API5 epitope and antibody specifically binding to API5 epitope

Abstract

The invention relates to an API5 antigen epitope and an antibody specifically binding to the API5 antigen epitope or an antigen binding fragment thereof. The API5 epitopes according to the present invention elicit resistance, tolerance or non-responsiveness of cancer to anti-cancer drugs more strongly than other regions or epitopes present in the API5 protein. Therefore, the antibody or the antigen-binding fragment thereof that binds to the API5 epitope has an excellent cancer cell killing effect and an excellent tumor proliferation or growth inhibition effect, and particularly has an excellent anti-cancer effect on cancers that are resistant, tolerant or non-responsive to existing anti-cancer drugs.

Description

TECHNICAL FIELD

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

[0002] Despite intensive research on cancer over the past several decades, cancer remains a major cause of death worldwide. Many anti-cancer treatment methods have been developed, but are not effective for all cancer types and all patients. Current methods for treating cancer are relatively non-selective. By surgically removing diseased tissue, reducing the size of a solid tumor through radiotherapy, or using chemotherapy to rapidly kill cancer cells. However, chemotherapy can cause drug resistance, and sometimes limit the dose administered. This can cause serious side effects, so they can exclude the possibility of using potentially effective drugs. Therefore, there is a need to develop more target-specific and effective cancer therapies.

[0003] In this regard, targeted anti-cancer drugs that specifically target only cancer cells or anti-cancer immunotherapy drugs that utilize the patient's immune system are being developed. For example, in Korea, there are currently about 30 targeted anti-cancer drugs approved, and anti-cancer immunotherapy drugs approved in Korea include nivolumab (ingredient name: nivolumab), Keytruda (ingredient name: pembrolizumab), Tecentriq (ingredient name: atezolizumab), Imfinzi (ingredient name: durvalumab), Yervoy (ingredient name: ipilimumab), etc., 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 ratio of such targeted anti-cancer drugs and immunological anti-cancer drugs increases, the ratio of patients who have resistance, tolerance, or inappropriateness to them also increases accordingly. Therefore, there is an urgent need in the art to discover new targets and develop drugs that utilize them to treat cancers that have resistance, tolerance, or inappropriateness to existing anti-cancer drugs. SUMMARY

[0005] Technical problems to be solved The present inventors identified a new target for an anti-cancer drug and conducted research to develop a drug that utilizes it to treat cancers that have resistance, tolerance, or inappropriateness to existing anti-cancer drugs. As a result, an antigenic epitope of API5 represented by the amino acid sequence of SEQ ID NO: 7 according to the present application was identified, and it was experimentally demonstrated that an antibody or an antigen-binding fragment thereof targeting the antigenic epitope can also exhibit an excellent anti-cancer effect on cancers that have resistance, tolerance, or inappropriateness to existing anti-cancer drugs, thereby completing the present application.

[0006] Technical solution for solving the problem One aspect of the present application provides an antibody or an antigen-binding fragment thereof that specifically binds to an epitope of API5 (Apoptosis inhibitor 5).

[0007] The term "antibody" used in the present specification refers to an immunoglobulin molecule that is immunoreactive with a specific antigen, or a protein molecule that functions as a receptor for specifically recognizing an antigen. Thus, "antibody" in the present application is construed to include a monoclonal antibody, a polyclonal antibody, a whole antibody (an antibody composed of at least two heavy chains and two light chains linked to each other by disulfide bonds), an antibody fragment, Fab, Fab', F(ab')2, Fv, a single-chain Fv (scFv), a diabody, a linear antibody, a bispecific antibody, a multispecific antibody, a chimeric antibody, a humanized antibody, a human antibody, or an antigen-binding portion of an antibody. The whole antibody includes IgA, IgD, IgE, IgM, and IgG, which can include IgG1, IgG2, IgG3, and IgG4 as subtypes.

[0008] The term "antigen-binding fragment" used in the present specification refers to an antibody fragment or an analog of an antibody that retains at least a part of the binding specificity of the parent antibody and includes a part (e.g., one or more CDRs) of the antigen-binding region or the variable region of the parent antibody. The antigen-binding fragment can be, for example, Fab, Fab', F(ab')2, Fv fragment, sc-Fv, unibody, diabody, linear antibody, nanobody, domain antibody, or a multispecific antibody formed of antibody fragments.

[0009] Also, the antibody or the antigen-binding fragment thereof can form an antibody-drug conjugate (ADC) by binding to a drug.

[0010] The term "heavy chain" used in the present specification refers to a whole heavy chain and a fragment thereof that includes a heavy chain variable region and a heavy chain constant region. The heavy chain has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types.

[0011] The term "light chain" used in the present specification refers to a whole light chain and a fragment thereof that includes a light chain variable region and a light chain constant region. The light chain has kappa (κ) and lambda (λ) types.

[0012] The term "complementarity determining region (CDR)" used in the present specification is a part of the variable region of an antibody produced by B cells and T cells, which refers to an amino acid sequence of a hypervariable region forming an antigen binding site. Three CDRs are discontinuously arranged in the amino acid sequences of heavy chains and light chains, i.e., heavy chain CDR H1 , CDR H2 , CDR H3 , and light chain CDR L1 , CDR L2 , CDR L3 . The CDRs are sites involved in the recognition of an antigen, and provide major contact residues when an antibody binds to an antigen or an epitope, thus playing a decisive role in the diversity of antigen specificity.

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

[0014] The term "API5 (apoptosis inhibitor 5)" used in the present specification refers to a protein encoded by an API5 gene, and the amino acid sequence constituting it is known in the existing database (NCBI Reference Sequence: XP_016873953.1, XP_01687354.1, XP_006718422.1, etc.). The API5 protein is expressed at a high level in tumors such as solid cancer and metastatic cancer, and its expression is increased in cancer cells having resistance, tolerance, or insensitivity to anticancer drugs (Cancer Sci. 2013, 104(9): 1388-1394). Han Sol Janget al. Exp Mol Med. 2017 Sep 8, 49(9): e374 It is known that the API5 protein is phosphorylated and activates ERK1 / 2, thereby inducing tumor progression through cell cycle arrest and apoptosis inhibition (Cancer Sci. 2013, 104(9): 1388-1394). Hanbyoul Cho et al. BMC Cancer. 2014 Jul 28, 14:545

[0015] The term "epitope" used in the present specification is an antigenic determinant, which refers to a specific part of an antigen that allows an antibody or an immune cell such as a B cell, a T cell, etc. to recognize an antigen.

[0016] ​The antigenic epitope of API5 according to the present application can be represented by the amino acid sequence of SEQ ID NO: 7. Also, the API5 antigenic epitope can 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 can be a part of a 504-mer amino acid sequence constituting the API5 protein. More specifically, the API5 antigenic epitope represented by the amino acid sequence of SEQ ID NO: 7 can be the 206th to 225th amino acid sequence of the API5 protein represented by the amino acid sequence of SEQ ID NO: 6.

