Antibodies that specifically bind to API5 protein

Abstract

The present invention relates to antibodies that specifically bind to apoptosis inhibitor 5 (API5) protein and uses thereof. The antibody or antigen-binding fragment thereof that specifically binds to API5 according to the present invention can exert excellent anti-cancer effects, such as inhibiting the phosphorylation of ERK in cancer cells and inhibiting cancer growth.In addition, the antibody or antigen-binding fragment thereof exhibits excellent anti-cancer effects against cancers that are resistant or refractory to anti-cancer drugs.Therefore, it is useful for the prevention or treatment of cancers that are resistant or refractory to anti-cancer drugs and general cancers.

Description

[Technical field]

[0001] The present invention relates to antibodies that specifically bind to apoptosis inhibitor 5 (API5) protein and uses thereof. [Background technology]

[0002] Despite intensive cancer research over the past decades, cancer remains a leading cause of death worldwide. Although numerous anticancer treatments have been developed, they are not effective for all cancer types and in all patients. Currently, the methods used to treat cancer are relatively nonselective: surgery to remove diseased tissue, radiation therapy to shrink solid tumors, or chemotherapy to rapidly kill cancer cells. However, chemotherapy can induce drug resistance and may be dose-limiting. It may also cause severe side effects that preclude the use of potentially effective drugs. Therefore, there is a need to develop more target-specific and effective cancer treatments.

[0003] Therefore, the development of anti-cancer targeted drugs that specifically target only cancer cells and anti-cancer immunotherapy drugs that utilize the patient's immune system is underway. For example, there are about 30 types of anti-cancer targeted drugs approved in Korea, including Opdivo (ingredient name: nivolumab), Keytruda (ingredient name: pembrolizumab), Tecentriq (ingredient name: atezolizumab), Imfinzi (ingredient name: durvalumab), and Yervoy (ingredient name: ipilimumab), which are used for various cancers such as non-small cell lung cancer, melanoma, renal cell carcinoma, Hodgkin's lymphoma, head and neck cancer, and bladder cancer.

[0004] However, continued use of the same anticancer drug or anticancer drug with the same mechanism may cause new problems such as anticancer drug resistance and intractability. Therefore, there is still a need in the art for new anticancer drugs, and it is necessary to discover cancer cell-specific targets and develop drugs that can be used to prevent or treat cancer. Summary of the Invention [Problem to be solved by the invention]

[0005] The present inventors have made extensive research efforts to develop a novel anticancer drug. As a result, the present inventors have produced a number of anti-API5 antibodies that specifically bind to API5 protein, which is a cancer cell-specific target, and have confirmed that these antibodies have high binding ability to API5 protein and exhibit excellent anticancer effects, thereby completing the present invention. [Means for solving the problem]

[0006] Each description and embodiment disclosed in the present invention is applicable to other descriptions and embodiments. That is, all combinations of the various elements disclosed herein are included within the scope of the present invention. In addition, the scope of the present invention should not be construed as being limited by the specific description described below.

[0007] Furthermore, terms not specifically defined herein will be understood to have the meanings commonly used in the art to which this invention pertains, and the singular includes the plural and the plural includes the singular, unless otherwise defined by context.

[0008]

[0009] One aspect of the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to API5 (apoptosis inhibitor 5).

[0010] The term "antibody" as used herein refers to an immunoglobulin molecule having immunological reactivity with a particular antigen, or a protein molecule that acts as a receptor to specifically recognize an antigen. In the present invention, "antibody" is used in a broad sense and includes polyclonal antibodies, monoclonal antibodies, whole antibodies (antibodies consisting of at least two heavy chains and two light chains linked by disulfide bonds), and antibody fragments. Whole antibodies include IgA, IgD, IgE, IgM, and IgG. Furthermore, IgG may include IgG1, IgG2, IgG3, and IgG4 as subtypes. The term "antibody fragment" refers to an antigen-binding fragment or analog of an antibody that retains at least a portion of the binding specificity of the parent antibody and includes a variable region or a portion (e.g., one or more CDRs) of the antigen-binding region of the parent antibody. Antibody fragments are, for example, Fab, Fab', F(ab')2, Fv fragments, scFv, unibodies, bispecific antibodies, linear antibodies, nanobodies, domain antibodies, or multispecific antibodies formed from antibody fragments.

[0011] The term "API5 (Apoptosis Inhibitor 5) Protein" as used herein refers to a protein encoded by the API5 gene, the amino acid sequence of which is known in existing databases (NCBI Reference Sequences: XP_016873953.1, XP_016873954.1, XP_006718422.1, etc.). API5 protein is expressed at high levels in tumors such as solid and metastatic cancers, and its expression is particularly increased in cancer cells that are resistant 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).

[0012] The term "anti-API5 antibody" as used herein refers to an antibody or antigen-binding fragment thereof that specifically binds to API5 protein and neutralizes or inhibits the activity of API5 protein. Specifically, the anti-API5 antibody may be a human antibody that targets human API5 protein, and also has reactivity with API5 protein from mouse or rat.

