API5 epitope and antibody that specifically binds to it

Abstract

This invention relates to API5 epitopes and antibodies or antigen-binding fragments that specifically bind to them. The API5 epitopes according to this invention induce stronger resistance, resistance, or refractory properties in cancer compared to other regions or epitopes of the API5 protein. Therefore, antibodies or antigen-binding fragments that bind to specific API5 epitopes are highly effective in killing cancer cells and inhibiting tumor proliferation or growth. In particular, their anticancer effects against cancers that exhibit resistance, resistance, or refractory properties to existing anticancer drugs are exceptionally strong.

Description

[Technical Field]

[0001] This invention relates to an API5 epitope and an antibody or antigen-binding fragment that specifically binds thereto. [Background technology]

[0002] Despite decades of diligent research into cancer, it remains the leading cause of death worldwide. While several anti-cancer treatments have been developed, they are not effective for all cancer types and for all patients. The methods currently used to treat cancer are relatively non-selective: lesions are surgically removed, solid tumors are reduced in size through radiation therapy, or cancer cells are rapidly killed using chemotherapy. However, chemotherapy can lead to drug resistance and may limit the doses that can be administered. Chemotherapy can also cause severe side effects, which may preclude the use of potentially effective drugs. Accordingly, there is a need to develop more target-specific and effective cancer therapies.

[0003] In this context, anti-cancer molecular targeted agents that specifically target only cancer cells, or anti-cancer immunotherapies that utilize the patient's immune system, are under development. For example, there are approximately 30 types of anti-cancer molecular targeted agents approved in South Korea, and anti-cancer immunotherapies approved in South Korea include Opdivo (active ingredient: nivolumab), Keytruda (active ingredient: pembrolizumab), Tecentriq (active ingredient: atezolizumab), Imfinzi (active ingredient: durvalumab), and Yervoy (active ingredient: ipilimumab), among others. These are used for various types of cancer, including non-small cell lung cancer, melanoma, renal cell carcinoma, Hodgkin lymphoma, head and neck cancer, and bladder cancer.

[0004] However, as the use of these anti-cancer molecular targeted agents and anti-cancer immunotherapies increases, so does the proportion of patients who already exhibit resistance, tolerance, or refractory to them. Therefore, there is a need in this field to develop therapeutic agents for cancers that exhibit resistance, tolerance, or refractory to existing anti-cancer drugs by discovering and utilizing novel targets. [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] The inventors have been working to identify novel targets for anticancer drugs and develop therapeutic agents for cancers that exhibit resistance, resistance, or refractory properties to existing anticancer drugs by using these targets. As a result, they have identified the API5 epitope represented by the amino acid sequence of SEQ ID NO: 7, and experimentally demonstrated that an antibody or antigen-binding fragment targeting this epitope can show excellent efficacy against cancers that exhibit resistance, resistance, or refractory properties to existing anticancer drugs, thereby completing the present invention. [Means for solving the problem]

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

[0007] As used herein, the term “antibody” refers to an immunoglobulin molecule that has immunological reactivity with a particular antigen, or a protein molecule that acts as a receptor that specifically recognizes an antigen. In this invention, “antibody” includes monoclonal antibodies, polyclonal antibodies, whole antibodies (antibodies consisting of at least two heavy chains and two light chains linked by disulfide bonds), antibody fragments, Fab, Fab', F(ab')2, Fv, single-chain Fv(scFv), diabodies, linear antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, or antigen-binding moieties of antibodies. Whole antibodies include IgA, IgD, IgE, IgM, and IgG. In addition, IgG may include IgG1, IgG2, IgG3, and IgG4 as subtypes.

[0008] As used herein, the term “antigen-binding fragment” refers to an antibody fragment or antibody analog that maintains at least some of the binding specificity of the parent antibody and includes the variable region or part thereof (e.g., one or more CDRs) of the antigen-binding region of the parent antibody. Antigen-binding fragments are, for example, Fab, Fab', F(ab')2, Fv fragments, scFv, unibody, diabody, linear antibody, nanobody, domain antibody, or multispecific antibody formed from antibody fragments.

[0009] In addition, antibodies or their antigen-binding fragments can be combined with drugs to form antibody-drug conjugates (ADCs).

[0010] As used herein, the term “heavy chain” refers to a complete heavy chain, including its variable and constant regions, as well as fragments thereof. Heavy chains include gamma (γ), muon (μ), alpha (α), delta (δ), and epsilon (ε) types.

[0011] As used herein, the term “light chain” refers to a complete light chain, including its variable and constant regions, as well as its fragments. Light chains include kappa (κ) type and lambda (λ) type.

