titer was isolated from the mice.

Figure 2B is a bar graph depicting serum anti-separation from mice after immunization of mice with polypeptides CH-1, CH-2 or CH-3, respectively, in accordance with an embodiment of the present disclosure. HSP27 antibody titer.

Figure 3 is data in accordance with an embodiment of the present disclosure, which illustrates the use of flow cytometry to analyze polypeptide CH-1 (panel a), polypeptide CH-2 (panel b) or polypeptide CH-3, respectively (Fig. c After immunization of the mouse, the binding specificity of the serum antibody to the sclerosing polypeptide HSP27 was isolated from the mouse.

Figure 4 is a bar graph showing an example of an embodiment of the present disclosure, which separately describes the isolation of serum antibody pairs from mice after immunization of mice with polypeptides CH-1, CH-2 or CH-3, respectively. Cytotoxic effects of HT29 cells (panel a), AGS cells (panel b) and HepG2 cells (panel c).

Figure 5 is a graph of data according to one embodiment of the present disclosure, which is characterized by flow cytometry analysis of polypeptide CH-1 (panel a), polypeptide CH-2 (panel b) or polypeptide CH-3, respectively (Fig. c After immunization of mice, the apoptosis-inducing effect of serum antibodies on HT29 cells was isolated from mice.

Figure 6 is a bar graph depicting an enhanced green fluorescent protein (EGFP) or polypeptide CH-4, CH-5 or CH-3, respectively, in accordance with an embodiment of the present disclosure. After immunization of mice, the cytotoxic effect of serum antibodies on HT29 cells was isolated from mice.

Figure 7 is a linear diagram showing an embodiment of the present disclosure, which is characterized in that serum antibodies are isolated from immunized mice after immunization of mice with polypeptide CH-1 or CH-3, respectively. The inhibitory effect of antibodies on tumor volume in mice.

Figure 8A is a line graph depicting the binding affinity of monoclonal antibody 1C4 for polypeptide CH-3, in accordance with one embodiment of the present disclosure.

Figure 8B is data in accordance with one embodiment of the present disclosure, which illustrates the use of flow cytometry to analyze the binding specificity of monoclonal antibody 1C4 to a particular cell line.

Figure 9 is a bar graph depicting the inhibitory effect of monoclonal antibody 1C4 on the proliferation rate of HT29 cells in accordance with an embodiment of the present disclosure.

Figure 10 is a bar graph depicting the inhibitory effect of the commercially available antibody HSPB1 on the proliferation rate of HT29 cells in accordance with an embodiment of the present disclosure.