[0013] The term "human antibody" as used herein refers to an antibody having variable regions in which the framework and CDR regions are derived from human immunoglobulin sequences, also referred to as a "fully human antibody". Human antibodies of the present invention may include amino acid residues not encoded by immunoglobulin sequences of human origin (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

[0014] In one embodiment according to the invention, an anti-API5 antibody may comprise a heavy chain CDR1 comprising the amino acid sequence GFTFSTYA of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence ISGSGGX1T of SEQ ID NO: 2, and a heavy chain CDR3 comprising the amino acid sequence AKLVLX2WX3YFSMDH of SEQ ID NO: 3, where X1, X2 and X3 are independently any amino acid. The amino acid may be arginine (R), histidine (H), lysine (K), aspartic acid (D), glutamic acid (E), serine (S), threonine (T), asparagine (N), glutamine (Q), cysteine ​​(C), selenocysteine ​​(U), glycine (G), proline (P), alanine (A), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), tyrosine (Y) or tryptophan (W). Preferably, X1 may be lysine (K) or leucine (L), X2 may be lysine (K) or tryptophan (W), and X3 may be tryptophan (W) or phenylalanine (F), but is not limited thereto.

[0015] The heavy chain CDR2 may be or may include the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5, but is not limited to such.

[0016] In addition, the heavy chain CDR3 may be any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 6 to SEQ ID NO: 10, or may contain any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 6 to SEQ ID NO: 10, but is not limited thereto.

[0017] In the present invention, the anti-API5 antibody comprises a heavy chain variable region, and the heavy chain variable region may be any one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 11 to 15, or may comprise any one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 11 to 15, but is not limited thereto.

[0018] In addition, the anti-API5 antibody comprises light chain CDR1-CDR3, in which the light chain CDR1 may be or may include the amino acid sequence of SEQ ID NO: 16, the light chain CDR2 may be or may include the amino acid sequence of SEQ ID NO: 17, and the light chain CDR3 may be or may include the amino acid sequence of SEQ ID NO: 18, but is not limited thereto.

[0019] In addition, the anti-API5 antibody comprises a light chain variable region, which may be or may include the amino acid sequence of SEQ ID NO:19, but is not limited to such.

[0020] The sequences disclosed in the present invention include sequences that are substantially identical to the sequences listed in the sequence listing. Substantially identical means that two sequences are aligned as closely as possible and analyzed using algorithms commonly used in the art, showing 80%, 90%, 95% or more homology between the sequences.

[0021] In addition, the antibody or antigen-binding fragment thereof according to the present invention may comprise the sequence of the antibody or antigen-binding fragment thereof described herein, a sequence showing substantial identity thereto, and biological equivalents thereof to the extent that they are capable of specifically recognizing and binding to the API5 protein. For example, it may comprise additional mutations in the sequence to improve the antibody binding affinity and / or biological properties, and additional mutations within the scope that do not alter the overall activity of the molecule.

[0022]

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

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

[0025] The term "nucleic acid" as used herein includes DNA (gDNA and cDNA) and RNA. The nucleotides that constitute the basic structural units in nucleic acid molecules include not only natural nucleotides but also analog nucleotides in which sugar or base moieties are modified (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Review, 90:543-584 (1990)).

[0026] Nucleic acid molecules encoding an antibody or antigen-binding fragment thereof according to the present invention include nucleotide sequences encoding the amino acid sequence constituting the antibody or antigen-binding fragment thereof, but are not limited to any particular nucleotide sequence.

[0027] Taking into account codon degeneracy, a nucleotide sequence includes nucleotide sequences that contain functionally equivalent codons or codons that code for the same amino acid (e.g., there are six codons for arginine or serine due to codon degeneracy), or codons that code for biologically equivalent amino acids.

[0028] Considering the above-mentioned mutations having biologically equivalent activity, the nucleic acid molecule of the present invention encoding the antibody or antigen-binding fragment thereof contains a sequence that shows substantial identity thereto. "Substantially identical" means that the two sequences are aligned as closely as possible and analyzed using an algorithm commonly used in the art, and show 80%, 90%, or 95% or more sequence homology.

[0029]

[0030] Another aspect of the invention provides a vector containing the nucleic acid and a cell transformed with the vector.

[0031] In the vectors and transformed cells according to the present invention, unless otherwise specified, the related terms are understood to have the same meaning as the terms stated above.

[0032] The term "vector" as used herein refers to a vector that can be inserted into a host cell and can replicate genes. Vectors include plasmids, linear nucleic acids, cosmids, RNA vectors, and viral vectors, including but not limited to retroviruses, adenoviruses, and adeno-associated viruses. The recombinant vector system of the present invention may be constructed by various methods known in the art. The vector of the present invention may also be constructed as a cloning or expression vector, and may be constructed using prokaryotic or eukaryotic cells as hosts.

[0033] Furthermore, the transformed cell may be a prokaryotic cell, a eukaryotic cell or an animal cell. An appropriately selected host cell may be transformed with a vector and used for expression and / or secretion of a target protein. The host cell may be an immortalized hybridoma cell, an N / SO myeloma cell, a 293 cell, a HuT 78 cell, a CHO cell, a HELA cell, a COS cell, etc. However, without being limited thereto, any host cell known in the art may be used as the host cell of the present invention.

[0034]

[0035] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the antibody or antigen-binding fragment thereof.

[0036] In the pharmaceutical compositions according to the present invention, unless otherwise specified, the related terms are understood to have the same meaning as those stated above.

[0037] The term "treatment" as used herein refers to any administration of a composition according to the invention that ameliorates, reverses or completely cures cancer. The term "prophylaxis" as used herein refers to any administration of a composition according to the invention that inhibits, delays or prevents the onset or recurrence of cancer.

[0038] In the present invention, cancer may show resistance, tolerance, or intractability to anticancer drugs. As used herein, the term "resistance to anticancer drugs" or "resistance to anticancer drugs" means that when anticancer drugs are used to treat a cancer patient, the treatment is ineffective from the start of treatment, or the treatment is initially effective, but the effect is reduced or lost during continuous treatment. Also, as used herein, "intractability to anticancer drugs" means that when anticancer drugs are used to treat a cancer patient, the treatment is ineffective from the start of treatment, or the response to treatment does not last for a long time.

