Api5 epitope and antibody specifically binding to the same

Abstract

The present invention relates to an API5 epitope and an antibody or antigen-binding fragment thereof that specifically binds thereto. The API5 epitope according to the present invention induces stronger resistance, tolerance, or refractory to anticancer drugs of cancer compared to other regions or epitopes of the API5 protein. Therefore, such antibodies or antigen-binding fragments thereof binding to the specific API5 epitope are very effective in killing cancer cells and inhibiting the proliferation or growth of tumors. In particular, the anticancer effect on cancer exhibiting resistance, tolerance, or refractory to existing anticancer drugs is very excellent.

Description

API5 EPITOPE AND ANTIBODY SPECIFICALLY BINDING TO THE SAME

[0001] The present invention relates to an API5 epitope and an antibody or antigen-binding fragment thereof that specifically binds thereto.

[0002] Despite intensive research on cancer over the past decades, the cancer is still a leading cause of death worldwide. A number of anti-cancer treatments have been developed, but are not effective for all cancer types and for all patients. Methods currently being used to treat cancer are relatively non-selective. Diseased tissue is removed through surgery, the size of solid tumors is reduced through radiation therapy, or chemotherapy is used to kill cancer cells rapidly. However, the chemotherapy may cause the drug resistance, and sometimes restricts the administrable dose. It causes severe side effects so that they may rule out the use of potentially effective agents. Accordingly, there is a need to develop more target-specific and effective cancer therapies.

[0003] In this regard, an anticancer targeted agent that specifically targets only cancer cells, or an anticancer immunotherapeutic agent that utilizes a patient's immune system is being developed. For example, there are about 30 types of anticancer targeted agent approved in Korea, and the anticancer immunotherapeutic agents approved in Korea include Opdivo (ingredient name: nivolumab), Keytruda (ingredient name: pembrolizumab), Tecentriq (ingredient name: atezolizumab), Imfinzi (ingredient name: durvalumab), Yervoy (ingredient name: ipilimumab), etc., which used for various types of cancer including non-small cell lung cancer, melanoma, renal cell carcinoma, Hodgkin's lymphoma, head and neck cancer, bladder cancer, etc.

[0004] However, as the use of these anticancer targeted agents and anticancer immunotherapeutic agents increases, the proportion of patients who already exhibit resistance, tolerance, or refractory to them is also increasing. Therefore, there is a demand in the art to develop a therapeutic agent for cancer that exhibits resistance, tolerance, or refractory to existing anticancer drugs by discovering a novel target and utilizing it.

[0005] The inventors have made research efforts to identify a novel target for anticancer drugs and develop a therapeutic agent for cancer exhibiting resistance, tolerance, or refractory to existing anticancer drugs by using it. As a result, we identified the epitope of API5 represented by the amino acid sequence of SEQ ID NO: 7 according to the present invention, and experimentally proven that the antibody or antigen-binding fragment thereof targeting the epitope can show excellent effects for cancer that exhibits resistance, tolerance, or refractory to existing anticancer drugs, thereby completing the present invention.

[0006] One aspect of the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to an epitope of apoptosis inhibitor 5 (API5).

[0007] The term "antibody" as used herein refers to an immunoglobulin molecule having immunological reactivity with a specific antigen, or a protein molecule serving as a receptor to specifically recognize an antigen. In the present invention, "antibody" includes monoclonal antibody, polyclonal antibody, whole antibody (antibody consisting of at least two heavy chains and two light chains linked by disulfide bonds), antibody fragment, Fab, Fab', F(ab')2, Fv, single chain Fv (scFv), diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody or antigen-binding portions of antibody. The whole antibody includes IgA, IgD, IgE, IgM and IgG. In addition, the IgG may include IgG1, IgG2, IgG3, and IgG4 as subtypes.

[0008] The term "antigen-binding fragment" as used herein refers to an antibody fragment or an antibody analog which retains at least some of the binding specificity of the parent antibody and comprises a variable region or a portion (for example, one or more CDRs) of the antigen binding region of the parent antibody. The antigen-binding fragment is, for example, Fab, Fab', F(ab')2, Fv fragment, scFv, unibody, diabody, linear antibody, nanobody, domain antibody, or multispecific antibody formed from the antibody fragment.

[0009] In addition, the antibody or antigen-binding fragment thereof may be combined with a drug to form an antibody-drug conjugate (ADC).

[0010] The term "heavy chain" as used herein refers to a whole heavy chain comprising a heavy chain variable region and a heavy chain constant region, and fragment thereof. The heavy chain includes gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types.

[0011] The term "light chain" as used herein refers to a whole light chain comprising a light chain variable region and a light chain constant region, and fragment thereof. The light chain includes kappa (κ) and lambda (λ) types.

[0012] The term "CDR (complementarity determining region)" as used herein refers to an amino acid sequence of hypervariable region that forms an antigen-binding site as part of the variable region of an antibody produced by B cells and T cells. The amino acid sequence of the heavy chain comprises three non-contiguously arranged CDRs: heavy chain CDRH1, CDRH2 and CDRH3, and the amino acid sequence of the light chain comprises three non-contiguously arranged CDRs: light chain CDRL1, CDRL2 and CDRL3. The CDRs are a region concerned with antigen recognition and play a crucial role in the diversity of antigen specificity by providing major contact residues in binding of an antibody to an antigen or epitope.

[0013] In the present invention, the antibody is a whole antibody or an antibody fragment having antigen-binding ability. The heavy chain may be any one of gamma (γ), mu (μ), alpha (α), delta (δ) or epsilon (ε) type, and the light chain may be kappa (κ) or lambda (λ) type.

[0014] The term "API5 (apoptosis inhibitor 5)" as used herein refers to a protein encoded by theAPI5gene, and the amino acid sequence constituting it is known in an existing database (NCBI Reference Sequence: XP_016873953.1, XP_016873954.1, XP_006718422.1, etc.). API5 protein is expressed at high levels in tumors such as solid carcinoma and metastatic carcinoma, and its expression is particularly increased in cancer cells having resistance, tolerance, or refractory to anticancer drugs (Han Sol Jang et al. Exp Mol Med. 2017 Sep 8, 49(9):e374). This API5 protein is known to phosphorylate and activate ERK1 / 2, thereby inducing tumor development through cell cycle arrest and apoptosis inhibition (Hanbyoul Cho et al. BMC Cancer. 2014 Jul 28, 14:545).

[0015] The term "epitope" as used herein refers to an antigenic determinant and means a specific part of an antigen that allows antibodies or immune cells such as B cells and T cells to recognize an antigen.