[0012] As used herein, the term "CDR (Complementarity-Determining Region)" refers to the amino acid sequence of the hypervariable region that forms the antigen-binding site as part of the variable region of antibodies produced by B cells and T cells. The amino acid sequence of the heavy chain includes three discontinuously arranged CDRs: heavy chain CDRH1, CDRH2, and CDRH3, and the amino acid sequence of the light chain includes three discontinuously arranged CDRs: light chain CDRL1, CDRL2, and CDRL3. CDRs are regions involved in antigen recognition and play a crucial role in the diversity of antigen specificity by supplying key contact residues for antibody binding to antigens or epitopes.

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

[0014] As used herein, the term "API5 (apoptosis inhibitor 5)" refers to the protein encoded by the API5 gene, the amino acid sequence of which is known from existing databases (NCBI reference sequences: XP_016873953.1, XP_016873954.1, XP_006718422.1, etc.). The API5 protein is expressed at high levels in tumors such as solid tumors and metastatic cancers, and its expression is particularly elevated in cancer cells that are resistant, resilient, 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] As used herein, the term “epitope” refers to an antigenic determinant, meaning a specific portion of an antigen that enables an antibody or immune cell, such as B cells and T cells, to recognize the 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 epitope of API5 may be represented by the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 7. Specifically, the amino acid sequence of SEQ ID NO: 7 may be a part of the amino acid sequence of 504 residues (mer) that constitute the API5 protein. More specifically, the epitope of API5 represented by the amino acid sequence of SEQ ID NO: 7 may be the sequence of amino acids 206 to 225 of the API5 protein represented by the amino acid sequence of SEQ ID NO: 6.

[0017] As used herein, the term "anti-API5 antibody" refers to an antibody or its antigen-binding fragment that specifically binds to API5 and neutralizes or inhibits the activity of the API5 protein. In addition, an anti-API5 antibody refers to an antibody or its antigen-binding fragment 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 can bind to the 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 the human API5 protein and is also reactive to API5 proteins derived from mice or rats.

[0019] As used herein, the term “human antibody” refers to an antibody whose framework region and CDR region have variable regions derived from a human immunoglobulin sequence. In the present invention, a human antibody may contain amino acid residues not encoded by a human immunoglobulin sequence (for example, mutations introduced by random or site-directed mutagenesis in vitro or somatic mutation in vivo).

[0020] The sequences disclosed in the present invention include sequences that exhibit substantial identity to the sequences described in the Sequence Listing. Substantial identity means that two sequences are aligned to correspond as much as possible and analyzed using algorithms commonly used in the art, resulting in a homology between the sequences of 80%, 90%, 95% or more.

[0021] In addition, the antibody or antigen-binding fragment thereof according to the present invention can include the sequence of the antibody or antigen-binding fragment thereof described herein, a sequence that exhibits substantial identity to the above sequence, and biological equivalents thereof as long as they can specifically recognize and bind to the epitope of API5. For example, the above antibody or antigen-binding fragment thereof may include additional mutations in the sequence and additional mutations within the range that does not change the overall activity of the molecule in order to improve the binding affinity and / or biological properties of the antibody.

[0022]

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

[0024] Unless otherwise specified, in the nucleic acids according to the present invention, related terms are understood to have the same meaning as the above terms.

[0025] As used herein, the term "nucleic acid" comprehensively includes DNA (gDNA and cDNA) as well as RNA. Nucleotides that make up the basic structural units in nucleic acid molecules include natural nucleotides and analog nucleotides having 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 its antigen-binding fragment according to the present invention includes a nucleotide sequence that exhibits substantial identity with the above-mentioned nucleotide sequence. Substantial identity means that, as a result of aligning the two sequences to correspond as closely as possible and analyzing them using algorithms commonly used in the art, a sequence homology of 80%, 90%, 95%, or more is shown.

[0027]

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

[0029] In the vectors and cells transformed with the vectors according to the present invention, unless otherwise specified, the relevant terms are understood to have the same meaning as the terms described above.

[0030] As used herein, the term “vector” refers to any device that can be inserted into a host cell and is capable of gene replication. Vectors include plasmids, linear nucleic acids, cosmids, RNA vectors, viral vectors, etc., and viral vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, etc. The recombinant vector systems of the present invention can be constructed through various methods known in the art. In addition, the vectors of the present invention can be constructed as vectors for cloning or expression, and can be constructed using prokaryotic or eukaryotic cells as hosts.

[0031] Furthermore, the cells may be prokaryotic, eukaryotic, or animal cells. Appropriately selected host cells can be transformed with a vector and used to express and / or secrete the 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, etc., and preferably CHO cells. However, the host cells are not limited to these, and any host cell known in the art can 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 an antibody or an antigen-binding fragment thereof.

[0034] In the pharmaceutical compositions according to the present invention, unless otherwise specified, the relevant terms are understood to have the same meaning as the terms described above.

[0035] As used herein, the term “treatment” refers to a condition in which cancer is improved, restored, or completely cured by the administration of a composition according to the present invention.

[0036] As used herein, the term “prevention” refers to the suppression, delay, or prevention of the development or recurrence of cancer by administration of a composition according to the present invention.