[0039] The API5 disclosed in the present invention shows increased expression in cancer cells and activates ERK signaling, conferring resistance or refractory to anticancer drugs such as chemotherapeutic drugs and immunotherapeutic drugs. Thus, the antibody or antigen-binding fragment thereof according to the present invention that specifically binds to the API5 protein can reduce or decrease the activity of the API5 protein, and therefore may be very useful in preventing or treating cancers that are resistant or refractory to existing anticancer drugs.

[0040] For example, the existing anti-cancer drug may be one that has been reported to have resistance or intractability, specifically, but not limited to, anti-cancer chemotherapeutic drugs such as 5-FU, methotrexate, gemcitabine, cytarabine, paclitaxel, vinorelbine, cisplatin, oxaliplatin, and irinotecan, anti-cancer targeted drugs such as bortezomib, cetuximab, crizotinib, dasatinib, gefitinib, imatinib, and vemurafenib, and / or anti-cancer immunotherapeutic drugs such as CTLA-4 inhibitors, PD-1 inhibitors, PD-L1 inhibitors, and ACT (Adoptive Cell Therapy).

[0041] The cancer may be one or more selected from the group consisting of lung cancer, non-small cell lung cancer, colon cancer, rectal cancer, proximal anal cancer, colon cancer, small intestine cancer, breast cancer, uterine cancer, ovarian cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, head cancer, neck cancer, thyroid cancer, parathyroid cancer, gastric cancer, liver cancer, pancreatic cancer, bone cancer, skin cancer, cutaneous or intraocular melanoma, 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 tumor, central nervous system lymphoma, spinal cord tumor, glioblastoma, brain stem glioma, and pituitary adenoma, but is not limited thereto.

[0042] The pharmaceutical composition of the invention comprising an effective amount of the antibody or antigen-binding fragment thereof can be administered to a subject in need of prevention or treatment of cancer.

[0043] The term "administration" as used herein refers to physically giving a composition to a subject using any of a variety of methods or delivery systems known to those skilled in the art. Methods of administration may include, but are not limited to, oral administration or parenteral administration, such as intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, etc., such as by injection or infusion. The number of administrations may be, for example, a single administration, multiple administrations, or one or more extended periods of time.

[0044] The term "subject" as used herein includes any non-human animal, which may be a human or a vertebrate such as a primate, dog, cow, horse, pig, etc., or a rodent such as a mouse, rat, guinea pig, etc. As used herein, "subject" is used interchangeably with "individual" and "patient."

[0045] The effective amount may be a "therapeutically effective amount" or a "prophylactically effective amount". The term "therapeutically effective amount" refers to any amount that can show a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease asymptomatic periods, or the prevention of damage or injury due to disease. The term "prophylactically effective amount" refers to any amount that inhibits the occurrence or recurrence of cancer in a subject. The level of the effective amount may be determined according to the subject's severity, age, sex, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment duration, concomitant drugs, and other factors well known in the medical field.

[0046] The dosage of the pharmaceutical composition may vary depending on the age, sex, and weight of the subject, but specifically, 0.1 to 100 mg / kg of the composition of the present invention may be administered once or several times a day, or at intervals of several days to several months, depending on the symptoms of the subject. The dosage may be increased or decreased depending on the administration route, severity of the disease, sex, weight, age, etc.

[0047] In addition, pharmaceutical composition may further comprise suitable carrier, excipient and diluent that are generally used in the preparation of pharmaceutical composition.The carrier, excipient and diluent that may be comprised in composition include, but are not limited to, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

[0048] Furthermore, the pharmaceutical composition may be administered in combination with other therapeutic agents. In this case, the pharmaceutical composition of the present invention and the other therapeutic agents may be administered simultaneously, sequentially, or separately. The other therapeutic agents may be, but are not limited to, drugs such as compounds or proteins that have the effect of preventing, treating, and alleviating cancer.

[0049] Furthermore, the pharmaceutical composition may be formulated to be administered simultaneously, sequentially or separately with other therapeutic agents. For example, the antibody or antigen-binding fragment thereof and the other therapeutic agent may be administered simultaneously in one formulation, or simultaneously, sequentially or separately in separate formulations. For simultaneous, sequential or separate administration, the antibody or antigen-binding fragment thereof and the other therapeutic agent included in the pharmaceutical composition of the present invention may be formulated separately in separate containers or together in the same container. In addition, they may have the same or different pharmacologic effective amount, administration time, administration interval, administration route, treatment period, etc.

[0050]

[0051] Another aspect of the invention provides a method of preventing or treating cancer comprising administering an antibody or antigen-binding fragment thereof.

[0052] In the prophylactic or therapeutic methods according to the present invention, unless otherwise specified, the relevant terms are understood to have the same meaning as the terms above.

[0053] Furthermore, in the method for preventing or treating cancer according to the present invention, the antibody or antigen-binding fragment thereof may be administered to a subject simultaneously, sequentially, or separately with other therapeutic agents.

[0054] "Concurrent" administration refers to administering the antibody or its antigen-binding fragment and the other therapeutic agent at the same time in one formulation, or administering the antibody or its antigen-binding fragment and the other therapeutic agent at the same time in separate formulations. In this case, the administration routes of the antibody or its antigen-binding fragment and the other therapeutic agent may be different from each other. Furthermore, "sequential" administration refers to relatively continuous administration of the antibody or its antigen-binding fragment and the other therapeutic agent, meaning that the administration interval is as short as possible. Furthermore, "separate" administration refers to administration of the antibody or its antigen-binding fragment and the other therapeutic agent at a predetermined time interval. The method of administering the antibody or its antigen-binding fragment and the other therapeutic agent may be appropriately selected by a physician or a person skilled in the art, taking into account the therapeutic efficacy and side effects of the subject.