[0016] The epitope of API5 according to the present invention may be represented by the amino acid sequence of SEQ ID NO: 7. In addition, the API5 epitope may be represented by a base sequence encoding the amino acid sequence of SEQ ID NO: 7. Specifically, the amino acid sequence of SEQ ID NO: 7 may be part of 504-mer amino acid sequence constituting the API5 protein. More specifically, the API5 epitope represented by the amino acid sequence of SEQ ID NO: 7 may be the 206th to 225th amino acid sequences of the API5 protein represented by the amino acid sequence of SEQ ID NO: 6.

[0017] The term "anti-API5 antibody" as used herein refers to an antibody or antigen-binding fragment thereof that specifically binds to the API5 and neutralizes or inhibits the activity of the API5 protein. In addition, the anti-API5 antibody refers to an antibody or antigen-binding fragment thereof that neutralizes or inhibits the activity of API5 by specifically binding to an epitope of API5. Specifically, the anti-API5 antibody according to the present invention may bind to an epitope represented by the amino acid sequence of SEQ ID NO: 7.

[0018] In addition, the anti-API5 antibody may be a human antibody that targets human API5 protein and also has reactivity to a API5 protein derived from mouse or rat.

[0019] The term "human antibody" as used herein refers to an antibody whose framework and CDR regions have variable regions derived from human immunoglobulin sequences. In the present invention, a human antibody may comprise amino acid residues that are not encoded by a human-derived immunoglobulin sequence (e.g., a mutation introduced by random or site-specific mutagenesis in vitro, or a mutation introduced by somatic mutation in vivo).

[0020] The sequence disclosed in the present invention includes a sequence that shows substantial identity to the sequence described in the sequence list. The substantial identity means that the two sequences are aligned to correspond as much as possible and analyzed using an algorithm commonly used in the art, and then show homology between sequences of 80%, 90%, 95% or more.

[0021] In addition, the antibody or antigen-binding fragment thereof according to the present invention may comprise the sequence of the antibody or antigen-binding fragment thereof described herein, a sequence showing substantial identity with the sequence, as well as a biological equivalent thereof to the extent that it can specifically recognize and bind to the API5 epitope. For example, it may comprise additional mutations in the sequence to improve antibody binding affinity and / or biological properties, and additional mutations within a range that does not alter the overall activity of the molecule.

[0022]

[0023] Another aspect of the present invention provides a nucleic acid encoding the antibody or antigen-binding fragment thereof.

[0024] In the nucleic acid according to the present invention, unless specifically stated otherwise, related terms are understood to have the same meaning as the terms described above.

[0025] The term "nucleic acid" as used herein includes comprehensively DNA (gDNA and cDNA) and RNA. The nucleotides that make up the basic structural unit in a nucleic acid molecule include natural nucleotides as well as analogue nucleotides with modified sugar or base sites (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Review, 90:543-584 (1990)).

[0026] The nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to the present invention includes a nucleotide sequence exhibiting substantial identity to the nucleotide sequence described above. The substantial identity means that 80%, 90%, or 95% or more of sequence homology is shown as a result of aligning two sequences so as to correspond as much as possible and then analyzing using an algorithm commonly used in the art.

[0027]

[0028] Another aspect of the present invention provides a vector comprising the nucleic acid and a cell transformed with the vector.

[0029] In the vector and the cell transformed with the same according to the present invention, unless specifically stated otherwise, related terms are understood to have the same meaning as the terms described above.

[0030] The term "vector" as used herein refers to any one that is inserted into a host cell and capable of gene replication. The vector includes plasmid, linear nucleic acid, cosmid, RNA vector, viral vector, etc., and the viral vector includes, but not limited to, retrovirus, adenovirus, adeno-associated virus, and the like. A recombinant vector system of the present invention may be constructed through various methods known in the art. In addition, the vector of the present invention may be constructed as a vector for cloning or expression, and may be constructed using prokaryotic or eukaryotic cells as a host.

[0031] Furthermore, the cells may be prokaryotic cells, eukaryotic cells or animal cells. An appropriately selected host cell may be transformed with the vector and used to express and / or secrete a target protein. The host cells may be immortalized hybridoma cells, N / SO myeloma cells, 293 cells, HuT 78 cells, CHO cells, HELA cells, COS cells, and the like, preferably CHO cells. However, they are not limited thereto, and any host cell known in the art may be used as the host cell of the present invention.

[0032]

[0033] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the antibody or antigen-binding fragment thereof.

[0034] In the pharmaceutical composition according to the present invention, unless specifically stated otherwise, related terms are understood to have the same meaning as the terms described above.

[0035] The term "treatment" as used herein refers to anything in which cancer is improved, reversed, or completely cured by administration of the composition according to the present invention.

[0036] The term "prevention" as used herein refers to anything that inhibits, delays, or blocks the occurrence or recurrence of cancer by administration of the composition according to the present invention.

[0037] In the present invention, the cancer may exhibit resistance, tolerance, or refractory to anticancer drugs. The term "resistance to anticancer drugs" or "tolerance to anticancer drugs" as used herein means that when treating cancer patients using anticancer drugs, there is no effect from the beginning of treatment, or there is a therapeutic effect at the beginning, but is reduced or lost during continuous treatment. In addition, the term "refractory to anticancer drugs" as used herein means that when treating cancer patients using anticancer drugs, there is no effect from the beginning of treatment, or the response to treatment is not sustained for a long time.

[0038] API5 disclosed in the present invention exhibits increased expression in cancer cells. In particular, the specific API5 epitope represented by the amino acid sequence of SEQ ID NO: 7 comprises amino acid residues that bind to the TLR4 protein. Accordingly, it induces activation of ERK signaling through interaction with the TLR4 protein to give resistance, tolerance, or refractory to anticancer agents such as chemotherapeutic or immunotherapeutic agents. Therefore, the antibody or antigen-binding fragment thereof according to the present invention that specifically binds to the specific API5 epitope can reduce or decrease the activity of the API5 protein, and accordingly it can be very useful for preventing or treating cancers having resistance, tolerance, or refractory to the existing anticancer agents.

[0039] For example, the existing anticancer agents may be those reported to have resistance, tolerance, or refractory, specifically anticancer chemotherapeutic agents such as 5-FU, methotrexate, gemcitabine, cytarabine, paclitaxel, vinorelbine, cisplatin, oxaliplatin, and irinotecan, anticancer targeted agents such as bortezomib, cetuximab, crizotinib, dasatinib, gefitinib, imatinib, and vemurafenib, and / or anticancer immunotherapeutic agents such as CTLA-4 inhibitors, PD-1 inhibitors, PD-L1 inhibitors, and ACT (Adoptive Cell Therapy), but are not limited thereto.

[0040] In addition, the cancer may be one or more selected from the group consisting of bone cancer, lung cancer, head cancer, neck cancer, thyroid cancer, parathyroid cancer, non-small cell lung cancer, stomach cancer, liver cancer, pancreatic cancer, skin cancer, skin or intraocular melanoma, small intestine cancer, colorectal cancer, rectal cancer, proximal anal cancer, colon cancer, uterine cancer, breast cancer, ovarian cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, endocrine cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, renal cancer, ureteric cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, central nervous system lymphoma, spinal cord tumor, glioblastoma, brainstem glioma, and pituitary adenoma, but is not limited thereto.