[0037] In this invention, cancer may exhibit resistance, refractory, or intractability to anticancer drugs. As used herein, the terms "resistance to anticancer drugs" or "intractability to anticancer drugs" mean that, when treating a cancer patient with anticancer drugs, there is no effect from the start of treatment, or the treatment is effective at the start but is reduced or lost during the course of continuous treatment. In addition, as used herein, the term "intractability to anticancer drugs" means that, when treating a cancer patient with anticancer drugs, there is no effect from the start of treatment, or the response to treatment does not last for a long period of time.

[0038] The API5 disclosed in this invention exhibits increased expression in cancer cells. In particular, the specific API5 epitope represented by the amino acid sequence of SEQ ID NO: 7 contains amino acid residues that bind to the TLR4 protein. Thereafter, API5 induces activation of ERK signaling through interaction with the TLR4 protein, thereby conferring resistance, resilience, or refractory status to anticancer drugs such as chemotherapeutic agents or immunotherapeutic agents. Therefore, an antibody or antigen-binding fragment according to the present invention that specifically binds to a specific API5 epitope can reduce or decrease the activity of the API5 protein, thereby potentially being very useful in preventing or treating cancers that are resistant, resilient, or refractory to existing anticancer drugs.

[0039] For example, existing anticancer drugs may include, but are not limited to, those reported to have resistance, refractory properties, specifically anticancer chemotherapy agents such as 5-FU, methotrexate, gemcitabine, cytarabine, paclitaxel, vinorelbine, cisplatin, oxaliplatin, and irinotecan; anticancer molecular targeted agents such as bortezomib, cetuximab, crizotinib, dasatinib, gefitinib, imatinib, and vemurafenib; and / or anticancer immunotherapy agents such as CTLA-4 inhibitors, PD-1 inhibitors, PD-L1 inhibitors, and ACT (adoptive cell therapy).

[0040] In addition, 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, melanoma of the skin or eyeball, small intestine cancer, colorectal 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 lymphomas, spinal cord tumors, glioblastomas, brainstem gliomas, and pituitary adenomas, but is not limited to these.

[0041] The pharmaceutical composition of the present invention, containing an effective amount of an antibody or its antigen-binding fragment, can be administered to subjects requiring the prevention or treatment of cancer.

[0042] As used herein, the term “administration” refers to physically delivering a composition to a subject using any of the various methods or delivery systems known to those skilled in the art. Administration may be, but is not limited to, oral administration, or other parenteral administration such as intravenous, intramuscular, subcutaneous, intraperitoneal, or spinal administration, or administration by injection or infusion. The number of administrations may be, for example, a single dose, multiple doses, or one or more doses over a long period.

[0043] As used herein, the term “subject” includes humans or any non-human animals, which may be vertebrates such as primates, dogs, cattle, horses, pigs, and rodents (e.g., mice, rats, guinea pigs, etc.). Herein, “subject” is used interchangeably with “individual” and “patient.”

[0044] The effective dose may be either a "therapeutic effective dose" or a "preventive effective dose." The term "therapeutic effective dose" refers to any amount that can produce a reduction in the severity of disease symptoms, an improvement in the frequency and duration of asymptomatic periods, or prevention of injury or disability resulting from having the disease. The term "preventive effective dose" refers to any amount that suppresses the development or recurrence of cancer in the subject. The level of the effective dose can be determined based on various factors, including the severity of the subject's condition, age, sex, drug activity, drug sensitivity, administration time, route of administration and excretion rate, duration of treatment, concomitant medications, and other factors well known in the medical field.

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

[0046] In addition, the pharmaceutical composition may further contain suitable carriers, excipients, and diluents commonly used in the preparation of pharmaceutical compositions. Carriers, excipients, and diluents that can be included in the composition include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0047] In addition, the pharmaceutical composition can 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, but are not limited to, drugs such as compounds or proteins that have the effect of preventing, treating, and alleviating cancer.

[0048] In addition, the pharmaceutical composition can be formulated to be administered simultaneously, sequentially, or separately with other therapeutic agents. For example, an antibody or its antigen-binding fragment and 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 its antigen-binding fragment and other therapeutic agents contained in the pharmaceutical composition of the present invention may be formulated separately in different containers, or together in the same container. In addition, their pharmaceutically effective dose, administration time, administration interval, administration route, treatment duration, etc., may be the same or different.

[0049]

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

[0051] In the methods for prevention or treatment according to the present invention, unless otherwise specified, the relevant 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 its antigen-binding fragment may be administered to the target sequentially or individually, simultaneously with other therapeutic agents.

[0053] "Simultaneous" administration refers to the administration of an antibody or its antigen-binding fragment and other therapeutic agents in a single formulation, or the administration of an antibody or its antigen-binding fragment and other therapeutic agents in separate formulations. In this case, the administration routes of the antibody or its antigen-binding fragment and the other therapeutic agents may be different.

[0054] "Sequential" administration refers to the relatively continuous administration of antibodies or their antigen-binding fragments and other therapeutic agents, which allows for the shortest possible time consumed between administrations.