[0055]

[0056] Another aspect of the invention provides a composition for diagnosing cancer comprising the antibody or antigen-binding fragment thereof.

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

[0058] 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 above.

[0059] The API5 protein disclosed in the present invention is known to be overexpressed in various types of cancer cells, including cancers resistant or intractable to anticancer drugs, as well as common cancers. Therefore, the antibody or antigen-binding fragment thereof according to the present invention, which binds to the API5 protein with high specificity and affinity, can be usefully used in the diagnosis of cancer as a disease associated with the expression level or presence of the API5 protein.

[0060] Specifically, cancer diagnosis according to the present invention may be performed by reacting an antibody or its antigen-binding fragment with a biological sample to determine the presence or absence of cancer, or the possibility of cancer occurrence, or preferably, by contacting an antibody or its antigen-binding fragment with a biological sample to determine whether an antigen-antibody complex has been formed.

[0061] As used herein, the term "biological sample" includes, but is not limited to, tissues, cells, blood, serum, plasma, tissue autopsy samples (brain, skin, lymph nodes, spinal cord), and the like.

[0062] The term "antigen-antibody complex" as used herein refers to a complex formed by binding between an API5 protein antigen in a sample and an antibody of the present invention or an antigen-binding fragment thereof that recognizes it.

[0063] The formation of such antigen-antibody complexes may be detected by any method, including colorimetry, electrochemical methods, fluorescence, luminometry, particle counting, visual evaluation, scintillation counting, etc., but is not limited thereto, and various applications and modifications are possible according to methods known in the art.

[0064] Specifically, the diagnostic composition or kit of the present invention may be adapted for various immunoassays or immunostaining methods, including but not limited to enzyme-linked immunosorbent assay (ELISA), immunofluorescence, Western blotting, immunohistochemical staining, flow cytometry, immunocytochemistry, radioimmunoassay (RIA), immunoprecipitation assay, and protein chips.

[0065] Furthermore, labels for qualitatively or quantitatively determining the formation of an antigen-antibody complex include, but are not limited to, enzymes, fluorescent substances, ligands, luminescent substances, fine particles, redox molecules, radioisotopes, and the like.

[0066]

[0067] Another aspect of the present invention provides an informative method for cancer diagnosis, comprising detecting API5 protein in a biological sample isolated from a subject suspected of having cancer by antigen-antibody reaction using an antibody or an antigen-binding fragment thereof, or determining the expression level of API5 protein.

[0068] 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 above.

[0069] In the present invention, the method for providing information for cancer diagnosis may be performed by reacting an antibody or its antigen-binding fragment with a biological sample to determine the presence or absence of cancer or the possibility of cancer occurrence, or preferably, by contacting an antibody or its antigen-binding fragment with a biological sample to determine whether an antigen-antibody complex has been formed.

[0070] In one embodiment of the present invention, the method for providing information for cancer diagnosis may be a method for diagnosing cancer, or may be a method for providing information for selecting cancer patients who are resistant or refractory to anticancer drugs, or may be a method for selecting cancer patients who are resistant or refractory to anticancer drugs.

[0071] Specifically, the method includes the steps of: (a) treating a biological sample isolated from a subject suspected of having cancer with an antibody or an antigen-binding fragment thereof, and detecting API5 protein by antigen-antibody reaction; and (b) comparing the level of API5 protein detected in step (a) with the level of a control group, and determining that the subject has cancer if the level of API5 protein is higher than that of the control group.

[0072] Here, the control group may be a biological sample isolated from a cancer-free healthy individual or an individual who has been cured of cancer.

[0073]

[0074] Another aspect of the present invention provides a method for screening an anti-cancer drug, comprising: (a) treating cancer cells with a candidate anti-cancer drug; (b) measuring the expression level of API5 protein in the cancer cells treated with the candidate anti-cancer drug using the antibody or antigen-binding fragment thereof; and (c) determining that the candidate anti-cancer drug treated in step (a) is an anti-cancer drug if the expression level of API5 protein in step (b) is lower than that in cancer cells not treated with the candidate anti-cancer drug.

[0075] In the method for screening an anticancer agent according to the present invention, unless otherwise specified, the relevant terms are understood to have the same meaning as the terms above.

[0076] Specifically, step (a) is a step of treating cancer cells with a candidate anticancer drug, which may be carried out using a method known in the art. For example, the candidate anticancer drug may be treated with cancer cells and co-cultured, or may be administered to a living body having cancer cells. However, the present invention is not limited thereto, and a person skilled in the art would be able to use a method suitable for the purpose of the present invention.

[0077] The term "anticancer agent" as used herein refers to an agent that exhibits a preventive or therapeutic effect against cancer, specifically an agent that can kill or inhibit the growth of cancer cells or tumors. In the present specification, "anticancer agent" may be used interchangeably with "drug", and "treatment" may be used interchangeably with "addition" or "administration".

[0078] As used herein, the term "candidate anti-cancer drug" refers to an agent that is expected to exhibit preventive or therapeutic effects against cancer, specifically, an agent that is expected to kill or inhibit the growth of cancer cells or tumors.