[0041] The pharmaceutical composition of the present invention, comprising the antibody or antigen-binding fragment thereof in an effective amount, can be administered to a subject in need of prevention or treatment of cancer.

[0042] The term "administration" as used herein refers to physically giving a composition to a subject using any of various methods or delivery systems known to those skilled in the art. The administration may be, for example, oral administration, or intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral administration, such as administration by injection or infusion, etc., but is not limited thereto. The number of administrations may be, for example, single, multiple, or over one or more extended periods of time.

[0043] The term "subject" as used herein includes a human or any non-human animal which may be a vertebrate such as a primate, a dog, a cow, a horse, a pig, a rodent such as a mouse, a rat, a guinea pig, and the like. In this specification, the "subject" is used interchangeably with "individual" and "patient".

[0044] The effective amount may be a "therapeutically effective amount" or a "prophylactically effective amount". The term "therapeutically effective amount" refers to any amount capable of exhibiting a decrease in the severity of symptoms of a disease, an increase in the frequency and duration of symptom-free periods of a disease, or a prevention of damage or disability due to the suffering of a disease. The term "prophylactically effective amount" refers to any amount that inhibits the occurrence or recurrence of cancer in a subject. The level of the effective amount may be determined depending on the factors including subject's severity, age, sex, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment period, concurrently used drugs, and other factors well known in the medical field.

[0045] In addition, the dose of pharmaceutical composition may vary depending on the age, sex, and weight of the subject, and specifically, 0.1 to 100 mg / kg of the composition of the present invention may be administered once or several times a day, or at intervals of several days to several months, depending on the subject's symptoms. In addition, the dose may be increased or decreased depending on the route of administration, severity of disease, sex, weight, age, and the like.

[0046] In addition, the pharmaceutical composition may further comprise suitable carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions. The carriers, excipients and diluents that may be comprised in the composition include, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, but are not limited thereto.

[0047] In addition, the pharmaceutical composition may be administered in combination with other therapeutic agents. In this case, the pharmaceutical composition of the present invention and the other therapeutic agents may be administered simultaneously, sequentially, or separately. The other therapeutic agents may be drugs such as a compound or protein having effects of preventing, treating, and alleviating cancer, but are not limited thereto.

[0048] In addition, the pharmaceutical composition may be formulated to be administered simultaneously, sequentially or separately with other therapeutic agents. For example, the antibody or antigen-binding fragment thereof and the other therapeutic agents may be administered simultaneously in one formulation, or simultaneously, sequentially or separately in separate formulations. For simultaneous, sequential or separate administration, the antibody or antigen-binding fragment thereof comprised in the pharmaceutical composition of the present invention and other therapeutic agents may be separately formulated in different containers or formulated together in the same container. In addition, they may have the same or different pharmaceutically effective amount, administration time, administration interval, administration route, treatment period, and the like.

[0049]

[0050] Another aspect of the present invention provides a method for preventing or treating cancer, comprising administering the antibody or antigen-binding fragment thereof.

[0051] In the prevention or treatment method according to the present invention, unless otherwise specified, related terms are understood to have the same meaning as the terms described above.

[0052] In addition, in the method for preventing or treating cancer according to the present invention, the antibody or antigen-binding fragment thereof may be administered to a subject simultaneously, sequentially or individually with other therapeutic agents.

[0053] The "simultaneous" administration refers to administration of the antibody or antigen-binding fragment thereof and the other therapeutic agents at one time in one formulation, or administration of the antibody or antigen-binding fragment thereof and the other therapeutic agents at one time in separate formulations. In this case, the route of administration of the antibody or antigen-binding fragment thereof and other therapeutic agents may be different from each other.

[0054] The "sequential" administration refers to relatively consecutive administration of the antibody or antigen-binding fragment thereof and the other therapeutic agents, allowing as little time as possible to be consumed in the administration interval.

[0055] The "individual" administration refers to administration of the antibody or antigen-binding fragment thereof and other therapeutic agents at predetermined time intervals. The method of administering the antibody or antigen-binding fragment thereof and other therapeutic agents may be appropriately chosen by a physician or expert in the art in consideration of therapeutic efficacy and side effects of a subject.

[0056]

[0057] Another aspect of the present invention provides a composition for diagnosing cancer comprising the antibody or antigen-binding fragment thereof.

[0058] Another aspect of the present invention provides a kit for diagnosing cancer comprising the antibody or antigen-binding fragment thereof.

[0059] In the composition and kit for diagnosing cancer according to the present invention, unless otherwise specified, related terms are understood to have the same meaning as the terms described above.

[0060] It is known that the API5 protein disclosed in the present invention is overexpressed in various types of cancer cells, including cancers that exhibits resistance, tolerance, or refractory to anticancer drugs, as well as general cancers. In particular, the specific API5 epitope represented by the amino acid sequence of SEQ ID NO: 7 comprises amino acid residues that bind to the TLR4 protein. Accordingly, it induces activation of ERK signaling, inhibition of apoptosis of cancer cells, or promotion of tumor growth through interaction with the TLR4 protein. Therefore, the antibody or antigen-binding fragment thereof that binds to the specific API5 epitope with high specificity and affinity can be usefully used for diagnosing cancer.

[0061] Specifically, cancer diagnosis according to the present invention may be performed by reacting the antibody or antigen-binding fragment thereof with a biological sample to determine whether or not cancer has occurred or is likely to develop, and preferably, by contacting the antibody or antigen-binding fragment thereof and the biological sample to determine whether an antigen-antibody complex is formed.

[0062] The term "biological sample" used herein includes tissue, cell, blood, serum, plasma, tissue autopsy sample (brain, skin, lymph node, spinal cord), etc., but is not limited thereto.

[0063] The term "antigen-antibody complex" used herein refers to a complex formed by the binding of an API5 protein antigen in a sample and an antibody or antigen-binding fragment thereof of the present invention recognizing it.

[0064] The formation of such an antigen-antibody complex may be detected by any method such as colorimetric method, electrochemical method, fluorescence method, luminometric method, particle counting method, visual assessment or scintillation counting method. However, it is not limited thereto, and various applications and modifications are possible according to methods known in the art.

[0065] Specifically, the diagnostic composition or kit of the present invention may be prepared to be suitable for various immunoassays or immunostaining. The immunoassay or immunostaining includes, but not limited to, enzyme-linked immunosorbent assay (ELISA), immunofluorescence, western blotting, immunohistochemistry staining, flow cytometry, immunocytochemistry, radioimmunoassay (RIA), immunoprecipitation assay, and protein chip.