[0017] The term "anti-API5 antibody" used in the present specification refers to an antibody or an antigen-binding fragment thereof that specifically binds to API5 and neutralizes or inhibits the activity of API5. Also, the anti-API5 antibody refers to an antibody or an antigen-binding fragment thereof that specifically binds to an antigenic epitope of API5 to neutralize or inhibit the activity of API5. Specifically, the anti-API5 antibody according to the present application can bind to the antigenic epitope represented by the amino acid sequence of SEQ ID NO: 7.

[0018] The anti-API5 antibody can be a human antibody targeting a human API5 protein, and also reactive to an API5 protein derived from a mouse or a rat.

[0019] The term "human antibody" used in the present specification refers to an antibody having a variable region of a framework and a CDR region derived from a human immunoglobulin sequence. The human antibody in the present application can include an amino acid residue not encoded by a human-derived immunoglobulin sequence (e.g., a mutation introduced by inducing random or site-specific mutation in vitro, or a mutation introduced by somatic mutation in vivo).

[0020] The sequences disclosed in the present application include sequences showing substantial identity to the sequences described in the sequence listing. The substantial identity refers to a result of analysis using an algorithm commonly used in the art, showing 80%, 90%, or 95% or more sequence homology when two sequences are maximally aligned.

[0021] In addition, the antibody or antigen-binding fragment thereof of the present application, within the range of being able to specifically recognize and bind to an API5 antigenic epitope, can include the sequence of the antibody or antigen-binding fragment described in the present specification, a sequence showing substantial identity to the sequence, and biological equivalents thereof. For example, it can include additional mutations in the sequence to improve antibody binding affinity and / or biological properties, and additional mutations within the range of not changing the overall activity of the molecule.

[0022] Another aspect of the present application provides a nucleic acid encoding the antibody or antigen-binding fragment thereof.

[0023] In the nucleic acid of the present application, unless otherwise specifically stated, the relevant terms are to be understood as having the same meaning as the aforementioned terms.

[0024] The term "nucleic acid" used in the present specification has the meaning encompassing DNA (gDNA and cDNA) and RNA. The nucleotides constituting the basic structural unit in the nucleic acid molecule include natural nucleotides as well as analogue nucleotides having a modified sugar or base Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Review, 90:543-584 (1990) ).

[0025] The nucleic acid molecule encoding the antibody or the antigen-binding fragment thereof of the present application includes a nucleotide sequence showing substantial identity to the nucleotide sequence. The substantial identity means that, after the two sequences are maximally aligned, the result of analysis using an algorithm commonly used in the art shows 80%, 90%, or 95% or more sequence homology.

[0026] Still another aspect of the present application provides a vector comprising the nucleic acid and a cell transformed with the vector.

[0027] The vector and the cell transformed with the vector according to the present application, unless otherwise specifically stated, the relevant terms are to be understood as having the same meaning as the aforementioned terms.

[0028] The term "vector" used in the present specification means any vector inserted into a host cell and capable of gene replication. The vector includes a plasmid, a linear nucleic acid, a cosmid, an RNA vector, a viral vector, etc., and the viral vector includes, but is not limited to, a retrovirus, an adenovirus, an adeno-associated virus, etc. The recombinant vector system of the present application can be constructed by various methods known in the art. In addition, the vector of the present application can be constructed as a vector for cloning or expression, and can be constructed using prokaryotic or eukaryotic cells as a host.

[0029] Also, in the present application, the cell can be a prokaryotic cell, an eukaryotic cell, or an animal cell. The vector can be used to transform a host cell appropriately selected and used for expression and / or secretion of a target protein. The host cell can be an immortal hybridoma cell, an N / SO myeloma cell, a 293 cell, a HuT 78 cell, a CHO cell, a HELA cell, a COS cell, etc., and preferably a CHO cell. However, it is not limited thereto, and any host cell known in the art can be used as the host cell of the present application.

[0030] Still another aspect of the present application provides a pharmaceutical composition for preventing or treating cancer, comprising the antibody or the antigen-binding fragment thereof.

[0031] In the pharmaceutical composition according to the present application, unless otherwise specifically stated, the relevant terms are understood to have the same meaning as the above-described terms.

[0032] The term "treatment" used in the present application means all cases of improvement, reversal, cure, etc. of cancer by administering the composition according to the present application.

[0033] The term "prevention" used in the present application means all cases of inhibition, delay, block, etc. of the occurrence or recurrence of cancer by administering the composition according to the present application.

[0034] In the present application, the cancer can exhibit resistance, tolerance, or insensitivity to anticancer drugs. At this time, the term "anticancer drug resistance" or "anticancer drug tolerance" means a case in which there is no effect from the initial stage of treatment when a cancer patient is treated with an anticancer drug, or there is an effect on treatment at the initial stage, but the effect of cancer treatment is reduced or lost during the treatment. Also, the term "anticancer drug insensitivity" means a case in which there is no effect from the initial stage of treatment when a cancer patient is treated with an anticancer drug, or the response to treatment cannot be sustained for a long time.

[0035] The API5 disclosed in the present application exhibits increased expression in cancer cells, and in particular, since a specific API5 antigen epitope represented by the amino acid sequence of SEQ ID NO: 7 contains an amino acid residue that binds to a TLR4 protein, ERK signaling is activated by interaction with the TLR4 protein, thereby exhibiting resistance, tolerance, or insensitivity to anticancer drugs such as a chemical anticancer drug or an immune anticancer drug. Accordingly, the antibody or antigen-binding fragment thereof of the present application that specifically binds to the specific API5 antigen epitope described above can reduce or decrease the activity of the API5 protein, and thus can be very useful for preventing or treating cancer that exhibits resistance, tolerance, or insensitivity to existing anticancer drugs.

[0036] For example, the existing anti-cancer drug can be an anti-cancer drug reported to have developed resistance, tolerance, or insensitivity, can be a chemical anti-cancer drug such as 5-FU, Methotrexate, Gemcitabine, Cytarabine, Paclitaxel, Vinorelbine, Cisplatin, Oxaliplatin, Irinotecan, etc.; a targeted anti-cancer drug such as Bortezomib, Cetuximab, Crizotinib, Dasatinib, Gefitinib, Imatinib, Vemurafenib, etc.; and / or an immune anti-cancer drug such as a CTLA-4 inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, ACT (Adoptive Cell Therapy), etc., but is not limited thereto.