[0079] Preferably, the anti-cancer drugs or anti-cancer drug candidates include, but are not limited to, compounds, proteins, fusion proteins, compound-protein conjugates, drug-protein conjugates, antibodies, compound-antibody conjugates, drug-antibody conjugates, amino acids, peptides, viruses, carbohydrates, lipids, nucleic acids, extracts, fractions, etc. For example, they may include, but are not limited to, compounds, peptides, peptidomimetics, fusion proteins, antibodies, aptamers, antibody-drug conjugates (ADCs), etc. Preferably, they may be antibodies or immunotumor drugs.

[0080] Step (b) is a step of measuring the expression level of API5 protein, and any method known to those skilled in the art may be used. For example, Western blot, co-immunoprecipitation assay, enzyme-linked immunosorbent assay (ELISA), tissue immunostaining, flow cytometry analysis, etc. may be used, but are not limited thereto, and those skilled in the art will be able to use a method suitable for the purpose of the present invention.

[0081] Step (c) is a step of judging whether the anticancer drug candidate can be used as an anticancer drug. API5 protein is overexpressed in cancer cells and promotes the proliferation, migration, invasion and growth of cancer cells, so anticancer drug candidates that reduce the expression level of API5 protein can be used as anticancer drugs. In addition, API5 protein is overexpressed in cancer cells that are resistant or refractory to anticancer drugs and promotes the proliferation, migration, invasion and growth of cancer cells that are resistant or refractory to anticancer drugs, so anticancer drug candidates that reduce the expression level of API5 protein can be used as anticancer drugs for preventing or treating cancers that are resistant or refractory to anticancer drugs.

[0082] The antibody or antigen-binding fragment thereof that specifically binds to API5 according to the present invention can exert excellent anti-cancer effects, such as inhibiting the phosphorylation of ERK in cancer cells and inhibiting cancer growth.In addition, the antibody or antigen-binding fragment thereof exhibits excellent anti-cancer effects against cancers that are resistant or refractory to anti-cancer drugs.Therefore, it is useful for the prevention or treatment of cancers that are resistant or refractory to anti-cancer drugs and general cancers. [Brief description of the drawings]

[0083] [Figure 1] 1 shows the results of Western blot analysis of phosphorylated ERK (pERK) and ERK, showing the effect of anti-API5 antibodies 1 to 5 having specific heavy chain CDR sequences according to the present invention (hereinafter referred to as "anti-API5 antibodies according to the present invention") on reducing ERK phosphorylation in TC-1 P3PD-1.

[0084] [Diagram 2]TC-1 is a graph showing the concentration-dependent ERK phosphorylation-reducing effect of anti-API5 antibodies 1 to 5 according to the present invention on P3PD-1, and relates to the ratio of ERK phosphorylation to total ERK.

[0085] [Diagram 3] 1 shows the results of Western blot analysis of phosphorylated ERK (pERK) and ERK, showing the effect of anti-API5 antibodies 1 to 5 according to the present invention on reducing ERK phosphorylation in TC-1 P3PD-1, MC38 P3PD-1, and CT26 P3PD-1.

[0086] [Figure 4] 1 shows a graph illustrating the apoptosis-enhancing effect of anti-API5 antibodies 3 to 5 according to the present invention on TC-1 P3PD-1 and CT26 P3PD-1.

[0087] [Diagram 5] 1 shows a graph showing the tumor growth inhibitory effect of anti-API5 antibodies 2 to 5 according to the present invention in an in vivo model of melanoma refractory to anti-cancer immunotherapy drugs, where **** means p-value<0.0001.

[0088] [Figure 6] 1 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 colon cancer or colon cancer refractory to anti-cancer immunotherapy drugs. * means p-value<0.05, **** means p-value<0.0001.

[0089] [Figure 7] Figure 2 shows a graph illustrating the tumor growth inhibitory effect of the anti-API5 antibody 1 according to the invention in an in vivo model of non-small cell lung cancer or cervical cancer.*** means p-value<0.001.

[0090] [Figure 8]1 shows graphs showing the tumor growth inhibitory effect of anti-API5 antibodies 1 to 5 according to the present invention in in vivo models of colon cancer or colon cancer refractory to anticancer immunotherapy drugs. * means p value < 0.05, *** means p value < 0.001, and **** means p value < 0.0001.

[0091] [Figure 9] 1 shows a graph showing the tumor growth inhibitory effect of anti-API5 antibody 1 according to the present invention, anti-PD-1 antibody (catalog number BE0146, purchased from BioXcell), or a combination of anti-API5 antibody 1 and anti-PD-1 antibody in an in vivo model of colon cancer refractory to anti-cancer immunotherapy drugs. **** means p-value<0.0001.

[0092] [Figure 10] 1 shows a graph showing the tumor growth inhibitory effect of anti-API5 antibodies 1 to 4 according to the present invention in an in vivo model of colon cancer refractory to anti-cancer immunotherapy drugs, where *** means p value < 0.001 and **** means p value < 0.0001.

[0093] [Figure 11] 1 shows a graph showing the tumor growth inhibition rate of anti-API5 antibodies 1 to 4 according to the present invention in an in vivo model of colon cancer refractory to anti-cancer immunotherapy drugs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES

[0094] The present invention will be described in more detail below with reference to examples. These examples are intended to explain the present invention in more detail, but are not intended to limit the scope of the present invention.

[0095]

[0096] Example 1. Preparation of anti-API5 antibody

[0097] The present inventors have experimentally demonstrated that a group of antibodies having specific heavy chain CDR sequences shown in Table 1 below have high affinity for API5.