[0066] In addition, labels for qualitatively or quantitatively determining the formation of antigen-antibody complexes include enzymes, fluorescent substances, ligands, luminous substances, microparticles, redox molecules, and radioisotopes, but are not limited thereto.

[0067]

[0068] Another aspect of the present invention provides a method for providing information for cancer diagnosis, comprising detecting the API5 protein or determining the expression or activity level of the API5 protein in a biological sample isolated from a subject suspected of having cancer through an antigen-antibody reaction using the antibody or antigen-binding fragment thereof.

[0069] In the method for providing information for cancer diagnosis according to the present invention, unless otherwise specified, related terms are understood to have the same meaning as the terms described above.

[0070] In the present invention, the method for providing information for cancer diagnosis may be performed by reacting the antibody or antigen-binding fragment thereof with a biological sample to determine whether or not cancer has occurred or is likely to develop, and preferably, by contacting the antibody or antigen-binding fragment thereof and the biological sample to determine whether an antigen-antibody complex is formed.

[0071] In one embodiment according to the present invention, the method for providing information for cancer diagnosis may be a cancer diagnosis method, a method for providing information for selecting a cancer patient who has resistance, tolerance, or refractory to anticancer drugs, or a method for selecting a cancer patient who has resistance, tolerance, or refractory to anticancer drugs.

[0072] Specifically, the method comprises (a) treating a biological sample isolated from a subject suspected of having cancer with the antibody or antigen-binding fragment thereof to detect API5 protein through an antigen-antibody reaction; and (b) comparing the expression or activity level of API5 protein detected in step (a) with that of the control group, and determining that the subject has cancer if the expression or activity level of API5 protein is higher than that of the control group.

[0073] Herein, the control group may be a biological sample isolated from a healthy person without cancer or a person who has been cured of cancer.

[0074]

[0075] Another aspect of the present invention provides a method for providing information for cancer treatment, comprising detecting the API5 protein or determining the expression or activity level of the API5 protein in a biological sample isolated from a cancer patient through an antigen-antibody reaction using the antibody or antigen-binding fragment thereof.

[0076] In the method for providing information for cancer treatment according to the present invention, unless otherwise specified, related terms are understood to have the same meaning as the terms described above.

[0077] In the present invention, the method for providing information for cancer treatment may be performed by reacting the antibody or antigen-binding fragment thereof with a biological sample to provide information about resistance, tolerance, or refractory to anticancer drugs, and preferably, by contacting the antibody or antigen-binding fragment thereof and the biological sample to determine whether an antigen-antibody complex is formed.

[0078] In one embodiment according to the present invention, the method for providing information for cancer treatment may be a method for providing information for selecting a cancer patient who has resistance, tolerance, or refractory to anticancer drugs, or a method for selecting a cancer patient who has resistance, tolerance, or refractory to anticancer drugs.

[0079] In one embodiment according to the present invention, the method comprises (a) treating a biological sample isolated from a cancer patient with the antibody or antigen-binding fragment thereof to detect API5 protein through an antigen-antibody reaction; and (b) comparing the expression or activity level of API5 protein detected in step (a) with that of the control group, and determining that the patient has resistance, tolerance, or refractory to anticancer drugs if the expression or activity level of API5 protein is higher than that of the control group.

[0080] In one embodiment according to the present invention, the method comprises (a) treating a biological sample isolated from a cancer patient with the antibody or antigen-binding fragment thereof to detect API5 protein through an antigen-antibody reaction; and (b) comparing the expression or activity level of API5 protein detected in step (a) with that of the control group, and determining that the cancer or tumor is more likely to have resistance, tolerance, or refractory to anticancer drugs if the expression or activity level of API5 protein is higher than that of the control group.

[0081] Herein, the control group may be a biological sample isolated from a healthy person without cancer, a person who has been cured of cancer, or a cancer patient determined not to have resistance, tolerance, or refractory to anticancer drugs.

[0082]

[0083] Another aspect of the present invention provides a method for screening anticancer drugs comprising (a) treating cancer cells with an anticancer drug candidate; (b) determining the expression or activity level of the API5 protein in cancer cells treated with the anticancer drug candidate using the antibody or antigen-binding fragment thereof; and (c) determining the anticancer drug candidate treated in step (a) as an anticancer drug when the API5 protein expression or activity level in step (b) is lower than that in cancer cells not treated with the anticancer drug candidate.

[0084] In the method for screening anticancer drugs according to the present invention, unless otherwise specified, related terms are understood to have the same meaning as the terms described above.

[0085] Specifically, step (a) is a step of treating cancer cells with an anticancer drug candidate, and may be performed using a method known in the art. For example, the anticancer drug candidate may be treated to cancer cells and cocultured, or administered to a living body having cancer cells. However, it is not limited thereto, and those skilled in the art will be able to use a method suitable for the purpose of the present invention.

[0086] The term "anticancer drug" used herein refers to an agent that exhibits a preventive or therapeutic effect on cancer, and specifically an agent capable of killing cancer cells or tumors, or inhibiting their growth. In the present specification, the "anticancer drug" may be used interchangeably with "drug", and the "treatment" may be used interchangeably with "addition" or "administration.

[0087] The term "anticancer drug candidate" used herein refers to an agent expected to exhibit a preventive or therapeutic effect on cancer, and specifically, an agent expected to kill cancer cells or tumors, or inhibit their growth.

[0088] Preferably, the anticancer drug or anticancer drug candidate includes, but not limited to, a compound, protein, fusion protein, compound-protein complex, drug-protein complex, antibody, compound-antibody complex, drug-antibody complex, amino acid, peptide, virus, carbohydrate, lipid, nucleic acid, extracts, fractions, etc. For example, they may include, but not limited to, a compound, peptide, peptide mimetics, fusion protein, antibody, aptamer, antibody-drug conjugate (ADC), etc. Preferably, they may be an antibody or an immuno-oncology agent.

[0089] Step (b) is a step of determining the expression or activity level of the API5 protein, and any method known to those skilled in the art may be used. For example, western blot, co-immunoprecipitation assay, enzyme linked immunosorbent assay (ELISA), tissue immunostaining, flow cytometry analysis, etc. may be used, but is not limited thereto, and those skilled in the art will be able to use a method suitable for the purpose of the present invention.

[0090] The step (c) is a step of determining whether the anticancer drug candidate can be used as an anticancer drug. API5 protein is overexpressed in cancer cells and the specific API5 epitope represented by the amino acid sequence of SEQ ID NO: 7 comprises amino acid residues that bind to the TLR4 protein. Accordingly, it promotes the proliferation, migration, invasion, and growth of cancer cells through interaction with the TLR4 protein. Therefore, the anticancer drug candidate that reduces the expression or activity level of API5 protein can be used as an anticancer drug. In addition, API5 protein is overexpressed in cancer cells that have resistance, tolerance, or refractory to anticancer drugs, and the specific API5 epitope represented by the amino acid sequence of SEQ ID NO: 7 comprises amino acid residues that bind to the TLR4 protein. Accordingly, it promotes the proliferation, migration, invasion, and growth of cancer cells that have resistance, tolerance, or refractory to anticancer drugs through interaction with the TLR4 protein. Therefore, the anticancer drug candidate that reduces the expression or activity level of API5 protein can be used as an anticancer drug for the prevention or treatment of cancers exhibiting resistance, tolerance, or refractory to anticancer drugs.