[0055] "Individualized" administration refers to the administration of antibodies or their antigen-binding fragments and other therapeutic agents at predetermined time intervals. The method of administration of antibodies or their antigen-binding fragments and other therapeutic agents may be appropriately selected by a physician or expert in the art, taking into consideration the therapeutic efficacy and side effects of the target patient.

[0056]

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

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

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

[0060] The API5 proteins disclosed in this invention are known to be overexpressed in various types of cancer cells, including cancers that exhibit resistance, refractory, or intractable properties to anticancer drugs, as well as common cancers. In particular, the specific API5 epitope represented by the amino acid sequence of SEQ ID NO: 7 contains amino acid residues that bind to the TLR4 protein. As a result, the API5 epitope induces activation of ERK signaling, inhibition of apoptosis in cancer cells, or promotion of tumor growth through interaction with the TLR4 protein. Therefore, antibodies or antigen-binding fragments that bind to specific API5 epitopes with high specificity and high affinity can be effectively used for diagnosing cancer.

[0061] Specifically, the cancer diagnosis according to the present invention can be performed by reacting an antibody or its antigen-binding fragment with a biological sample to determine whether cancer has occurred or whether there is a possibility of cancer developing, preferably by contacting an antibody or its antigen-binding fragment with a biological sample to determine whether an antigen-antibody complex has been formed.

[0062] As used herein, the term "biological sample" includes, but is not limited to, tissues, cells, blood, serum, plasma, and autopsy tissue samples (brain, skin, lymph nodes, spinal cord).

[0063] As used herein, the term "antigen-antibody complex" refers to a complex formed by the binding of an API5 protein antigen in a sample to the antibody of the present invention that recognizes it or its antigen-binding fragment.

[0064] The formation of such antigen-antibody complexes can be detected by any method, including colorimetric, electrochemical, fluorescence, luminometry, particle counting, visual evaluation, or scintillation counting. However, the method is not limited to these, and various applications and modifications are possible according to methods known in the art.

[0065] Specifically, the diagnostic compositions or kits of the present invention can be prepared to be suitable for various immunoassays or immunostaining methods. These immunoassays or immunostaining methods include, but are not limited to, enzyme-linked immunosorbent assays (ELISA), immunofluorescence, Western blotting, immunohistochemical staining, flow cytometry, immunocytochemistry, radioimmunoassays (RIA), immunoprecipitation assays, and protein chips.

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

[0067]

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

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

[0070] In the present invention, a method for providing information for cancer diagnosis can be carried out by reacting an antibody or its antigen-binding fragment with a biological sample to determine whether cancer has occurred or whether there is a possibility of cancer developing, preferably by contacting an antibody or its antigen-binding fragment with a biological sample to determine whether an antigen-antibody complex has been formed.

[0071] In one embodiment of the present invention, the method for providing information for cancer diagnosis may be a method for diagnosing cancer, a method for providing information for selecting cancer patients who have resistance, resistance or refractory properties to anticancer drugs, or a method for selecting cancer patients who have resistance, resistance or refractory properties to anticancer drugs.

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

[0073] Here, the control group may consist of biological samples isolated from healthy individuals without cancer, or biological samples isolated from individuals whose cancer has been cured.

[0074]

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

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

[0077] In the present invention, a method for providing information for cancer treatment can be carried out by reacting an antibody or its antigen-binding fragment with a biological sample to provide information on resistance, resistance, or refractory status to anticancer drugs, preferably by contacting an antibody or its antigen-binding fragment with a biological sample to determine whether an antigen-antibody complex has been formed.

[0078] In one embodiment of the present invention, the method for providing information for cancer treatment may be a method for providing information for selecting cancer patients who have resistance, resistance or refractory properties to anticancer drugs, or a method for selecting cancer patients who have resistance, resistance or refractory properties to anticancer drugs.

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

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

[0081] Here, the control group may be biological samples isolated from healthy individuals without cancer, biological samples isolated from individuals whose cancer has been cured, or biological samples isolated from cancer patients who have been determined not to have resistance, resistance, or refractory properties 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 level of expression or activity of the API5 protein in the cancer cells treated with the anticancer drug candidate using an antibody or an antigen-binding fragment thereof; and (c) determining the anticancer drug candidate treated in step (a) as an anticancer drug if the level of expression or activity of the API5 protein in step (b) is lower than the level 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, the relevant 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 a candidate anticancer drug, which can be carried out using methods known in the art. For example, the candidate anticancer drug may be used to treat cancer cells and co-culture them, or it may be administered to a living organism having cancer cells. However, this is not limited to these, and those skilled in the art will be able to use a method suitable for the purpose of the present invention.

[0086] As used herein, the term “anti-cancer drug” refers to a drug that exhibits a preventive or therapeutic effect against cancer, specifically a drug that can kill cancer cells or tumors, or inhibit their growth. In this specification, “anti-cancer drug” may be used interchangeably with “drug,” and “treatment” may be used interchangeably with “addition” or “administration.”