[0098] Specifically, the antibody expression vector was constructed by synthesizing the heavy and light chain variable domain base sequences encoding the anti-API5 antibody and inserting them into the pCDNA3.4 vector expressing the IgG4 frame. The heavy and light chain expression vectors were introduced into ExpiCHO cells and transiently expressed to secrete the antibody into the culture medium. The antibody secreted into the culture medium was purified using protein A affinity chromatography. The protein concentration and purity were determined by measuring the absorbance at 280 nm and SDS-PAGE, respectively.

[0099] On the other hand, among various sequences constituting an antibody, six CDR sequences, including three CDR sequences in the light chain variable region and three CDR sequences in the heavy chain variable region, are known to be important for antigen binding. In addition, CDR3, the third CDR sequence in the heavy chain variable region, which is responsible for the diversity of the antibody structure, is known to be a particularly important part among them (Brian D. Weitzner et al., Structure., 2015 Feb 3, 23(2):302-311). Therefore, manipulating CDR3 in the heavy chain variable region is a necessary technique for antibody development, and therefore, the anti-API5 antibody according to the present invention was produced by designing only the sequences of the heavy chain CDR2 and the heavy chain CDR3 to be different. The anti-API5 antibody produced in this way has the sequence shown in Table 1 below.

[0100] [Table 1]

[0101] In Table 1, X1, X2, and X3 are independently any amino acid. In the present specification, five representative antibodies (anti-API5 antibodies 1 to 5) among the anti-API5 antibodies having the sequences shown in Table 1 were used.

[0102] Specifically, anti-API5 antibody 1 has a heavy chain CDR1 represented by SEQ ID NO: 3, a heavy chain CDR2 represented by SEQ ID NO: 4, and a heavy chain CDR3 represented by SEQ ID NO: 6, and has a heavy chain variable region represented by SEQ ID NO: 11, and a light chain variable region represented by SEQ ID NO: 19.

[0103] Furthermore, anti-API5 antibody 2 has a heavy chain CDR1 represented by SEQ ID NO: 3, a heavy chain CDR2 represented by SEQ ID NO: 4, and a heavy chain CDR3 represented by SEQ ID NO: 7, and has a heavy chain variable region represented by SEQ ID NO: 12, and a light chain variable region represented by SEQ ID NO: 19.

[0104] Furthermore, anti-API5 antibody 3 has a heavy chain CDR1 represented by SEQ ID NO: 3, a heavy chain CDR2 represented by SEQ ID NO: 4, and a heavy chain CDR3 represented by SEQ ID NO: 8, and has a heavy chain variable region represented by SEQ ID NO: 13, and a light chain variable region represented by SEQ ID NO: 19.

[0105] Furthermore, anti-API5 antibody 4 has a heavy chain CDR1 represented by SEQ ID NO: 3, a heavy chain CDR2 represented by SEQ ID NO: 4, and a heavy chain CDR3 represented by SEQ ID NO: 9, and has a heavy chain variable region represented by SEQ ID NO: 14, and a light chain variable region represented by SEQ ID NO: 19.

[0106] Furthermore, anti-API5 antibody 5 has a heavy chain CDR1 represented by sequence number 3, a heavy chain CDR2 represented by sequence number 5, and a heavy chain CDR3 represented by sequence number 10, and has a heavy chain variable region represented by sequence number 15 and a light chain variable region represented by sequence number 19.

[0107]

[0108] Example 2. Binding activity of anti-API5 antibodies to API5 protein

[0109] To confirm the binding ability of the anti-API5 antibody prepared in Example 1 to API5, the binding kinetics was analyzed.

[0110] Specifically, the binding ability of purified anti-API5 antibodies to API5 protein was analyzed individually using Biacore T200. API5 was immobilized on a sensor chip, and anti-API5 antibodies were used as analytes to measure the association rate constant (ka) and dissociation rate constant (kd) values. The data of dissociation rate constant (kd) and association rate constant (ka) were calculated using Biacore evaluation software, and the equilibrium dissociation constant (KD) was calculated from the ratio of kd and ka. The association rate constant (ka) represents the complex formation rate, i.e., the number of antigen-antibody complexes formed per second in a 1 molar solution of API5 and anti-API5 antibodies. The unit of ka is 1 / Ms. The dissociation rate constant (kd) represents the stability of the complex, i.e., the dissociation rate of the complex per second. The unit of kd is 1 / s. For example, kd=0.01 1 / s means that 1% of the complex disintegrates per second. The equilibrium dissociation constant (KD) calculated from the ratio of kd to ka is 1×10 -9 If the binding affinity of the antibody to the antigen protein is M or less, it is determined that the antibody has excellent binding affinity to the antigen protein.

[0111] [Table 2]

[0112] As a result, as shown in Table 2 above, it was confirmed that all of the anti-API5 antibodies 1 to 5 have excellent binding ability to the API5 protein.

[0113]

[0114] Example 3. Cancer treatment effect of anti-API5 antibody

[0115] Example 3-1. Reduction of phosphorylated ERK (pERK)

[0116] In order to confirm the anti-cancer effect of the anti-API5 antibody prepared in Example 1, the effect of the anti-API5 antibody in reducing ERK activation (i.e., ERK phosphorylation) in cancer cells was analyzed. ERK is mainly activated by growth factors involved in cell proliferation, differentiation, and survival. ERK activation is achieved by phosphorylation of ERK protein, and sustained activation of ERK plays an important role in tumor formation. ERK activation is known to be enhanced in various types of tumors, such as pancreatic cancer, colon cancer, lung cancer, ovarian cancer, kidney cancer, and breast cancer.