[0091] The API5 epitope according to the present invention induces stronger resistance, tolerance, or refractory to anticancer drugs of cancer compared to other regions or epitopes of the API5 protein. Therefore, such antibodies or antigen-binding fragments thereof binding to the API5 epitope are very effective in killing cancer cells and inhibiting the proliferation or growth of tumors. In particular, the anticancer effect on cancer exhibiting resistance, tolerance, or refractory to existing anticancer drugs is very excellent.

[0092] FIG. 1 is an image showing the API5 epitope.

[0093] FIG. 2 is a graph showing the binding ability of peptides containing each region of the API5 protein and five anti-API5 antibodies #1 to #5. API5 Protein refers to a full-length API5 protein, and the untreated group refers to the group not treated with the protein or peptide. In addition, API5 1-50 refers to a peptide composed of amino acids 1 to 50 of the API5 protein, API5 51-100 refers to a peptide composed of amino acids 51 to 100, API5 101-150 refers to a peptide composed of amino acids 101 to 150, API5 151-200 refers to a peptide composed of amino acids 151 to 200, API5 201-220 refers to a peptide composed of amino acids 201 to 220, API5 206-225 refers to a peptide composed of amino acids 206 to 225, API5 251-300 refers to a peptide composed of amino acids 251 to 300, API5 301-350 refers to a peptide composed of amino acids 301 to 350, API5 340-359 refers to a peptide composed of amino acids 340 to 359, API5 401-430 refers to a peptide composed of amino acids 401 to 430, API5 431-470 refers to a peptide composed of amino acids 431 to 470, and API5 471-504 refers to a peptide composed of amino acids 471 to 504.

[0094] FIG. 3 is western blot results for phosphorylated ERK (pERK) and ERK, and shows the ERK phosphorylation reduction effect of five anti-API5 antibodies #1 to #5 according to the present invention and the anti-API5 antibodies Novus, D-1 and Mybio in the comparative experimental group on TC-1 P3PD-1, MC38 P3PD-1, and CT26 P3PD-1.

[0095] FIG. 4 is western blot results for phosphorylated ERK (pERK) and ERK, and shows the ERK phosphorylation reduction effect of anti-API5 antibody #1 according to the present invention on TC-1 P3PD-1, MC38 P3PD-1, and CT26 P3PD-1.

[0096] FIG. 5 represents graphs showing the apoptosis increasing effect of anti-API5 antibodies #3, #4 and #5 according to the present invention and the anti-API5 antibodies Novus, D-1 and Mybio in the comparative experimental group on TC-1 P3PD-1and CT26 P3PD-1.

[0097] FIG. 6 represents graphs showing the tumor growth inhibitory effect of the anti-API5 antibody #1 according to the present invention in thein vivomodel of colorectal cancer or colorectal cancer refractory to anticancer immunotherapeutic agents. * means p-value < 0.05, and **** means p-value < 0.0001.

[0098] FIG. 7 is a graph showing the tumor growth inhibitory effect of the anti-API5 antibody #1 according to the present invention in thein vivomodel of non-small cell lung cancer or cervical cancer. *** means p-value < 0.001.

[0099] FIG. 8 represents graphs showing the tumor growth inhibitory effect of the anti-API5 antibody #1 according to the present invention in thein vivomodel of colorectal cancer or colorectal cancer refractory to anticancer immunotherapeutic agents. * means p-value < 0.05, *** means p-value < 0.001, and **** means p-value < 0.0001.

[0100] FIG. 9 is a graph showing the tumor growth inhibitory effect of the anti-API5 antibody #1 according to the present invention, an anti-PD-1 antibody, or a combination of the anti-API5 antibody #1 and the anti-PD-1 antibody in thein vivomodel of colorectal cancer refractory to anticancer immunotherapeutic agents. **** means p-value < 0.0001.

[0101] FIG. 10 is a graph showing the tumor growth inhibitory effect of anti-API5 antibodies #1 to #4 according to the present invention and the anti-API5 antibodies D-1 and Mybio in the comparative experimental group in thein vivomodel of colorectal cancer refractory to anticancer immunotherapeutic agents. *** means p-value < 0.001, and **** means p-value < 0.0001.

[0102] FIG. 11 is a graph showing the tumor growth inhibition rate of the anti-API5 antibodies #1 to #4 according to the present invention and the anti-API5 antibodies D-1 and Mybio in the comparative experimental group in thein vivomodel of colorectal cancer refractory to anticancer immunotherapeutic agents.

[0103] Hereinafter, the present invention will be explained in more detail through examples. These examples are intended to explain the present invention in more detail, and the scope of the present invention is not limited by these examples.

[0104]

[0105] Example 1. Identification of specific API5 epitope

[0106] The inventors identified for the first time that a specific API5 epitope, corresponding to amino acids 206th to 225th of the API5 protein, plays a major role in causing cancer resistance, tolerance, or refractory to anticancer drugs. Additionally, we discovered for the first time that the specific API5 epitope comprises amino acid residues involved in binding to the TLR4 protein, which is a receptor for the API5 protein.

[0107] To verify this, we analyzed modeling suggesting amino acid residues involved in the binding of the API5 protein to its receptor, the TLR4 protein. FIG. 1 shows that the API5 protein binds to the TLR4 protein through its 206th to 225th amino acid residues. Particluar binding residue pairs of API5 protein and TLR4 protein are shown in Table 1 below.

[0108] API5TLR4S208N22Q211E20Q212E20Q212L21E215M19E219M19

[0109]

[0110] Example 2. Anti-API5 antibodies binding to specific API5 epitope

[0111] Example 2-1. Preparation of anti-API5 antibodies

[0112] The inventors prepared antibodies binding to the API5 protein. Specifically, antibodies that specifically bind to the amino acid residue serving as an epitope in the API5 protein were prepared. To discover an antibody that binds to API5, a human synthetic Fab phage library was used, and the API5 gene expression vector was transiently expressed in Expi293 cells (Thermo Fisher Scientific), and then the purified API5 protein was used. Antibodies exhibiting binding ability to API5 were selected through a phage display technology. As a result, a Fab antibody phage clone was selected, and then anti-API5 antibodies (#1 to #5) were prepared by transplanting it into an IgG4(S228P) backbone. The anti-API5 antibody #1 comprises a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 1 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 8. The anti-API5 antibody #2 comprises a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 2 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 9. The anti-API5 antibody #3 comprises a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 3 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 10. The anti-API5 antibody #4 comprises a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 4 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 11. The anti-API5 antibody #5 comprises a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 5 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 12.