[0087] As used herein, the term "candidate anticancer drug" refers to a drug that is expected to exhibit a preventive or therapeutic effect against cancer, and more specifically, a drug that is expected to kill cancer cells or tumors or inhibit their growth.

[0088] Preferably, anticancer drugs or anticancer drug candidates include, but are 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, fractions, etc. For example, anticancer drugs or anticancer drug candidates may include, but are not limited to, compounds, peptides, peptide mimes, fusion proteins, antibodies, aptamers, antibody-drug conjugates (ADCs), etc. Preferably, anticancer drugs or anticancer drug candidates may be antibodies or tumor immunotherapy agents.

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

[0090] Step (c) is a step to determine whether a candidate anticancer drug can be used as an anticancer drug. The API5 protein is overexpressed in cancer cells, and the specific API5 epitope represented by the amino acid sequence of SEQ ID NO: 7 contains amino acid residues that bind to the TLR4 protein. Thus, the specific API5 epitope promotes the proliferation, migration, invasion, and growth of cancer cells through interaction with the TLR4 protein. Therefore, a candidate anticancer drug that reduces the level of expression or activity of the API5 protein can be used as an anticancer drug. In addition, the API5 protein is overexpressed in cancer cells that are resistant, resilient, or refractory to anticancer drugs, and the specific API5 epitope represented by the amino acid sequence of SEQ ID NO: 7 contains amino acid residues that bind to the TLR4 protein. Thus, the specific API5 epitope promotes the proliferation, migration, invasion, and growth of cancer cells that are resistant, resilient, or refractory to anticancer drugs through interaction with the TLR4 protein. Therefore, anticancer drug candidates that reduce the expression or activity level of the API5 protein can be used as anticancer drugs for the prevention or treatment of cancers that exhibit resistance, resistance, or refractory properties to conventional anticancer drugs. [Effects of the Invention]

[0091] The API5 epitope according to the present invention induces stronger resistance, resistance, or refractory properties in cancer cells compared to other regions or epitopes of the API5 protein. Therefore, antibodies or antigen-binding fragments that bind to the API5 epitope are highly effective in killing cancer cells and inhibiting tumor proliferation or growth. In particular, their anticancer effects are excellent against cancers that exhibit resistance, resistance, or refractory properties to existing anticancer drugs. [Brief explanation of the drawing]

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

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

[0094] [Figure 3] Figure 3 shows the Western blot results for phosphorylated ERK (pERK) and ERK, illustrating the ERK phosphorylation reduction effects of the five anti-API5 antibodies #1 to #5 according to the present invention against TC-1 P3PD-1, MC38 P3PD-1, and CT26 P3PD-1, as well as the anti-API5 antibodies Novus, D-1, and Mybio in the comparative experimental group.

[0095] [Figure 4] Figure 4 shows the Western blot results for phosphorylated ERK (pERK) and ERK, demonstrating the ERK phosphorylation-reducing effect of anti-API5 antibody #1 according to the present invention against TC-1 P3PD-1, MC38 P3PD-1, and CT26 P3PD-1.

[0096] [Figure 5] Figure 5 is a graph showing the apoptosis-enhancing effects of anti-API5 antibodies #3, #4, and #5 according to the present invention against TC-1 P3PD-1 and CT26 P3PD-1, as well as the effects of the anti-API5 antibodies Novus, D-1, and Mybio in the comparative experimental group.

[0097] [Figure 6] Figure 6 is a graph showing the tumor growth inhibitory effect of anti-API5 antibody #1 according to the present invention in an in vivo model of colorectal cancer or colorectal cancer refractory to anti-cancer immunotherapy agents. * indicates a p-value < 0.05, and **** indicates a p-value < 0.0001.

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

[0099] [Figure 8]Figure 8 shows a graph illustrating the tumor growth inhibitory effect of anti-API5 antibody #1 according to the present invention in an in vivo model of colorectal cancer or colorectal cancer refractory to anti-cancer immunotherapy agents. * indicates a p-value < 0.05, *** indicates a p-value < 0.001, and **** indicates a p-value < 0.0001.

[0100] [Figure 9] Figure 9 is a graph showing the tumor growth inhibitory effect of the anti-API5 antibody #1, anti-PD-1 antibody, or a combination of anti-API5 antibody #1 and anti-PD-1 antibody according to the present invention in an in vivo model of refractory colorectal cancer to anti-cancer immunotherapy agents. **** indicates a p-value < 0.0001.

[0101] [Figure 10] Figure 10 is a graph showing the tumor growth inhibitory effects of anti-API5 antibodies #1 to #4 according to the present invention, as well as anti-API5 antibodies D-1 and Mybio in the comparative experimental group, in an in vivo model of colorectal cancer refractory to anti-cancer immunotherapy agents. *** indicates a p-value < 0.001, and **** indicates a p-value < 0.0001.