[0037] Also, the cancer can be 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, cutaneous or intraocular melanoma, small intestinal cancer, large intestinal cancer, rectal cancer, anal proximal cancer, colon cancer, uterine cancer, breast cancer, ovarian cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, endocrine cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvis cancer, central nervous system tumor, central nervous system lymphoma, spinal cord tumor, glioblastoma, brain stem neuroglioma, and pituitary adenoma, but is not limited thereto.

[0038] The pharmaceutical composition of the present application includes an effective amount of the antibody or antigen-binding fragment thereof, and can be administered to a subject in need of prevention or treatment of cancer.

[0039] The term "administration" as used herein refers to the physical introduction of a composition into a subject using any of a variety of methods and delivery systems known to those of ordinary skill in the relevant art. The administration can be, for example, oral administration, or intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral administration, for example, by injection or infusion-based administration, etc., but is not limited thereto. The number of administrations can be, for example, single, multiple, or performed over one or more extended periods of time.

[0040] As used in this specification, the term "subject" includes humans or any non-human animal, such as vertebrates like primates, dogs, cattle, horses, pigs, and rodents like mice, rats, and guinea pigs. In this specification, "subject" may be used interchangeably with "individual" and "patient."

[0041] Furthermore, the effective amount can be a "therapeutic effective amount" or a "preventive effective amount." The term "therapeutic effective amount" refers to any amount, when used alone or in combination with other therapeutic agents, that reduces the severity of disease symptoms, increases the frequency and duration of symptom-free periods, or exhibits protection against damage or impairment caused by disease-related pain. The term "preventive effective amount" refers to any amount that inhibits the occurrence or recurrence of cancer in a subject. The level of the effective amount can be determined based on factors including the subject's severity of illness, age, sex, drug activity, drug sensitivity, time of administration, route of administration and excretion rate, treatment duration, factors of concomitant medications, and factors well known in the medical field.

[0042] Furthermore, the dosage of the pharmaceutical composition can vary according to the subject's age, sex, and weight. Specifically, the composition of the present invention can be administered once or more daily at a dose of 0.1 to 100 mg / kg, or every few days or months, depending on the subject's symptoms. The dosage can also be increased or decreased based on the route of administration, the severity of the disease, sex, weight, age, etc.

[0043] Furthermore, the pharmaceutical composition may also contain suitable carriers, excipients, and diluents commonly used in the preparation of pharmaceutical compositions. Carriers, excipients, and diluents that may be included in the composition may include, for example, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil, but are not limited thereto.

[0044] Furthermore, the pharmaceutical composition can be administered in combination with other therapeutic agents. In this case, the pharmaceutical composition of the present invention and other therapeutic agents can be administered simultaneously, sequentially, or individually. The other therapeutic agents may be compounds, proteins, or other drugs with preventative, therapeutic, or cancer-improving effects, but are not limited thereto.

[0045] Furthermore, the pharmaceutical composition can be formulated for simultaneous, sequential, or individual administration with other therapeutic agents. For example, the antibody or its antigen-binding fragment and other therapeutic agents can be administered simultaneously using a single formulation, or simultaneously, sequentially, or individually using separate formulations. For simultaneous, sequential, or individual administration, the antibody or its antigen-binding fragment and other therapeutic agents contained in the pharmaceutical composition of the present invention can be separately formulated in separate containers, or formulated together in the same container. Furthermore, the antibody or its antigen-binding fragment and other therapeutic agents contained in the pharmaceutical composition of the present invention can have the same or different pharmaceutically effective amounts, administration times, administration intervals, routes of administration, treatment periods, etc.

[0046] Another aspect of the present invention provides a method for preventing or treating cancer, comprising the step of administering the antibody or an antigen-binding fragment thereof.

[0047] In the prevention or treatment methods according to the present invention, unless otherwise specified, the relevant terms shall be understood to have the same meaning as those described above.

[0048] Furthermore, in the method for preventing or treating cancer according to the present invention, the antibody or its antigen-binding fragment may be administered to the subject simultaneously, sequentially, or alone with other therapeutic agents.

[0049] The term "simultaneous" administration means administering the antibody or its antigen-binding fragment and other therapeutic agents once using a single formulation, or administering the antibody or its antigen-binding fragment and other therapeutic agents once using separate formulations, wherein the route of administration of the antibody or its antigen-binding fragment and the other therapeutic agents may be different from each other.

[0050] The term "sequential" administration refers to administering antibodies or their antigen-binding fragments and other therapeutic agents in a relatively continuous manner, while allowing as little time as possible to be consumed between administration intervals.

[0051] "Administering alone" means administering the antibody or its antigen-binding fragment and other therapeutic agents at predetermined time intervals. The method of administration of the antibody or its antigen-binding fragment and other therapeutic agents may be appropriately selected by a physician or expert in the field, taking into account the therapeutic efficacy, side effects, etc., of the subject.

[0052] Another aspect of the present invention provides a composition for diagnosing cancer, comprising the antibody or an antigen-binding fragment thereof.

[0053] Another aspect of the present invention provides a kit for diagnosing cancer, comprising the antibody or an antigen-binding fragment thereof.

[0054] In the compositions and kits for diagnosing cancer according to the present invention, unless otherwise specified, the relevant terms shall be understood to have the same meaning as those described above.

[0055] The API5 protein disclosed in this invention is known to be overexpressed in a variety of cancer cell types, including not only general cancers but also cancers exhibiting resistance, tolerance, or insensitivity to anticancer drugs. In particular, because the specific API5 antigenic epitope represented by the amino acid sequence of SEQ ID NO: 7 contains amino acid residues that bind to the TLR4 protein, it participates in the activation of ERK signaling in cancer cells, inhibition of cancer cell killing, and promotion of tumor growth through interaction with the TLR4 protein. Therefore, antibodies or antigen-binding fragments thereof that bind to the aforementioned specific API5 antigenic epitope with high specificity and affinity can be usefully used for cancer diagnosis.

[0056] Specifically, the cancer diagnosis according to the present invention can be performed by reacting the antibody or its antigen-binding fragment with a biological sample to confirm whether or not cancer has occurred or the likelihood of cancer occurring. Preferably, it can be performed by contacting the antibody or its antigen-binding fragment with a biological sample to confirm whether an antigen-antibody complex has formed.

[0057] The term "biological sample" as used in this specification includes, but is not limited to, tissues, cells, blood, serum, plasma, autopsy samples (brain, skin, lymph nodes, spinal cord), etc.

[0058] As used in this specification, the term "antigen-antibody complex" refers to a combination of the API5 protein antigen in a sample and the antibody of the present invention or its antigen-binding fragment that recognizes it.

[0059] The formation of such antigen-antibody complexes can be detected by any method, such as colorimetric, electrochemical, fluorescent, luminescent, particle counting, visual assessment, or scintillation counting. However, it is not limited to these methods, and various applications and uses can be made based on methods known in the art.