[0113] In the comparative experimental group described later, we used commercially available antibodies, specifically "NOVUS" antibody available from NOVUS that binds to the region consisting of the amino acids 1st to 50th of the API5 protein (1-50), "D-1" antibody available from Santa Cruz Biotechnology that binds to the region consisting of the amino acids 72th to 84th of the API5 protein (72-84), and "Mybio" antibody available from MyBioSource that binds to the region consisting of the amino acids 487th to 504th of the API5 protein (487-504).

[0114]

[0115] Example 2-2. Analysis of binding activity of anti-API5 antibodies to specific API5 epitope

[0116] The inventors confirmed whether the anti-API5 antibodies prepared in Example 2-1 binds to the specific API5 epitope identified as a new target for anticancer drugs through Example 1.

[0117] Specifically, the full length API5 protein composed of 504-mer amino acid was divided into regions composed of about 20 to 50-mer amino acid, and then peptide fragments containing the region were prepared. After that, the binding level of the anti-API5 antibodies prepared in Example 2-1 and each API5 region was determined.

[0118] In Table 2 below, the entire amino acid sequence of the API5 protein consisting of 504-mer amino acid is represented by SEQ ID NO: 6.

[0119] TypeAmino acid sequenceNumber of sequenceAPI5 proteinMPTVEELYRNYGILADATEQVGQHKDAYQVILDGVKGGTKEKRLAAQFIPKFFKHFPELADSAINAQLDLCEDEDVSIRRQAIKELPQFATGENLPRVADILTQLLQTDDSAEFNLVNNALLSIFKMDAKGTLGGLFSQILQGEDIVRERAIKFLSTKLKTLPDEVLTKEVEELILTESKKVLEDVTGEEFVLFMKILSGLKSLQTVSGRQQLVELVAEQADLEQTFNPSDPDCVDRLLQCTRQAVPLFSKNVHSTRFVTYFCEQVLPNLGTLTTPVEGLDIQLEVLKLLAEMSSFCGDMEKLETNLRKLFDKLLEYMPLPPEEAENGENAGNEEPKLQFSYVECLLYSFHQLGRKLPDFLTAKLNAEKLKDFKIRLQYFARGLQVYIRQLRLALQGKTGEALKTEENKIKVVALKITNNINVLIKDLFHIPPSYKSTVTLSWKPVQKVEIGQKRASEDTTSGSPPKKSSAGPKRDARQIYNPPSGKYSSNLGNFNYERSLQGKSEQ ID NO: 6

[0120] Specifically, peptides containing the region consisting of the amino acids 1 to 50 of the API5 protein (API5 1-50), the region consisting of the amino acids 51 to 100 (API5 51-100), the region consisting of the amino acids 101 to 150 (API5 101-150), the region consisting of the amino acids 151 to 200 (API5 151-200), the region consisting of the amino acids 201 to 220 (API5 201-220), the region consisting of the amino acids 206 to 225 (API5 206-225), the region consisting of the amino acids 251 to 300 (API5 251-300), the region consisting of the amino acids 301 to 350 (API5 301-350), the region consisting of the amino acids 340 to 359 (API5 340-359), the region consisting of the amino acids 401 to 430 (API5 401-430), the region consisting of the amino acids 431 to 470 (API5 431-470), or the region consisting of the amino acids 471 to 504 (API5 471-504) were prepared, respectively.

[0121] Then, ELISA was performed to detect the binding between the peptide and the anti-API5 antibody. A 96-well ELISA microtiter plate was coated overnight at 4 ℃ with streptavidin at a concentration of 5 μg / ml dissolved in PBS. The plate was washed twice and blocked with 3% MPBS for 1 hour at 37 ℃. Thereafter, 100 μl of each peptide at a concentration of 10 μg / ml dissolved in PBS was added to the plate and incubated at 37 ℃ for 1 hour. After washing the plate three times, 100 μl of anti-API5 antibody was added and incubated for 2 hours at a room temperature. After washing, HRP-conjugated anti-mouse IgG (H&L) was added to the plate and incubated at 37 ℃ for 30 minutes. The plate was further washed six times, and the reaction proceeded for 1 minute by adding a substrate solution to the wells. Absorbance was measured at 450 nm using a spectrometer.

[0122] As a result, FIG. 2 shows that all of the anti-API5 antibodies #1 to #5 showed excellent binding to the peptide containing API5 206-225 among API5 peptides containing each region.

[0123]

[0124] Furthermore, the binding affinity between the specific API5 epitope identified as a new target for anticancer drugs in Example 1 and the anti-API5 antibody prepared in Example 2-1 was confirmed through Surface Plasmon Resonance (SPR) analysis.

[0125] Specifically, the binding ability of the purified anti-API5 antibody that binds to the specific API5 epitope was individually analyzed using Biacore T200. API5 was immobilized on a sensor chip and the binding rate was measured using the anti-API5 antibody as an analyte. Data on dissociation rate constant (kd) and association rate constant (ka) rate constants were determined using Biacore evaluation software. The association rate constant (ka) represents the rate of complex formation, i.e., the number of antigen-antibody complexes formed per second in a 1 molar solution of a specific API5 epitope and anti-API5 antibody, and its unit is 1 / Ms. The dissociation rate constant (kd) indicates the stability of the complex, i.e. the rate at which the complex dissociates per second, and its unit is 1 / s. For example, kd=0.01 1 / s means that 1% of the complex collapses in 1 second. When the equilibrium dissociation constant (KD) calculated from the ratio of kd to ka is 1 x 10-9M or less, the binding ability of the antibody to the antigen protein is assessed to be excellent.

[0126] Analyteka (l / Ms)kd (1 / s)KD (M)Anti-API5 antibody #11.91 x 1051.29 x 10-46.76 x 10-10Anti-API5 antibody #21.79 x 1051.08 x 10-46.02 x 10-10Anti-API5 antibody #32.38 x 1051.07 x 10-44.51 x 10-10Anti-API5 antibody #43.13 x 1067.59 x 10-42.43 x 10-11Anti-API5 antibody #55.43 x 1061.47 x 10-42.71 x 10-11

[0127] As a result, as shown in Table 3 above, it was confirmed that anti-API5 antibodies #1 to #5 had excellent binding ability to the specific API5 epitope (API5 206-225) described as SEQ ID NO: 7.