[0117] Specifically, TC-1 P3 of non-small cell lung cancer or uterine cancer that is refractory to anti-PD-1 antibody treatment PD-1 Cell line, colon cancer MC38 P3 PD-1 Cell lines, and colon cancer CT26 P3 PD-1 Cell lines were cultured at 1.5 × 10 cells per well in RPMI medium containing 5% FBS. 5 The cells were seeded in a 12-well plate. After 2 hours, the medium was replaced with RPMI medium containing 0.1% FBS, and treated with each anti-API5 antibody at a concentration of up to 150 ng / ml.

[0118] After 24 hours, the cells were washed with 1x PBS buffer and placed in 150 μl of RIPA buffer at 4°C for 10 minutes to induce cell lysis. Then, the cells were centrifuged at 13,000 rpm at 4°C for 15 minutes to obtain cell extracts as supernatants. Proteins were quantified by Bradford assay, and the levels of ERK protein and its phosphorylation levels were measured by SDS-PAGE Western blot for the same amount of protein. The measured ERK protein levels and phosphorylation levels of ERK protein were quantified using Image J (densitometer), and the ratio of phosphorylated ERK protein levels to total ERK protein levels was calculated.

[0119] Here, IgG4 S228P Antibody treatment or no treatment served as control groups (Con).

[0120] "TC-1 P3 PD-1", MC38 P3 PD-1 " or "CT26 P3 PD-1 " refers to cells refractory to anti-PD-1 antibody treatment, obtained by culturing the parent cancer cells "TC-1 P0," "MC38 P0," or "CT26 P0" three times with anti-PD-1 antibody.

[0121] As a result, IgG4 S228P FIG. 1 shows that ERK phosphorylation was significantly reduced in the experimental groups treated with anti-API5 antibodies 1 to 5 according to the invention compared to the antibody-treated control group.

[0122] Furthermore, Figure 2 shows that ERK phosphorylation was significantly reduced in the experimental groups treated with anti-API5 antibodies 1 to 5 compared to the negative control group treated with IgG, resulting in a decrease in the ratio of ERK phosphorylation to total ERK. It was confirmed that such effects were concentration-dependent.

[0123] 3 also shows that in all cancer cell lines, the ERK phosphorylation rate relative to the total ERK in the experimental group treated with anti-API5 antibodies 1 to 5 was reduced to about half compared to the control group not treated with the antibody. These effects were concentration-dependent and similar for all of the exemplified anti-API5 antibodies 1 to 5.

[0124] The above results suggest that the anti-API5 antibody having a specific heavy chain CDR sequence according to the present invention can reduce ERK activation in cancer cells and exhibit excellent anti-cancer effects.

[0125]

[0126] Example 3-2. Increase in apoptosis

[0127] In order to confirm the anti-cancer effect of the anti-API5 antibody prepared in Example 1, the effect of the anti-API5 antibody in increasing apoptosis of cancer cells was analyzed.

[0128] TC-1 P3 prepared in Example 3-1 PD-1Non-small cell lung cancer or cervical cancer cell lines and CT26 P3 PD-1 Colon cancer cell lines were cultured in 12-well plates at 3 × 10 5 Cells were seeded at 1000 x g / well in RPMI medium containing 5% FBS. After 2 hours, the medium was replaced with RPMI medium containing 0.1% FBS and treated with each anti-API5 antibody at a concentration up to 150 ng / ml. After 24 hours, the percentage of apoptotic dead cells was measured using FACS.

[0129] Here, the group not treated with the antibody served as the control group.

[0130] As a result, in the experimental groups treated with anti-API5 antibodies 3 to 5 according to the present invention, apoptosis of cancer cells was increased by about 10 to 20% compared to the control group not treated with the antibodies, and these effects were observed equally in all cancer cell lines, as shown in Figure 4.

[0131] The above results suggest that the anti-API5 antibody having a specific heavy chain CDR sequence according to the present invention can increase the apoptosis sensitivity of cancer cells and exhibit excellent anti-cancer effects.

[0132]

[0133] Example 3-3. Inhibition of tumor growth in vivo

[0134] To confirm the anti-cancer effect of the anti-API5 antibody prepared in Example 1, the extent of tumor growth after administration of the antibody to mice was analyzed.

[0135] In the experiment, B16 P3, which is refractory to anti-PD-1 antibody treatment, PD-1 Melanoma cell line, MC-38 P0 Colon cancer cell line, MC-38 P3 refractory to anti-PD-1 antibody treatment PD-1 Colon cancer cell line, MC-38 P3, refractory to anti-PD-L1 antibody treatment PD-L1 Colon cancer cell line, TC-1 P0 non-small cell lung cancer or uterine cancer cell line, CT26 P0 colon cancer cell line, CT26 P3 refractory to anti-PD-1 antibody treatment PD-1 A colon cancer cell line was used.

[0136] Specifically, 5 × 10 cells were 5 Tumors were formed by transplanting the cells into BALB / c mice. The average tumor size was about 60-75 mm. 3 (30-40mm for MC-38) 3 When the tumor size reached 100 μg / kg, 8 to 9 mice were allocated to each experimental group so that the tumor size was distributed as uniformly as possible, and each antibody was administered. The antibody dose was 200 μg, and the mice were intraperitoneally injected three times a week, a total of five times, starting from the day antibody administration began (day 1).

[0137] Thereafter, the length of the tumor was measured using a caliper at intervals of 2 or 3 days from the day of starting antibody administration, and the tumor size was calculated as follows: tumor size = {(long axis length x short axis length)} 2 Tumor size was calculated using the formula:} / 2. Furthermore, tumor growth inhibition rate (TGI, %) was calculated from tumor size according to the formula: 100-(100 x tumor size in experimental group / tumor size in IgG antibody-treated control group).