[0060] Specifically, the diagnostic compositions or kits of the present invention can be formulated to be suitable for a variety of immunoassays or immunostainings. These immunoassays or immunostainings include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), immunofluorescence, Western blotting, immunohistochemistry staining, flow cytometry, immunocytochemistry, radioenergy Acid (RIA), immunoprecipitation assay, and protein chips.

[0061] Furthermore, labels used for qualitative or quantitative measurement of whether antigen-antibody complexes are formed include, but are not limited to, enzymes, fluorescent substances, ligands, luminescent substances, microparticles, redox molecules, and radioactive isotopes.

[0062] Another aspect of the present invention provides a method for providing information for cancer diagnosis, comprising the steps of: detecting API5 protein or measuring the expression level or activity of API5 protein in a biological sample isolated from a subject suspected of having cancer by performing an antigen-antibody reaction using the antibody or an antigen-binding fragment thereof.

[0063] In the method for providing information for cancer diagnosis according to the present invention, unless otherwise specified, the relevant terms shall be understood to have the same meaning as those described above.

[0064] In this invention, the method for providing information for cancer diagnosis can be performed by reacting the antibody or its antigen-binding fragment with a biological sample to provide information related to the occurrence or likelihood of cancer. Preferably, it can be performed by contacting the antibody or its antigen-binding fragment with a biological sample to confirm the formation of an antigen-antibody complex.

[0065] In a specific embodiment of the present invention, the method for providing information for cancer diagnosis may be a cancer diagnosis method, a method for providing information for screening cancer patients who have resistance, tolerance or indifference to anticancer drugs, and a method for screening cancer patients who have resistance, tolerance or indifference to anticancer drugs.

[0066] Specifically, the method may include the following steps: (a) treating a biological sample isolated from a subject suspected of having cancer with the antibody or its antigen-binding fragment to detect API5 protein by antigen-antibody reaction; and (b) comparing the expression level or activity of API5 protein detected in step (a) with the expression level or activity of a control group, and identifying the subject as a cancer patient when the expression level or activity of API5 protein is higher than that of the control group.

[0067] In this case, the control group can be a biological sample isolated from a healthy person who has never had cancer or a person who has recovered from cancer.

[0068] Another aspect of the present invention provides a method for providing information for cancer treatment, comprising the steps of: detecting API5 protein or measuring the expression level or activity of API5 protein in a biological sample isolated from a cancer patient by performing an antigen-antibody reaction using the antibody or an antigen-binding fragment thereof.

[0069] In the method of providing information for cancer treatment according to the present invention, unless otherwise specified, the relevant terms shall be understood to have the same meaning as those described above.

[0070] In this invention, the method for providing information for cancer treatment can be performed by reacting the antibody or its antigen-binding fragment with a biological sample to provide information related to resistance, tolerance, or insensitivity to anticancer drugs. Preferably, it can be performed by contacting the antibody or its antigen-binding fragment with a biological sample to confirm whether an antigen-antibody complex has formed.

[0071] In a specific embodiment of the invention, the method of providing information for cancer treatment may be a method of providing information for screening cancer patients who are resistant, tolerant, or unresponsive to anticancer drugs, and may be a method of screening cancer patients who are resistant, tolerant, or unresponsive to anticancer drugs.

[0072] In another specific embodiment of the invention, the method may include the following steps: (a) treating a biological sample isolated from a cancer patient with the antibody or its antigen-binding fragment to detect API5 protein by antigen-antibody reaction; and (b) comparing the expression level or activity of the API5 protein detected in step (a) with the expression level or activity of a control group, wherein when the expression level or activity of the API5 protein is higher than that of the control group, the cancer patient is identified as a cancer patient with resistance, tolerance, or insensitivity to anticancer drugs.

[0073] In another specific embodiment of the present invention, the method may include the following steps: (a) treating a biological sample isolated from a cancer patient with the antibody or its antigen-binding fragment to detect API5 protein by antigen-antibody reaction; and (b) comparing the expression level or activity of the API5 protein detected in step (a) with the expression level or activity of a control group, wherein when the expression level or activity of the API5 protein is higher than that of the control group, it is determined that the cancer or tumor is more likely to have resistance, tolerance or insensitivity to anticancer drugs.

[0074] At this time, the control group may be a healthy person who has never had cancer, a recovered person who has recovered from cancer, or a biological sample isolated from a cancer patient who has been identified as having no resistance, tolerance, or incompatibility with anticancer drugs.

[0075] Another aspect of the present invention provides a method for screening anticancer drugs, comprising the following steps: (a) treating cancer cells with an anticancer drug candidate; (b) measuring the expression level or activity of API5 protein in cancer cells treated with the anticancer drug candidate using the antibody or its antigen-binding fragment; and (c) identifying the anticancer drug candidate treated in step (a) as an anticancer drug when the expression level or activity of API5 protein in step (b) is lower than that of API5 protein in cancer cells not treated with the anticancer drug candidate.

[0076] In the method for screening anticancer drugs according to the present invention, unless otherwise specified, the relevant terms shall be understood to have the same meaning as those described above.

[0077] Specifically, step (a) is the step of treating cancer cells with an anticancer drug candidate, which can be performed using methods known in the art. For example, the anticancer drug candidate can be treated by co-culturing the cancer cells with the anticancer drug candidate, or by administering the anticancer drug candidate to an in vivo organism containing cancer cells, but it is not limited thereto, and those skilled in the art can use methods suitable for the purposes of this invention.

[0078] As used in this specification, the term "anticancer drug" refers to a substance that exhibits preventive or therapeutic effects against cancer, specifically a substance capable of killing cancer cells or tumors or inhibiting their growth. In this specification, the term "anticancer drug" may be used interchangeably with "drug," and the term "treatment" may be used interchangeably with "addition" or "administration."

[0079] The term "cancer drug candidate" as used in this specification refers to a substance that is expected to have a preventive or therapeutic effect on cancer, specifically a substance that is expected to kill cancer cells or tumors or inhibit their growth.

[0080] Preferably, the anticancer drug or anticancer drug candidate includes, but is not limited to, compounds, proteins, fusion proteins, compound-protein complexes, drug-protein complexes, antibodies, compound-antibody complexes, drug-antibody complexes, amino acids, peptides, viruses, carbohydrates, lipids, nucleic acids, extracts, fragments, etc. For example, the anticancer drug or anticancer drug candidate may include compounds, peptides, peptide mimics, fusion proteins, antibodies, aptamers, antibody-drug complexes (ADCs), etc., but is not limited to these. Preferably, the anticancer drug or anticancer drug candidate may be an antibody or an immunotherapeutic anticancer drug.