[0128]

[0129] Example 3. Cancer treatment effect of anti-API5 antibody binding to the specific API5 epitope

[0130] Example 3-1. Reduction of phosphorylated ERK (pERK)

[0131] In order to check the anti-cancer effect of the specific API5 epitope identified in Example 1, the effect of reducing ERK activation (i.e., ERK phosphorylation) in cancer cells by the anti-API5 antibodies binding to the epitope was analyzed. ERK is mainly activated by growth factors involved in cell proliferation, differentiation, and survival. Activation of ERK is achieved by phosphorylation of ERK protein, and continuous activation of ERK plays an important role in tumorigenesis. It is known that the activation of ERK is increased in various types of tumors such as pancreatic cancer, colorectal cancer, lung cancer, ovarian cancer, kidney cancer, and breast cancer.

[0132] Specifically, TC-1 P3PD-1cell line for non-small cell lung cancer or uterine cancer, MC38 P3PD-1cell line for colorectal cancer, and CT26 P3PD-1cell line for colorectal cancer, which are refractory to anti-PD-1 antibody treatment, were seeded in a 12-well plate at 1.5 x 105cells per well, using RPMI medium containing 5% FBS. After two hours, the medium was replaced with RPMI medium containing 0.1% FBS, and treated with each anti-API5 antibody at a concentration of up to 150 ng / ml.

[0133] After 24 hours, the cells were washed with a 1X PBS buffer solution, and placed at 4 ℃ for 10 minutes in 150 μl of a RIPA buffer solution to induce cell lysis. And then, centrifugation was performed at 4 ℃ for 15 minutes at a speed of 13,000 rpm to obtain a cell extract as a supernatant. Protein was quantified using the Bradford assay, and the level of ERK protein and its phosphorylation was measured through the SDS-PAGE western blot for the same amount of protein. The measured ERK protein level and ERK protein phosphorylation level were quantified using Image J (densitometer), and the ratio of phosphorylated ERK protein level to total ERK protein level was calculated.

[0134] Herein, the group not treated with the anti-API5 antibody was set as a control group (Con). As antibodies in the comparative experimental group, "NOVUS" antibody available from NOVUS that binds to the region (1-50) consisting of the amino acids 1 to 50 of the API5 protein, "D-1" antibody available from Santa Cruz Biotechnology that binds to the region (72-84) consisting of the amino acids 72 to 84 of the API5 protein, and "Mybio" antibody available from MyBioSource that binds to the region (487-504) consisting of the amino acids 487 to 504 of the API5 protein were used.

[0135] The "TC-1 P3PD-1", "MC38 P3PD-1" or "CT26 P3PD-1" refers to cells refractory to anti-PD-1 antibody treatment, survived from three times subculturing the parent cancer cells "TC-1 P0", "MC38 P0" or "CT26 P0" with an anti-PD-1 antibody.

[0136] As a result, FIG. 3 shows that ERK phosphorylation was significantly reduced in all cancer cell lines by the anti-API5 antibodies binding to the specific API5 epitope represented by the amino acid sequence of SEQ ID NO: 7, and the ratio of ERK phosphorylation to total ERK reduced by up to half compared to the control group. These results were the same for all of the anti-API5 antibodies #1 to #5. On the contrary, the anti-API5 antibodies (Novus, D-1 and Mybio) in the comparative experimental group that do not bind to the specific API5 epitope represented by the amino acid sequence of SEQ ID NO: 7 significantly increased ERK phosphorylation in all cancer cell lines, and the ratio of ERK phosphorylation to total ERK was increased by up to 2 times compared to the control group.

[0137] In addition, as shown in FIG. 4, ERK phosphorylation was significantly reduced in all cancer cell lines by the anti-API5 antibodies binding to the specific API5 epitope represented by the amino acid sequence of SEQ ID NO: 7. On the contrary, when the anti-API5 antibody and the peptide having the specific API5 epitope sequence represented by the amino acid sequence of SEQ ID NO: 7 were simultaneously treated to prevent the anti-API5 antibody from targeting API5, the effect of reducing ERK phosphorylation was not shown.

[0138] The above results suggest that the specific API5 epitope represented by the amino acid sequence of SEQ ID NO: 7 according to the present invention comprises amino acid residues that bind to the TLR4 protein, which is a receptor for the API5 protein. Thus, it involves ERK activation in cancer cells through interaction with the TLR4 protein. Therefore, it suggests that anti-API5 antibodies binding to the specific API5 epitope can reduce ERK activation in cancer cells, thereby exhibiting excellent anticancer effects.

[0139]

[0140] Example 3-2. Increased apoptosis

[0141] In order to check the anticancer effect of the specific API5 epitope identified in Example 1, the effect of increasing apoptosis of cancer cells by the anti-API5 antibodies binding to the epitope was analyzed.

[0142] TC-1 P3PD-1non-small cell lung cancer or cervical cancer cell line and CT26 P3PD-1colorectal cancer cell line prepared in Example 3-1 were seeded in a 12-well plate at 3 x 105cells per well, and RPMI medium containing 5% FBS was used. After two hours, the medium was replaced with RPMI medium containing 0.1% FBS, and each anti-API5 antibody was treated at a maximum concentration of 150 ng / ml. After 24 hours, the percentage of dead cells by apoptosis was determined using FACS.

[0143] Herein, as antibodies in the comparative experimental group, "NOVUS" antibody available from NOVUS that binds to the region (1-50) consisting of the amino acids 1 to 50 of the API5 protein, "D-1" antibody available from Santa Cruz Biotechnology that binds to the region (72-84) consisting of the amino acids 72 to 84 of the API5 protein, and "Mybio" antibody available from MyBioSource that binds to the region (487-504) consisting of the amino acids 487 to 504 of the API5 protein were used.

[0144] As a result, FIG. 5 shows that the proportion of cells killed by the anti-API5 antibodies binding to the specific API5 epitope represented by the amino acid sequence of SEQ ID NO: 7 increased by about 10 to 20% compared to the group untreated with an anti-API5 antibody, and these effects were observed equally in all cancer cell lines. On the contrary, when treated with the anti-API5 antibodies (Novus, D-1 and Mybio) in the comparative experimental group that do not bind to the specific API5 epitope represented by the amino acid sequence of SEQ ID NO: 7, the ratio of apoptotic cells in all cancer cell lines was rather reduced by about 10 to 60%.

[0145] The above results suggest that the specific API5 epitope represented by the amino acid sequence of SEQ ID NO: 7 according to the present invention comprises amino acid residues that bind to the TLR4 protein, which is a receptor for the API5 protein. Thus, it involves apoptosis of cancer cells through interaction with the TLR4 protein. Therefore, it suggests that anti-API5 antibodies binding to the specific API5 epitope can increase the sensitivity of cancer cells to apoptosis, thereby exhibiting excellent anticancer effects.

[0146]

[0147] Example 3-3. Inhibition of tumor growthin vivo

[0148] In order to check the anticancer effect of the specific API5 epitope identified in Example 1, thein vivogrowth inhibitory effect of the anti-API5 antibodies binding to the specific API5 epitope represented by the amino acid sequence of SEQ ID NO: 7 was analyzed.