[0102] [Figure 11] Figure 11 is a graph showing the tumor growth inhibition rates of anti-API5 antibodies #1 to #4 according to the present invention, as well as anti-API5 antibodies D-1 and Mybio in the comparative experimental group, in an in vivo model of colorectal cancer refractory to anti-cancer immunotherapy agents. [Modes for carrying out the invention]

[0103] The present invention will be described in more detail below with reference to examples. These examples are intended to illustrate the present invention in more detail, and the scope of the present invention is not limited by these examples.

[0104] [Examples]

[0105] Example 1. Identification of a specific API5 epitope.

[0106] The inventors have for the first time confirmed that a specific API5 epitope, corresponding to amino acids 206-225 of the API5 protein, plays a major role in causing cancer resistance, refractory disease, or intractability to anticancer drugs. Furthermore, the inventors have for the first time discovered that this specific API5 epitope contains amino acid residues involved in the binding of the API5 protein to the TLR4 protein, which is its receptor.

[0107] To verify this, the inventors analyzed a model suggesting the amino acid residues involved in the binding of the API5 protein to its receptor, the TLR4 protein. Figure 1 shows that the API5 protein binds to the TLR4 protein through amino acid residues 206 to 225. Table 1 below shows the specific binding residue pairs of the API5 protein and the TLR4 protein.

[0108] [Table 1]

[0109]

[0110] Example 2. Anti-API5 antibody that binds to a specific API5 epitope

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

[0112] The inventors prepared antibodies that bind to the API5 protein. Specifically, they prepared antibodies that specifically bind to amino acid residues that act as epitopes in the API5 protein. To discover antibodies that bind 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), followed by the use of purified API5 protein. Antibodies exhibiting the ability to bind to API5 were selected through phage display technology. As a result, Fab antibody phage clones were selected, and then anti-API5 antibodies (#1 to #5) were prepared by transplanting them into an IgG4 (S228P) backbone. Anti-API5 antibody #1 includes 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. Anti-API5 antibody #2 includes 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. Anti-API5 antibody #3 contains 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. Anti-API5 antibody #4 contains 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. Anti-API5 antibody #5 contains 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 experiments described later, the inventors used commercially available antibodies, specifically the "NOVUS" antibody available from NOVUS that binds to the region consisting of amino acids 1 to 50 of the API5 protein (1 to 50), the "D-1" antibody available from Santa Cruz Biotechnology that binds to the region consisting of amino acids 72 to 84 of the API5 protein (72 to 84), and the "Mybio" antibody available from MyBioSource that binds to the region consisting of amino acids 487 to 504 of the API5 protein (487 to 504).

[0114]

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

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

[0117] Specifically, the full-length API5 protein, consisting of 504 amino acid residues (mers), was divided into regions consisting of approximately 20 to 50 amino acid residues (mers), and then peptide fragments containing these regions were prepared. Subsequently, the binding levels of the anti-API5 antibody prepared in Example 2-1 to each API5 region were determined.

[0118] The complete amino acid sequence of the API5 protein, consisting of 504 amino acid residues (mer), is represented by Sequence ID No. 6 in Table 2 below.

[0119] [Table 2]

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

[0121] Next, ELISA was performed to detect binding between the peptides and the anti-API5 antibody. A 96-well ELISA microtiter plate was coated overnight at 4°C with streptavidin at a concentration of 5 μg / ml dissolved in PBS. The plate was washed twice and blocked at 37°C for 1 hour. Then, 100 μl of each peptide at a concentration of 10 μg / ml dissolved in PBS was added to the plate and incubated at 37°C for 1 hour. After washing the plate 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 the reaction was allowed to proceed for 1 minute by adding the substrate solution to the wells. Absorbance was measured at 450 nm using a spectrometer.

[0122] As a result, Figure 2 shows that all anti-API5 antibodies #1 to #5 exhibited excellent binding affinity to the API5 peptides containing API5 206 to 225, among the API5 peptides containing their respective regions.

[0123]

[0124] Furthermore, the binding affinity between the specific API5 epitope identified as a novel 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 purified anti-API5 antibodies to specific API5 epitopes was individually analyzed using Biacore T200. API5 was immobilized on a sensor chip, and the binding rate was measured using anti-API5 antibodies as the analyte. Data on the rate constants of dissociation rate constant (kd) and binding rate constant (ka) were determined 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 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 dissociates per second. The equilibrium dissociation constant (KD), calculated from the ratio of kd to ka, is 1 × 10⁻⁶ -9 When the value is M or less, the antibody's ability to bind to the antigen protein is considered excellent.

[0126] [Table 3]

[0127] As a result, as shown in Table 3 above, anti-API5 antibodies #1 to #5 were confirmed to have excellent binding ability to specific API5 epitopes (API5 206 to 225) as described in Sequence ID No. 7.

[0128]

[0129] Example 3. Cancer therapeutic effect of anti-API5 antibodies that bind to specific API5 epitopes.