Sequence Listing <110> Kanghe Biomedical Technology Co., Ltd. <120> Synthetic polypeptide, composition containing the same, antibody produced thereby and use thereof <130> P2970-PCT<150> US 62/436,405<151> 2016-12-19<160> 15<170> BiSSAP 1.3<210> 1<211> 30<212> PRT<213> Artificial sequence <220> <223> Synthetic peptide <400> 1Pro Arg Leu Pro Glu Glu Trp Ser Gln Trp Leu Gly Gly Ser Ser Trp 1 5 10 15 Pro Gly Tyr Val Arg Pro Leu Pro Pro Ala Ala Ile Glu Ser 20 25 30 <210> 2<211> 30<212> PRT<213> Artificial Sequence <220> < 223> Synthetic polypeptide <400> 2Leu Gly Gly Ser Ser Trp Pro Gly Tyr Val Arg Pro Leu Pro Pro Ala 1 5 10 15 Ala Ile Glu Ser Pro Ala Val Ala Ala Pro Ala Tyr Ser Arg 20 25 30 <210> 3<211 > 30<212> PRT<213> Artificial sequence <220> <223> Synthetic peptide <400> 3Arg Pro Leu Pro Pro Ala Ala Ile Glu Ser Pro Ala Val Ala Ala Pro 1 5 10 15 Ala Tyr Ser Arg Ala Leu Ser Arg Gln Leu Ser Ser Gly Val 20 25 30 <210> 4<211> 30<212> PRT<213> Artificial Sequence <220> <223> Synthetic Peptide <400> 4Val Pro Phe Ser Leu Leu Arg Gly Pro Ser Trp Asp Pro Phe Arg Asp 1 5 10 15 Trp Tyr Pro His Ser Arg Leu Phe Asp Gln Ala Phe Gly Leu 20 25 30 <210> 5<211> 30<212> PRT<213> Artificial Sequence <220> <223> Synthetic Peptide <400> 5Ser Glu Ile Arg His Thr Ala Asp Arg Trp Arg Val Ser Leu Asp Val 1 5 10 15 Asn His Phe Ala Pro Asp Glu Arg Thr Val Lys Thr Lys Asp 20 25 30 <210> 6<211> 13<212> PRT<213> Artificial Sequence <220> <223> Fusion tumor cells <400> 6Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Tyr Met His 1 5 10 <210> 7<211> 11<212> PRT<213> Artificial sequence <220> <223> Fusion tumor Cell <400> 7Gly Arg Val Asn Pro Asn Asn Gly Gly Thr Ser 1 5 10 <210> 8<211> 14<212> PRT<213> Artificial sequence <220> <223> Fusion tumor cell <400> 8Ala Tyr Tyr Tyr Gly Ser Ser Tyr Tyr Ala Met Asp Tyr Trp 1 5 10 <210> 9<211> 12<212> PRT<213> Artificial Sequence <220> <223> Fusion Cell <400> 9Arg Ala Ser Ser Ser Val Ser Ser Ser Tyr Leu His 1 5 10 <210> 10<211> 14<212> PRT<213> Artificial sequence <220> <223> Fusion tumor cell <400> 10Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile 1 5 10 <210> 11<211> 11<212> PRT<213> Artificial sequence <220> <223> Synthetic cells <400> 11Gln Gln Tyr Ser Gly Tyr Pro Leu Thr Phe Gly 1 5 10 <210> 12<211> 112<212> PRT<213> Artificial sequence <220> <223> Fusion tumor cells <400> 12Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe 1 5 10 15 Thr Gly Tyr Tyr Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu 20 25 30 Glu Trp Ile Gly Arg Val Asn Pro Asn Asn Gly Gly Thr Ser Tyr Asn 35 40 45 Gln Lys Phe Lys Gly Lys Ala Ile Leu Thr Val Asp Lys Ser Ser Ser 50 55 60 Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val 65 70 75 80 Tyr Tyr Cys Ala Tyr Tyr Tyr Gly Ser Ser Tyr Tyr Ala Met Asp Tyr 85 90 95 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Glu Ser Gln Ser Phe 100 105 110 <210> 13<211> 103<212> PRT<213> Labor Sequence <220> <223> Fusion tumor cell <400> 13Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser 1 5 10 15 Ser Val Ser Ser Ser Tyr Leu His Trp Tyr Gln Gln Lys Ser Gly Ala 20 25 30 Ser Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val 35 40 45 Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr 50 55 60 Ile Ser Ser Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln 65 70 75 80 Tyr Ser Gly Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 85 90 95 Lys Arg Ala Asp Ala Ala Pro 100 <210> 14<211> 336<212> DNA<213> Artificial sequence <220> <223> Fusion tumor cell<400> 14cctggggctt cagtgaagat atcctgcaag gcttctggtt actcattcac tggctactac 60atgcactggg tgaagcagag ccatggaaag agccttgagt ggattggacg tgttaatcct 120aacaatggtg gtactagcta caaccagaag ttcaagggca aggccatatt aactgtagac 180aagtcatcca gcacagccta catggagctc cgcagcctga catctgagga ctctgcggtc 240tattactgtg cttattacta cggtagtagc tactatgcta tggactactg gggtcaagga 300acctcagtca ccgtctcctc agagagtcag tccttc 336 <210> 15 <211> 309 <212> DNA <213> artificial sequence <220> <223> hybridoma cells <400 > 15tctgcatctc caggggaaaa ggtcaccatg acctgcaggg ccagctcaag tgtaagttcc 60agttacttgc actggtacca gcagaagtca ggtgcctccc ccaaactctg gatttatagc 120acatccaact tggcttctgg agtccctgct cgcttcagtg gcagtgggtc tgggacctct 180tactctctca caatcagcag tgtgg Aggct gaagatgctg ccacttatta ctgccagcag 240tacagtggtt acccactcac gttcggaggg gggaccaagc tggaaataaa acgggctgat 300gctgcacca 309

Claims (29)