[0081] Step (b) is a step of measuring the expression level or activity of the API5 protein, which can be performed using any method known to those skilled in the art. Specific examples include Western blot, co-immunoprecipitation assay, enzyme-linked immunosorbent assay (ELISA), immunostaining, and flow cytometry, but the method is not limited thereto. Those skilled in the art can use methods suitable for the purposes of this invention.

[0082] Step (c) is a step of determining whether the candidate anticancer drug can be used as an anticancer drug. API5 protein is overexpressed in cancer cells. In particular, since the specific API5 antigenic epitope represented by the amino acid sequence of SEQ ID NO: 7 contains amino acid residues that bind to the TLR4 protein, it promotes the proliferation, migration, invasion, and growth of cancer cells through interaction with the TLR4 protein. Therefore, an anticancer drug candidate that reduces the expression level or activity of API5 protein can be used as an anticancer drug. Furthermore, API5 protein is overexpressed in cancer cells that are resistant, tolerant, or unresponsive to anticancer drugs. In particular, since the specific API5 antigenic epitope represented by the amino acid sequence of SEQ ID NO: 7 contains amino acid residues that bind to the TLR4 protein, it promotes the proliferation, migration, invasion, and growth of cancer cells that are resistant, tolerant, or unresponsive to anticancer drugs through interaction with the TLR4 protein. Therefore, an anticancer drug candidate that reduces the expression level or activity of API5 protein can be used as an anticancer drug for the prevention or treatment of cancers that are resistant, tolerant, or unresponsive to anticancer drugs.

[0083] Technical effect The API5 antigenic epitope of the present invention, together with other regions or antigenic epitopes present in the API5 protein, more strongly induces drug resistance, tolerance, or refractory behavior in cancer cells. Therefore, antibodies binding to the API5 antigenic epitope or their antigen-binding fragments exhibit excellent effects in killing cancer cells and inhibiting tumor proliferation or growth. In particular, it demonstrates excellent anticancer effects against cancers that exhibit resistance, tolerance, or refractory behavior to existing anticancer drugs. Attached Figure Description

[0084] Figure 1 This is an image showing the API5 antigenic epitope.

[0085] Figure 2 This is a graph showing the binding energies of peptides containing different regions of the API5 protein to five anti-API5 antibodies (anti-API5 antibodies 1 through 5). API5 protein refers to the full-length API5 protein, and the untreated group indicates the untreated protein or peptide. Furthermore, API5 1-50 represents a peptide composed of amino acids 1 to 50 of the API5 protein; API5 51-100 represents a peptide composed of amino acids 51 to 100; API5 101-150 represents a peptide composed of amino acids 101 to 150; API5 151-200 represents a peptide composed of amino acids 151 to 200; API5 201-220 represents a peptide composed of amino acids 151 to 200; API5 206-225 represents a peptide composed of amino acids 206 to 225; API5 251-300 represents a peptide composed of amino acids 251 to 300; API5 301-350 represents a peptide composed of amino acids 301 to 350; API5 340-359 represents a peptide composed of amino acids 340 to 359; API5 401-430 represents a peptide composed of amino acids from position 401 to 430, API5 431-470 represents a peptide composed of amino acids from position 431 to 470, and API5 471-504 represents a peptide composed of amino acids from position 471 to 504.

[0086] Figure 3 This demonstrates the effects of five anti-API5 antibodies (anti-API5 antibodies 1 to 5) and comparative anti-API5 antibodies (Novus, D-1, and Mybio) on TC-1 P3 according to the present invention. PD-1 MC38 P3 PD-1 CT26 P3 PD-1 The graph shows the effect of reducing ERK phosphorylation, which is the result of Western blot analysis targeting ERK phosphorylation (pERK) and ERK.

[0087] Figure 4 This illustrates the effect of anti-API5 antibody No. 1 according to the present invention on TC-1 P3. PD-1 MC38 P3 PD-1 CT26 P3 PD-1 The graph shows the effect of reducing ERK phosphorylation, which is the result of Western blot analysis targeting ERK phosphorylation (pERK) and ERK.

[0088] Figure 5 This demonstrates the efficacy of anti-API5 antibodies No. 3, 4, and 5 according to the present invention, and comparative anti-API5 antibodies (Novus, D-1, and Mybio) against TC-1 P3. PD-1 CT26 P3 PD-1 The curve showing the effect of increasing apoptosis.

[0089] Figure 6 This is a graph showing the inhibitory effect of the No. 1 anti-API5 antibody according to the present invention on tumor growth in an in vivo model of colorectal cancer or colorectal cancer unresponsive to immunotherapy. * indicates p-value < 0.05, **** indicates p-value < 0.0001.

[0090] Figure 7 This is a graph showing the inhibitory effect of the anti-API5 antibody No. 1 according to the present invention on tumor growth in in vivo models of non-small cell lung cancer or uterine cancer. *** indicates p-value < 0.001.

[0091] Figure 8 This is a graph showing the inhibitory effect of the No. 1 anti-API5 antibody according to the present invention on tumor growth in an in vivo model of colorectal cancer or colorectal cancer resistant to immunotherapy. * indicates p-value < 0.05, **** indicates p-value < 0.0001.

[0092] Figure 9 This is a graph showing the tumor growth inhibition effect of the anti-API5 antibody, anti-PD-1 antibody, or a combination of the anti-API5 antibody and anti-PD-1 antibody according to the present invention on an in vivo model of colorectal cancer resistant to immunotherapy. **** indicates a p-value < 0.0001.

[0093] Figure 10 This is a graph showing the inhibitory effects of anti-API5 antibodies No. 1 to No. 4 according to the present invention and the comparative anti-API5 antibodies (D-1 and Mybio) on tumor growth in an in vivo model of colorectal cancer resistant to immunotherapy. *** indicates p-value < 0.001, **** indicates p-value < 0.0001.

[0094] Figure 11This is a graph showing the tumor growth inhibition rate of anti-API5 antibodies No. 1 to No. 4 according to the present invention and the comparative group of anti-API5 antibodies (Novus, D-1 and Mybio) in an in vivo model of colorectal cancer that is resistant to immunotherapy. Detailed Implementation

[0095] The present invention will now be described in more detail through embodiments. However, these embodiments are for illustrative purposes only, and the scope of the invention is not limited to these embodiments.

[0096] Example 1. Identification of specific API5 antigen epitopes The inventors have, for the first time, identified a specific API5 antigenic epitope corresponding to amino acids 206 to 225 of the API5 protein that plays a major role in inducing resistance, tolerance, or insensitivity to anticancer drugs in cancer. Furthermore, they have, for the first time, identified that this specific API5 antigenic epitope contains amino acid residues involved in binding to the TLR4 protein, which acts as a receptor for the API5 protein.