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

[0131] To check the anticancer effect of the specific API5 epitope identified in Example 1, we analyzed the effect of an anti-API5 antibody that binds to the epitope on reducing ERK activation (i.e., ERK phosphorylation) in cancer cells. ERK is primarily activated by growth factors involved in cell proliferation, differentiation, and survival. ERK activation occurs through phosphorylation of the ERK protein, and continuous ERK activation plays a crucial role in tumorigenesis. ERK activation is known to be elevated in various types of tumors, including pancreatic cancer, colorectal cancer, lung cancer, ovarian cancer, kidney cancer, and breast cancer.

[0132] Specifically, for non-small cell lung cancer or uterine cancer, TC-1 P3 PD-1 For cell lines, specifically for colorectal cancer, use MC38 P3. PD-1 For cell lines and colorectal cancer, see CT26 P3. PD-1 Cell lines (these cell lines are refractory to anti-PD-1 antibody therapy) were cultured in RPMI medium containing 5% FBS, at a rate of 1.5 × 10⁶ per well in a 12-well plate. 5 Cells were seeded individually. After 2 hours, the medium was replaced with RPMI medium containing 0.1% FBS, and each cell was treated with an anti-API5 antibody at a maximum concentration of 150 ng / ml.

[0133] After 24 hours, the cells were washed with 1×PBS buffer solution and lysed in 150 μl of RIPA buffer solution at 4°C for 10 minutes. The cells were then centrifuged at 13,000 rpm at 4°C for 15 minutes to obtain the cell extract as the supernatant. Protein quantification was performed using the Bradford assay, and the levels of ERK protein and its phosphorylation were measured for the same amount of protein by SDS-PAGE Western blotting. The measured ERK protein levels and ERK protein phosphorylation levels were quantified using Image J (densitometer), and the ratio of phosphorylated ERK protein level to total ERK protein level was calculated.

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

[0135] "TC-1 P3 PD-1 "MC38 P3 PD-1 " or "CT26 P3 PD-1 "Cells that are refractory to anti-PD-1 antibody therapy" refers to cells that survived being passaged three times with anti-PD-1 antibodies from the parent cancer cells "TC-1 P0", "MC38 P0", or "CT26 P0".

[0136] As a result, Figure 3 shows that ERK phosphorylation was significantly reduced in all cancer cell lines by anti-API5 antibodies that bind to a specific API5 epitope represented by the amino acid sequence of SEQ ID NO: 7, and the ratio of ERK phosphorylation to total ERK was reduced by up to half compared to the control group. These results were the same for all anti-API5 antibodies #1 to #5. Conversely, the comparative group of anti-API5 antibodies (Novus, D-1, and Mybio) 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 increased by up to twofold compared to the control group.

[0137] In addition, as shown in FIG. 4, ERK phosphorylation was significantly reduced in all cancer cell lines by an anti-API5 antibody that binds to a specific API5 epitope represented by the amino acid sequence of SEQ ID NO: 7. On the other hand, when the anti-API5 antibody and a peptide having a specific API5 epitope sequence represented by the amino acid sequence of SEQ ID NO: 7 were treated simultaneously 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 contains an amino acid residue that binds to the TLR4 protein, which is a receptor of the API5 protein. Therefore, the specific API5 epitope is involved in ERK activation in cancer cells through interaction with the TLR4 protein. Thus, this suggests that an anti-API5 antibody that binds to the specific API5 epitope can reduce ERK activation in cancer cells, thereby exhibiting an excellent anti-cancer effect.

[0139]

[0140] Example 3-2. Improvement of apoptosis

[0141] To check the anti-cancer effect of the specific API5 epitope identified in Example 1, the effect of improving apoptosis of cancer cells by an anti-API5 antibody that binds to the above epitope was analyzed.

[0142] TC-1 P3 prepared in Example 3-1 PD-1 Non-small cell lung cancer cell line or cervical cancer cell line and CT26 P3 PD-1 Colorectal cancer cell lines were seeded in 12-well plates at 3×10 5 cells per well and RPMI medium containing 5% FBS was used. After 2 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, FACS was used to determine the percentage of dead cells due to apoptosis.

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

[0144] As a result, Figure 5 shows that the percentage of cells killed by anti-API5 antibodies that bind to the specific API5 epitope represented by the amino acid sequence of SEQ ID NO: 7 increased by approximately 10–20% compared to the untreated group, and these effects were equally observed in all cancer cell lines. Conversely, when treated with the comparative group of anti-API5 antibodies (Novus, D-1, and Mybio) 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 reduced by approximately 10–60%.

[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 contains amino acid residues that bind to the TLR4 protein, which is the receptor for the API5 protein. Therefore, the specific API5 epitope is involved in apoptosis of cancer cells through interaction with the TLR4 protein. Consequently, this suggests that an anti-API5 antibody that binds to the specific API5 epitope can enhance the sensitivity of cancer cells to apoptosis, thereby exhibiting excellent anti-cancer effects.