[0149] In the experiments, MC-38 P0 colorectal cancer cell line, MC-38 P3PD-1colorectal cancer cell line refractory to anti-PD-1 antibody treatment, MC-38 P3PD-L1colorectal cancer cell line refractory to anti-PD-L1 antibody treatment, TC-1 P0 non-small cell lung cancer or uterine cancer cell line, CT26 P0 colorectal cancer cell line, CT26 P3PD-1colorectal cancer cell line refractory to anti-PD-1 antibody treatment were used.

[0150] Specifically, tumors were formed by transplanting each cell into a balb / c mouse at a cell number of 5 x 105. When the size of the tumor reached an average of about 60 to 75 mm3(30 to 40 mm3for MC-38), 8 to 9 mice per experimental group were distributed so that the size of the tumor was distributed as uniformly as possible in each group, and administered with the anti-API5 antibodies #1 to #4 that bind to the epitope represented by the amino acid sequence of SEQ ID NO: 7 or the anti-API5 antibodies (D-1 or Mybio) that do not bind to the epitope represented by the amino acid sequence of SEQ ID NO: 7. The dose of antibody was 200 μg, and was intraperitoneally injected three times a week, a total of 5 times, based on the antibody administration start day (Day 1). Thereafter, the length of the tumor was measured using calipers at intervals of 2 or 3 days on the basis of the start day of antibody administration, and the size of the tumor was calculated using the formula: tumor size = {(long axis length x short axis length)2} / 2. In addition, the tumor growth inhibition rate (TGI, %) was calculated from the tumor size according to the formula: 100 - (100 x tumor size in the experimental group / tumor size in the IgG antibody-treated control group).

[0151] The above results were analyzed by assuming normality of the data and using parametric multiple comparison procedures. If the results of the parametric two-way ANOVA were significant, a post-hoc test was performed using Sidak's multiple comparison test. Statistical analysis was performed using Prism 8.0.1 (GraphPad Software Inc., San Diego, CA, USA), and a P value less than 0.05 was determined to be statistically significant.

[0152] As a result, as shown in FIGs. 6 to 8, tumor growth could be remarkably inhibited by the anti-API5 antibodies that bind to the specific API5 epitope represented by the amino acid sequence of SEQ ID NO: 7. The tumor growth inhibitory effect was increased by about 1.5 to 4 times in the experimental group compared to the control group treated with IgG antibody. This effect was not only shown for cancer cells that are refractory to existing immuno-anticancer drugs, but also for various cancers such as colorectal cancer, non-small cell lung cancer, or cervical cancer.

[0153] In particular, FIG. 9 shows that when the anti-API5 antibody is administered in combination with the anti-PD-1 antibody, it also exhibited an excellent growth inhibitory effect against cancer cells that exhibit tolerance, resistance, or refractory to anti-PD-1 antibody treatment, and this effect was synergistic.

[0154] Furthermore, as shown in FIGs. 10 and 11, the tumor growth inhibition rate (TGI) of anti-API5 antibodies #1 to #4 was 30.1%, 49.6%, 59.2% and 55.2%, respectively, and tumor growth was remarkably suppressed by at least 30% compared to the control IgG antibody. On the contrary, the anti-API5 antibodies (D-1 and Mybio) in the comparative experimental group that do not bind to the specific API5 epitope represented by the amino acid sequence of SEQ ID NO: 7 did not show tumor growth inhibitory effect.

[0155] The above results show that the specific API5 epitope represented by the amino acid sequence of SEQ ID NO: 7 according to the present invention comprises amino acid residues binding to the TLR4 protein, which is a receptor for the API5 protein, and accordingly it involves promoting the growth of cancer cells through interaction with the TLR4 protein. Also, since SEQ ID NO: 7 is an essential epitope sequence for the binding between the API5 protein and its target antibody, it is suggested that anti-API5 antibodies binding to the specific API5 epitope can exhibit excellent anticancer effects against cancers that are refractory to anticancer immunotherapeutic agents, and also exhibit excellent synergistic effects with drugs that have been used as immunotherapeutic agents.

[0156] Attached as an electronic file

Claims

1.An antibody or antigen-binding fragment thereof that specifically binds to an epitope of apoptosis inhibitor 5 (API5) represented by the amino acid sequence of SEQ ID NO: 7.2.A nucleic acid encoding the antibody or antigen-binding fragment thereof according to claim 1.3.A vector comprising the nucleic acid according to claim 2.4.A cell transformed with the vector according to claim 3.5.A pharmaceutical composition for preventing or treating cancer, comprising the antibody or antigen-binding fragment thereof according to claim 1.6.The pharmaceutical composition according to claim 5, wherein the cancer exhibits resistance, tolerance, or refractory to an anticancer drug.7.The pharmaceutical composition according to claim 6, wherein the anticancer drug is one or more selected from the group consisting of an anticancer chemotherapeutic agent, an anticancer targeted agent and an anticancer immunotherapeutic agent.8.The pharmaceutical composition according to claim 5, wherein the cancer is selected from the group consisting of bone cancer, lung cancer, head cancer, neck cancer, thyroid cancer, parathyroid cancer, non-small cell lung cancer, stomach cancer, liver cancer, pancreatic cancer, skin cancer, skin or intraocular melanoma, small intestine cancer, colorectal cancer, rectal cancer, proximal anal cancer, colon cancer, uterine cancer, breast cancer, ovarian cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, endocrine cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, renal cancer, ureteric cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, central nervous system lymphoma, spinal cord tumor, glioblastoma, brainstem glioma, and pituitary adenoma.9.A composition for diagnosing cancer, comprising the antibody or antigen-binding fragment thereof according to claim 1.10.A kit for diagnosing cancer, comprising the antibody or antigen-binding fragment thereof according to claim 1.11.A method for providing information for cancer diagnosis, comprising:detecting the API5 protein or determining the expression or activity level of the API5 protein in a biological sample isolated from a subject suspected of having cancer through an antigen-antibody reaction using the antibody or antigen-binding fragment thereof according to claim 1.12.A method for providing information for cancer treatment, comprising:detecting the API5 protein or determining the expression or activity level of the API5 protein in a biological sample isolated from a cancer patient through an antigen-antibody reaction using the antibody or antigen-binding fragment thereof according to claim 1.13.A method for screening an anticancer drug, comprising:(a) treating cancer cells with an anticancer drug candidate;(b) determining the expression or activity level of the API5 protein in cancer cells treated with the anticancer drug candidate using the antibody or antigen-binding fragment thereof according to claim 1; and(c) determining the anticancer drug candidate treated in step (a) as an anticancer drug when the API5 protein expression or activity level in step (b) is lower than that in cancer cells not treated with the anticancer drug candidate.

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