[0097] To validate this, modeling analysis was performed on the amino acid residues in the API5 protein that are involved in binding to the TLR4 protein, which acts as its receptor. Figure 1 The API5 protein binds to the TLR4 protein via its amino acid residues from position 206 to 225. The specific binding residue pairs of the API5 and TLR4 proteins are shown in Table 1 below.

[0098] Table 1

[0099] Example 2. Anti- API5 antibodies binding to specific API5 antigen epitopes Example 2-1. Preparation of anti- API5 antibodies The inventors prepared antibodies that bind to the API5 protein. Specifically, antibodies that specifically bind to amino acid residues in the API5 protein that serve as antigenic epitopes were fabricated. To discover antibodies that bind to API5, a human synthetic Fab phage library was used as the fixed antigen. API5 protein purified after transient expression of the API5 gene expression vector in Expi293 cells (Thermo Fisher Scientific) was used, and antibodies exhibiting binding energy to API5 were screened using phage display technology. As a result, Fab antibody phage clones were screened, and anti-API5 antibodies 1 through 5 were prepared by transplanting them into the IgG4 (S228P) backbone. The first anti-API5 antibody comprises the heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 1 and the light chain variable region represented by the amino acid sequence of SEQ ID NO: 8; the second anti-API5 antibody comprises the heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 2 and the light chain variable region represented by the amino acid sequence of SEQ ID NO: 9; the third anti-API5 antibody comprises the heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 3 and the light chain variable region represented by the amino acid sequence of SEQ ID NO: 10; the fourth anti-API5 antibody comprises the heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 4 and the light chain variable region represented by the amino acid sequence of SEQ ID NO: 11; and the fifth anti-API5 antibody comprises the heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 5 and the light chain variable region represented by the amino acid sequence of SEQ ID NO: 12.

[0100] The comparative antibodies mentioned in the experiments described later were commercially available antibodies. Specifically, the comparative antibodies used were NOVUS antibody from NOVUS Corporation, which binds to the region (1-50) of the API5 protein composed of amino acids 1 to 50; D-1 antibody from Santa Cruz Biotechnology Corporation, which binds to the region (72-84) of the API5 protein composed of amino acids 72 to 84; and Mybio antibody from MyBioSource Corporation, which binds to the region (487-504) of the API5 protein composed of amino acids 487 to 504.

[0101] Example 2-2. Analysis of binding activity of anti- API5 antibodies to specific API5 antigen epitopes It was confirmed whether the anti-API5 antibody prepared in Example 2-1 binds to a specific API5 antigenic epitope of a novel target identified as an anticancer drug in Example 1.

[0102] Specifically, the API5 protein (full length), composed of amino acids in 504-mers, was divided into regions to consist of approximately 20 to 50 amino acids in mers, and peptide fragments including the corresponding regions were prepared. The binding levels of the anti-API5 antibodies prepared in Example 2-1 to each API5 region were then confirmed.

[0103] In Table 2 below, the complete amino acid sequence of the API5 protein, which is composed of amino acids from the 504-mer, is represented by SEQ ID NO: 6.

[0104] Table 2

[0105] Specifically, regions consisting of amino acids 1 to 50 (API51-50), 51 to 100 (API551-100), 101 to 150 (API55101-150), 151 to 200 (API55151-200), 201 to 220 (API55201-220), 206 to 225 (API55206-225), 251 to 300 (API55251-300), 301 to 350 (API55301-350), and 340 to 359 (API5555) were prepared. Peptides consisting of amino acids 340-359, 401-430 (API5401-430), 431-470 (API5431-470), and 471-504 (API5471-504).

[0106] Then, an ELISA was performed to detect the binding between the peptides and the anti-API5 antibody. A 96-well ELISA microtiter plate was coated overnight at 4°C with streptavidin dissolved in PBS at a concentration of 5 μg / ml. The plate was washed twice and blocked with 3% MPBS at 37°C for 1 hour. Then, 100 μl of each peptide dissolved in PBS at a concentration of 10 μg / ml was added to the plate and incubated at 37°C for 1 hour. After washing three times, 100 μl of anti-API5 antibody was added and incubated at room temperature for 2 hours. After washing, HRP-conjugated anti-mouse IgG (H&L) was added to the plate and incubated at 37°C for 30 minutes. The plate was washed six more times, and substrate solution was added to the plate and reacted for 1 minute. The absorbance was measured at 450 nm using a spectrophotometer.

[0107] The result, such as Figure 2 As shown, all anti-API5 antibodies 1 through 5 exhibited excellent binding to peptides containing API5 206-225 in API5 peptides, including each region.

[0108] Furthermore, surface plasmon resonance (SPR) analysis confirmed the binding affinity between a specific API5 antigenic epitope, identified as a novel target for anticancer drugs in Example 1, and the anti-API5 antibody prepared in Example 2-1.

[0109] Specifically, the binding affinity of purified anti-API5 antibodies to the specific API5 epitope was analyzed individually using a Biacore T200 sensor. API5 was immobilized on the sensor chip, and the anti-API5 antibody was used as the analyte. The binding rate constant (ka) and dissociation rate constant (kd) were calculated using Biacore evaluation software. The binding rate constant (ka) represents the rate of complex formation, i.e., the number of antigen-antibody complexes formed per second in 1 mole of the specific API5 epitope and anti-API5 antibody solution; ka is expressed in units of 1 / Ms. The dissociation rate constant (kd) represents the stability of the complex, i.e., the ratio of complexes dissociating per second; kd is expressed in units of 1 / s. For example, kd = 0.01 1 / s means that 1% of the complex disintegrates within 1 second. The equilibrium dissociation constant (KD) calculated based on the ratio of kd to ka is 1 × 10⁻⁶. -9 When M is below a certain value, it indicates that the corresponding antibody has excellent binding affinity to the antigen protein.

[0110] Table 3

[0111] The results, as shown in Table 3 above, confirmed that all anti-API5 antibodies 1 through 5 exhibited excellent binding affinity to specific API5 antigenic epitopes.

[0112] Example 3. Cancer treatment effect of anti- API5 antibodies binding to specific API5 antigen epitopes Example 3-1. Reduction of ERK phosphorylation (pERK) To confirm the anticancer effect of the specific API5 antigenic epitope identified in Example 1, the reduction effect of ERK activation (i.e., ERK phosphorylation) in cancer cells based on anti-API5 antibodies binding to the antigenic epitope was analyzed. ERK is mainly activated by growth factors involved in cell proliferation, differentiation, and survival. ERK activation is achieved through ERK protein phosphorylation, and such sustained ERK activation plays an important role in tumorigenesis. ERK activation is known to be increased in various types of cancer, including pancreatic cancer, colorectal cancer, lung cancer, ovarian cancer, kidney cancer, and breast cancer.