[0138] The above results were analyzed using a parametric multiple comparison procedure, assuming normality of the data. If the results of the parametric two-way ANOVA were significant, post-hoc tests were 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 of less than 0.05 was considered statistically significant.

[0139] Here, the group treated with IgG antibody served as the control group.

[0140] As a result, as shown in Figures 5 to 8, tumor growth was significantly inhibited in the experimental group administered with anti-API5 antibodies 1 to 5 according to the present invention. The tumor growth inhibitory effect in the experimental group was increased by about 1.5 to 4 times compared to the control group treated with an IgG antibody, and this effect was demonstrated not only in cancer cells refractory to existing immune anticancer drugs, but also in various cancers such as melanoma, colon cancer, non-small cell lung cancer, and cervical cancer.

[0141] In particular, Figure 9 shows that when the anti-API5 antibody of the present invention is administered in combination with an anti-PD-1 antibody, it exhibits excellent growth inhibitory effects even against cancer cells that are resistant, refractory, or intractable to treatment with the anti-PD-1 antibody, and this effect is synergistic.

[0142] 10 and 11, tumor growth was significantly inhibited in the experimental groups administered with anti-API5 antibodies 1 to 4 according to the present invention. The tumor growth inhibitory effect in the experimental groups was increased by about 1.3 to 2 times compared to the control group treated with an IgG antibody, and the tumor growth inhibition rate (TGI) was increased by 30.1%, 49.6%, 59.2%, and 55.2%, respectively, compared to the control group.

[0143] From the above results, it was revealed that the anti-API5 antibody having a specific heavy chain CDR sequence according to the present invention exhibits excellent anticancer effects, such as inhibiting the growth of cancer cells in vivo. In particular, it is suggested that the antibody can exhibit excellent anticancer effects against cancers that are resistant or intractable to anticancer drugs, and also exhibits excellent synergistic effects with drugs that have been used as immunotherapeutic agents.

Claims

1. An antibody or its antigen-binding fragment that specifically binds to apoptosis inhibitor 5 (API5), Heavy chain CDR1 containing the amino acid sequence GFTFSTYA of SEQ ID NO: 1, Amino acid sequence of SEQ ID NO: 2 ISGSGGX １ T-containing heavy chain CDR2, and Amino acid sequence of SEQ ID NO: 3: AKLVLX ２ WX ３ Contains heavy chain CDR3 including YFSMDH, X １ , X ２ and X ３ An antibody or its antigen-binding fragment is an independent of any amino acid.

2. The antibody or antigen-binding fragment according to claim 1, wherein the heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO:

5.

3. The antibody or antigen-binding fragment according to claim 1, wherein the heavy chain CDR3 comprises any one amino acid sequence selected from the group consisting of SEQ ID NOs: 6 to SEQ ID NOs:

10.

4. The antibody or antigen-binding fragment according to claim 1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region containing any one amino acid sequence selected from the group consisting of SEQ ID NOs: 11 to 15.

5. The antibody or antigen-binding fragment according to claim 1, wherein the antibody or antigen-binding fragment comprises a light chain CDR1 containing the amino acid sequence of SEQ ID NO: 16, a light chain CDR2 containing the amino acid sequence of SEQ ID NO: 17, and a light chain CDR3 containing the amino acid sequence of SEQ ID NO: 18, wherein the antibody or antigen-binding fragment is not one in which X1 is L, X2 is E, and X3 is F.

6. The antibody or antigen-binding fragment according to claim 1, wherein the antibody or antigen-binding fragment includes a light chain variable region containing the amino acid sequence of SEQ ID NO:

19.

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

8. A vector comprising the nucleic acid described in claim 7.

9. Cells transformed with the vector described in claim 8.

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

11. The pharmaceutical composition according to claim 10, wherein the cancer is resistant to or refractory to anticancer drugs.

12. The pharmaceutical composition according to claim 11, wherein the anticancer agent is one or more selected from the group consisting of anticancer chemotherapy agents, anticancer targeted agents, and anticancer immunotherapy agents.

13. The pharmaceutical composition according to claim 11, wherein the cancer is selected from the group consisting of lung cancer, non-small cell lung cancer, colorectal cancer, rectal cancer, proximal anal cancer, colon cancer, small intestine cancer, breast cancer, uterine cancer, ovarian cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, head cancer, cervical cancer, thyroid cancer, parathyroid cancer, gastric cancer, liver cancer, pancreatic cancer, bone cancer, skin cancer, melanoma of the skin or eye, 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.

14. A composition for cancer diagnosis comprising the antibody or antigen-binding fragment described in claim 1.

15. A kit for cancer diagnosis comprising the antibody or antigen-binding fragment described in claim 1.

16. A method for providing information for cancer diagnosis, A method comprising detecting the API5 protein in a biological sample isolated from a subject suspected of having cancer, or determining the expression level of the API5 protein, by an antigen-antibody reaction using the antibody or antigen-binding fragment described in claim 1.

17. A screening method for anticancer drugs, (a) A step of treating cancer cells with a candidate anticancer drug, (b) A step of measuring the expression level of the API5 protein in cancer cells treated with the candidate anticancer drug using the antibody or antigen-binding fragment described in claim 1, and A method for screening anticancer drugs, comprising: (c) determining that the anticancer drug candidate treated in step (a) is an anticancer drug if the expression level of the API5 protein in step (b) is lower than that of cancer cells not treated with the anticancer drug candidate.

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