[0146]

[0147] Example 3-3. Inhibition of tumor growth in vivo.

[0148] To check the anticancer effect of the specific API5 epitope identified in Example 1, the in vivo growth inhibitory effect of an anti-API5 antibody that binds to the specific API5 epitope represented by the amino acid sequence of SEQ ID NO: 7 was analyzed.

[0149] In the experiment, the MC-38 P0 colorectal cancer cell line and MC-38 P3 cells that were refractory to anti-PD-1 antibody treatment were used. PD-1 MC-38 P3, a colorectal cancer cell line that is refractory to anti-PD-L1 antibody therapy. PD-L1 Colorectal cancer cell lines, TC-1 P0 non-small cell lung cancer or uterine cancer cell lines, CT26 P0 colorectal cancer cell lines, CT26 P3 cells refractory to anti-PD-1 antibody therapy. PD-1 A colon cancer cell line was used.

[0150] Specifically, each cell has a cell count of 5 × 10 5 Tumors were induced by transplanting individual cells into balb / c mice. The average tumor size was approximately 60-75 mm. 3 (For MC-38, 30-40mm) 3 When the tumor size reached a certain level, 8-9 mice were distributed per experimental group to ensure that the tumor size was distributed as uniformly as possible among the groups. These mice were administered either anti-API5 antibodies #1-#5 that 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. The antibody dose was 200 μg, administered intraperitoneally three times a week for a total of five times, based on the day antibody administration began (day 1). Subsequently, the length of the tumor was measured using calipers at 2- or 3-day intervals based on the day antibody administration began, and the formula for tumor size was: Tumor size = [(long axis length × short axis length)] 2 Tumor size was calculated using ] / 2. In addition, tumor growth inhibition rate (TGI, %) was calculated from the tumor size according to the formula: 100 - (100 × tumor size of the experimental group / tumor size of the IgG antibody treatment control group).

[0151] The above results were analyzed assuming normality of the data and using parametric multiple comparison methods. 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 considered statistically significant.

[0152] As a result, as shown in Figures 6-8, tumor growth was significantly inhibited by an anti-API5 antibody that binds to a specific API5 epitope represented by the amino acid sequence of SEQ ID NO: 7. Compared to the control group treated with IgG antibodies, the tumor growth inhibition effect was improved by approximately 1.5 to 4 times in the experimental group. This effect was demonstrated not only for cancer cells that are refractory to existing immunosuppressant drugs, but also for various cancers such as colorectal cancer, non-small cell lung cancer, and cervical cancer.

[0153] In particular, Figure 9 shows that when anti-API5 antibodies are administered in combination with anti-PD-1 antibodies, they exhibit excellent growth inhibitory effects even against cancer cells that are resistant, refractory, or intractable to anti-PD-1 antibody therapy, and that this effect was synergistic.

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

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

[0156] Sequence listing free text Attached as an electronic file

Claims

1. An antibody or its antigen-binding fragment that specifically binds to the epitope of apoptosis inhibitor 5 (API5), represented by the amino acid sequence of Sequence ID No.

7.

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

3. A vector comprising the nucleic acid described in claim 2.

4. Cells transformed with the vector described in claim 3.

5. A pharmaceutical composition for preventing or treating cancer, comprising the antibody or antigen-binding fragment described in claim 1.

6. The pharmaceutical composition according to claim 5, wherein the cancer exhibits resistance, tolerance, or refractory properties to anticancer drugs.

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

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, melanoma of the skin or eyeball, small intestine cancer, colorectal 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 lymphomas, spinal cord tumors, glioblastomas, brainstem gliomas, and pituitary adenomas.

9. A composition for diagnosing cancer, comprising the antibody or antigen-binding fragment described in claim 1.

10. A kit for diagnosing cancer, comprising the antibody or antigen-binding fragment described in claim 1.

11. A method for providing information for cancer diagnosis: Through an antigen-antibody reaction using the antibody or antigen-binding fragment described in claim 1, To detect the API5 protein in a biological sample isolated from a subject suspected of having cancer, or to determine the expression or activity level of the API5 protein. Methods that include...

12. A method for providing information for cancer treatment, Through an antigen-antibody reaction using the antibody or antigen-binding fragment described in claim 1, To detect the API5 protein in a biological sample isolated from a cancer patient, or to determine the level of expression or activity of the API5 protein. Methods that include...

13. A method for screening anticancer drugs: (a) Treating cancer cells with a candidate anticancer drug; (b) Determining the level of expression or activity of the API5 protein in cancer cells treated with the anticancer drug candidate substance using the antibody or antigen-binding fragment described in claim 1; and (c) If the level of expression or activity of the API5 protein in step (b) is lower than the level in cancer cells that have not been treated with the candidate anticancer drug, the candidate anticancer drug treated in step (a) is determined to be an anticancer drug. Methods that include...

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