[0113] Specifically, anti-PD-1 antibody therapy will be used to treat non-small cell lung cancer or uterine cancer with TC-1 P3. PD-1 Cancer cell line, colorectal cancer MC38 P3 PD-1 Cancer cell lines and colorectal cancer CT26 P3 PD-1 Cancer cell lines were cultured at 1.5 × 10⁻⁶ cells per well. 5 Cells were seeded in 12-well plates using RPMI medium containing 5% FBS. Two hours later, the medium was replaced with RPMI medium containing 0.1% FBS, and each anti-API5 antibody was treated with a maximum concentration of 150 ng / ml.

[0114] After 24 hours, cells were washed with 1X PBS buffer and incubated in 150 μl of RIPA buffer at 4°C for 10 minutes to induce cell lysis. Cells were then centrifuged at 13,000 rpm at 4°C for 15 minutes to obtain the supernatant. Protein levels were quantified using the Bradford assay, and ERK protein levels and phosphorylation levels were measured for the same amount of protein using SDS-PAGE. The measured ERK protein levels and phosphorylation levels were numerically converted using Image J (densitometer), and the ratio of phosphorylated ERK protein to total ERK protein was calculated.

[0115] At this point, the group not treated with anti-API5 antibody was set as the control group (Con). The antibodies used as the comparison group were NOVUS antibody, which binds to the region (1-50) of the API5 protein composed of amino acids 1 to 50; D-1 antibody, which binds to the region (72-84) of the API5 protein composed of amino acids 72 to 84; and MyBioSource antibody, which binds to the region (487-504) of the API5 protein composed of amino acids 487 to 504.

[0116] Additionally, the "TC-1 P3" PD-1 “MC38 P3” PD-1 "and CT26 P3" PD-1 "" refers to cells obtained by passaged three times through anti-PD-1 antibody therapy on parental cancer cells that are unresponsive ("TC-1 P0", "MC38 P0", or "CT26 P0").

[0117] The result, such as Figure 3 As shown, in the presence of anti-API5 antibodies binding to the specific API5 antigenic epitope represented by the amino acid sequence of SEQ ID NO: 7, ERK phosphorylation was significantly reduced in all cancer cell lines, with a reduction of up to half the rate of ERK phosphorylation relative to total ERK compared to the control group. This result was consistent with all anti-API5 antibodies 1 through 5. Conversely, in the presence of comparative group anti-API5 antibodies (Novus, D-1, and Mybio) that did not bind to the specific API5 antigenic epitope represented by the amino acid sequence of SEQ ID NO: 7, ERK phosphorylation was significantly increased in all cancer cell lines, with a maximum increase of two-fold in the rate of ERK phosphorylation relative to total ERK compared to the control group.

[0118] And, as Figure 4 As shown, ERK phosphorylation was significantly reduced in all cancer cell lines when treated with an anti-API5 antibody that binds to the specific API5 antigenic epitope represented by the amino acid sequence of SEQ ID NO: 7. Conversely, no reduction in ERK phosphorylation was observed when both the anti-API5 antibody and a peptide having the specific API5 antigenic epitope sequence represented by the amino acid sequence of SEQ ID NO: 7 were treated simultaneously, preventing the anti-API5 antibody from targeting API5.

[0119] The results indicate that the specific API5 antigenic epitope represented by the amino acid sequence of SEQ ID NO: 7 according to the present invention, as it contains amino acid residues that bind to the TLR4 protein, which is the receptor for the API5 protein, participates in ERK activation in cancer cells through interaction with the TLR4 protein. Therefore, anti-API5 antibodies that bind to the specific API5 antigenic epitope can reduce ERK activation in cancer cells and thereby exhibit excellent anti-cancer effects.

[0120] Example 3-2. Increase of apoptosis To confirm the anticancer effect of the specific API5 antigenic epitope identified in Example 1, the apoptosis-increasing effect of anti-API5 antibodies binding to the antigenic epitope was analyzed.

[0121] According to Example 3-1, non-small cell lung cancer or uterine cancer TC-1 P3 PD-1 Cancer cell lines and colorectal cancer CT26 P3 PD-1 Cancer cell lines were planted at 3 × 10⁻⁶ cells per well. 5 Cells were seeded in 12-well plates using RPMI medium containing 5% FBS. After 2 hours, the medium was replaced with RPMI medium containing 0.1% FBS, and each cell was treated with anti-API5 antibody at a maximum concentration of 150 ng / ml. After 24 hours, the percentage of cells killed by apoptosis was confirmed using FACS.

[0122] At this time, the comparative group antibodies used were NOVUS antibody, which binds to the region (1-50) of the API5 protein composed of amino acids 1 to 50; D-1 antibody, which binds to the region (72-84) of the API5 protein composed of amino acids 72 to 84; and MyBioSource antibody, which binds to the region (487-504) of the API5 protein composed of amino acids 487 to 504.

[0123] The result, such as Figure 5 As shown, compared to the untreated group, the percentage of cells killed by the anti-API5 antibody, which binds to the specific API5 antigenic epitope represented by the amino acid sequence of SEQ ID NO: 7, increased by approximately 10% to 20%, and this effect was observed in all cancer cell lines. On the other hand, in the case of the comparative group of anti-API5 antibodies (Novus, D-1, and Mybio) that did not bind to the specific API5 antigenic epitope represented by the amino acid sequence of SEQ ID NO: 7, the percentage of cells killed in all cancer cell lines decreased by approximately 10% to 60%.

[0124] The results indicate that the specific API5 antigenic epitope represented by the amino acid sequence of SEQ ID NO: 7 according to the present invention, as it contains amino acid residues that bind to the TLR4 protein, which is the receptor for the API5 protein, participates in the apoptosis of cancer cells through interaction with the TLR4 protein. Therefore, anti-API5 antibodies that bind to the specific API5 antigenic epitope can increase the sensitivity of cancer cells to apoptosis and thereby exhibit excellent anti-cancer effects.

[0125] Example 3-3. Inhibition of tumor growth in vivo To confirm the anticancer effect of the specific API5 antigenic epitope identified in Example 1, the in vivo growth inhibition effect of anti-API5 antibodies binding to the specific API5 antigenic epitope represented by the amino acid sequence of SEQ ID NO: 7 was analyzed.

[0126] At this time, the MC-38 P0 colorectal cancer cell line and anti-PD-1 antibody were used to treat refractory MC38 P3 colorectal cancer. PD-1 Cancer cell line, MC38 P3 colorectal cancer that is unresponsive to anti-PD-L1 antibody therapy PD-L1 Cancer cell lines, TC-1 P0 cancer cell lines for non-small cell lung cancer or uterine cancer, CT26 P0 cancer cell lines for colorectal cancer, and CT26 P3 colorectal cancer cells that are unresponsive to anti-PD-1 antibody therapy. PD-1 Cancer cell line.