A synthetic polypeptide useful for preventing and/or treating a cancer, comprising a first polypeptide, wherein the amino acid sequence of the first polypeptide has a sequence similarity to sequence number: 1, 2 or 3 of at least 85%. The synthetic polypeptide of claim 1, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 1, 2 or 3. The synthetic polypeptide according to claim 1, wherein: the N-terminus of the synthetic polypeptide is modified by acetylation, saccharification or formazanization, and/or the C-terminus of the synthetic polypeptide is glycosylated or saccharified. Modification. The synthetic polypeptide according to claim 1, further comprising a second polypeptide which is located at the N-terminus or C-terminus of the first polypeptide, wherein the second polypeptide is selected from the group consisting of ovalbumin and fetal bovine serum white. A group consisting of protein, keyhole limpet hemocyanin, beta-galactosidase, thyroglobulin, and combinations thereof. A vaccine composition for use in the prevention and/or treatment of a cancer comprising the synthetic polypeptide of claim 1 and a pharmaceutically acceptable adjuvant. The vaccine composition of claim 5, wherein the pharmaceutically acceptable adjuvant is selected from the group consisting of fatty alcohol polyoxyethylene ether-D, aluminum hydroxide, Freund's incomplete adjuvant, Freund's complete adjuvant, and Endotoxin-related adjuvant, mineral oil, mineral oil and surfactant, Ribi adjuvant, gold adjuvant, Syntax adjuvant preparation, aluminum salt adjuvant, nitrocellulose adsorption antigen, immunostimulating complex, Gebru adjuvant, Overloading agent, copolymer of ethylene and vinyl acetate, L-tyrosine acid, oil-based emulsion, Adju prime adjuvant, squalene, sodium phthalocyanine, calcium phosphate, saponin and cell wall dipeptide The group consisting of. The vaccine composition of claim 5, wherein the synthetic polypeptide further comprises a second polypeptide located at the N-terminus or C-terminus of the first polypeptide, wherein the second polypeptide is selected from the group consisting of ovalbumin, A group consisting of fetal bovine serum albumin, keyhole limpet hemocyanin, beta-galactosidase, thyroglobulin, and combinations thereof. An antibody or fragment thereof having binding affinity to the synthetic polypeptide of claim 1, comprising: a heavy chain variant region comprising an amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8. A light chain variant region comprising the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11. The antibody of claim 8, wherein the amino acid sequence of the heavy chain variant region has a sequence similarity to SEQ ID NO: 12 of at least 85%, and the amino acid sequence of the light chain variant region is SEQ ID NO: 13 Has a sequence similarity of at least 85%. The antibody of claim 9, wherein the heavy chain variant region has the amino acid sequence of SEQ ID NO: 12, and the light chain variant region has the amino acid sequence of SEQ ID NO: 13. A use of the synthetic polypeptide of claim 1 for the preparation of a medicament for preventing or treating cancer in an individual in need thereof. The use of claim 11, wherein the medicament is administered to the individual, thereby producing from 0.8 micrograms to 80 milligrams of the synthetic polypeptide of claim 1 per kilogram of body weight per dose. The use of claim 12, wherein the medicament is administered to the individual, thereby producing from 8 micrograms to 8 milligrams of the synthetic polypeptide of claim 1 per kilogram of body weight per dose. The use of claim 13, wherein the medicament is administered to the individual to produce a synthetic polypeptide of claim 1 in a dose of from 80 micrograms to 800 micrograms per kilogram of body weight per dose. The use of claim 11, wherein the agent is administered to the individual at least twice during the vaccination session. The use of claim 15, wherein the individual is administered the drug 6 to 10 times during the vaccination session. The use according to claim 11, wherein the cancer is selected from the group consisting of gastric cancer, lung cancer, bladder cancer, breast cancer, pancreatic cancer, renal cancer, colorectal cancer, cervical cancer, ovarian cancer, brain tumor, prostate cancer, and hepatocellular carcinoma. , a group of melanoma, esophageal cancer, multiple myeloma, and squamous cell carcinoma of the head and neck. The use of claim 11 wherein the system is human. An use of the antibody of claim 8 for the preparation of a medicament for treating cancer in an individual in need thereof. The use of claim 19, wherein the medicament is administered to the individual, whereby an antibody of claim 8 is produced in an amount of from 0.8 micrograms to 80 milligrams per kilogram of body weight per dose. The use of claim 20, wherein the medicament is administered to the individual, thereby producing from 8 micrograms to 8 milligrams of the antibody of claim 8 per kilogram of body weight per dose. The use of claim 21, wherein the medicament is administered to the individual, thereby producing an antibody of claim 8 in an amount of from 80 micrograms to 800 micrograms per kilogram of body weight per dose. The use of claim 19, wherein at least the drug is administered to the individual twice. The use of claim 23, wherein the individual is administered the drug 6 to 10 times. The use according to claim 19, wherein the cancer is selected from the group consisting of gastric cancer, lung cancer, bladder cancer, breast cancer, pancreatic cancer, renal cancer, colorectal cancer, cervical cancer, ovarian cancer, brain tumor, prostate cancer, and hepatocellular carcinoma. , a group of melanoma, esophageal cancer, multiple myeloma, and squamous cell carcinoma of the head and neck. The use of claim 19, wherein the system is human. The isolated monoclonal antibody of claim 8, wherein the anti-system humanized antibody, chimeric antibody, scFv antibody, Fab antibody or Fab2 antibody. The isolated monoclonal antibody of claim 8, wherein the anti-systematic human antibody. A pharmaceutical composition comprising any of the monoclonal antibodies or binding fragments thereof according to claim 8 and a pharmaceutically acceptable carrier.