[0127] Specifically, each cell was divided into 5 × 10 5 Cells were transplanted into Balb / c mice and formed tumors. The tumors reached an average size of approximately 60 to 75 mm. 3 (However, in the case of MC-38, it is 30 to 40 mm) 3 Eight to nine mice were assigned to each experimental group to ensure that tumor size was distributed as evenly as possible across groups. Then, anti-API5 antibodies 1 through 4, which bind to the epitope represented by the amino acid sequence of SEQ ID NO: 7, or 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, were administered intraperitoneally three times a week for a total of five times, starting from Day 1. Tumor size was then measured every two or three days using calipers, starting from Day 1, and calculated using the following formula: Tumor size = {(major axis length × minor axis length)} 2} / 2. Furthermore, the tumor growth inhibition rate (TGI, %) was calculated based on the tumor size using the following formula: 100 - (100 × experimental group tumor size / IgG antibody-treated control group tumor size).

[0128] The results were analyzed using parametric multiple comparison procedures, assuming the data followed a normal distribution. Post-hoc tests were performed using Sidak's multiple comparison test, provided the parametric two-way ANOVA results were significant. 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 considered statistically significant.

[0129] The result, such as Figure 6 to Figure 8 As shown, the anti-API5 antibody binding to the specific API5 antigenic epitope represented by the amino acid sequence of SEQ ID NO: 7 significantly inhibits tumor growth, exhibiting an approximately 1.5- to 4-fold increase in tumor growth inhibition compared to the control group IgG antibody. Furthermore, this effect is not only observed in cancer cells resistant to existing immunotherapeutic drugs, but also in a variety of cancers arising from different cancer cells, such as colorectal cancer, non-small cell lung cancer, or uterine cancer.

[0130] In particular, such as Figure 9 As shown, when the anti-API5 antibody is administered in combination with the anti-PD-1 antibody, it also exhibits excellent growth inhibition effects on cancer cells that are resistant, tolerable, or unresponsive to anti-PD-1 antibody treatment, and this effect is synergistic.

[0131] Furthermore, such as Figure 10 and Figure 11 As shown, the tumor growth inhibition rates (TGI) of anti-API5 antibodies 1 to 4 were 30.1%, 49.6%, 59.2%, and 55.2%, respectively, significantly inhibiting tumor growth by at least 30% compared to the control group IgG antibody. On the other hand, the comparative group anti-API5 antibodies (D-1 and Mybio) that did not bind to the specific API5 antigenic epitope represented by the amino acid sequence of SEQ ID NO: 7 failed to show any tumor growth inhibition effect.

[0132] The results indicate that the specific API5 antigenic epitope represented by the amino acid sequence of SEQ ID NO: 7 according to the present invention, as it contains amino acid residues that bind to the TLR4 protein, which is the receptor for the API5 protein, participates in the promotion of cancer cell growth through interaction with the TLR4 protein. Furthermore, since the amino acid sequence represented by the amino acid sequence of SEQ ID NO: 7 is the antigenic epitope sequence necessary for its targeting antibody to bind to the API5 protein, the anti-API5 antibody bound to the specific API5 antigenic epitope can inhibit the growth of cancer cells. It not only exhibits excellent anti-cancer effects against cancers resistant to immunotherapy, but also shows excellent synergistic effects with existing drugs used as immunotherapy drugs.

[0133] sequence list The sequence listing is found in the attached electronic file. Example 1. Identification of specific API5 antigen epitopes Example 2. Anti- API5 antibodies binding to specific API5 antigen epitopes Example 2-1. Preparation of anti- API5 antibodies Example 2-2. Analysis of binding activity of anti- API5 antibodies to specific API5 antigen epitopes Example 3. Cancer treatment effect of anti- API5 antibodies binding to specific API5 antigen epitopes Example 3-1. Reduction of ERK phosphorylation (pERK) Example 3-2. Increase of apoptosis Example 3-3. Inhibition of tumor growth in vivo Example 1. Identification of specific API5 antigen epitopes Example 2. Anti- API5 antibodies binding to specific API5 antigen epitopes Example 2-1. Preparation of anti- API5 antibodies Example 2-2. Analysis of binding activity of anti- API5 antibodies to specific API5 antigen epitopes Example 3. Cancer treatment effect of anti- API5 antibodies binding to specific API5 antigen epitopes Example 3-1. Reduction of ERK phosphorylation (pERK) Example 3-2. Increase of apoptosis Example 3-3. Inhibition of tumor growth in vivo Example 1. Identification of specific API5 antigen epitopes Example 2. Anti- API5 antibodies binding to specific API5 antigen epitopes Example 2-1. Preparation of anti- API5 antibodies Example 2-

Claims

1. An antibody or an antigen-binding fragment thereof that specifically binds to an antigenic epitope of apoptosis inhibitor factor 5 (API5), represented by the amino acid sequence of SEQ ID NO:

7.

2. A nucleic acid encoding the antibody or an antigen-binding fragment thereof according to claim 1.

3. A vector comprising the nucleic acid according to claim 2.

4. A cell transformed using the vector according to claim 3.

5. A pharmaceutical composition for the prevention or treatment of cancer, comprising the antibody or antigen-binding fragment thereof according to claim 1.

6. The pharmaceutical composition according to claim 5, wherein, The cancer has resistance, tolerance, or insensitivity to anticancer drugs.

7. The pharmaceutical composition according to claim 6, wherein, The anticancer drug is selected from one or more of the group consisting of chemical anticancer drugs, targeted anticancer drugs, and immunotherapy anticancer drugs.

8. The pharmaceutical composition according to claim 5, wherein, The cancers selected are those comprising 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, melanoma of the skin or eye, small intestine cancer, large intestine cancer, rectal cancer, proximal anal cancer, colon cancer, uterine cancer, breast cancer, ovarian cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, endocrine cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvis cancer, central nervous system tumors, central nervous system lymphoma, spinal cord tumors, 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 the following steps: The API5 protein can be detected or its expression level or activity can be measured from biological samples isolated from subjects suspected of having cancer by using an antigen-antibody reaction with the antibody or its antigen-binding fragment as described in claim 1.

12. A method for providing information for cancer treatment, comprising the following steps: The API5 protein can be detected or its expression level or activity can be measured from biological samples isolated from cancer patients by using the antibody or its antigen-binding fragment as described in claim 1 for an antigen-antibody reaction.

13. A method for screening anticancer drugs, comprising the following steps: (a) Treating cancer cells with anticancer drug candidates; (b) Measuring the expression level or activity of 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) When the expression level or activity of API5 protein in step (b) is lower than that of API5 protein in cancer cells that have not been treated with the anticancer drug candidate, the anticancer drug candidate treated in step (a) is identified as an anticancer drug.

Images