Method for Activating T Cells for Cancer Treatment

Abstract

The present invention relates to a cancer-specific neoepitope represented by any one of SEQ ID NOs: 1 to 214, an antigen-presenting cell loaded with the neoepitope, and a method for activating T cells for cancer treatment using the antigen-presenting cell. The present invention provides an antigen-presenting cell loaded with a cancer-specific epitope, i.e., a dendritic cell, which can rapidly and effectively induce the differentiation and proliferation of cancer antigen-specific T cells, preferably memory T cells. The activated memory T cells can evade the defense mechanisms of cancer cells to treat cancer or neoplastic conditions or prevent the recurrence, progression, or metastasis of cancer. [Selected Figure] Figure 1

Description

Technical Field

[0001] The present invention relates to cancer-specific neo-epitopes, antigen-presenting cells loaded with the neo-epitopes, and a method for activating T cells for cancer treatment by the antigen-presenting cells.

Background Art

[0002] The onset of gastric cancer is known as a malignant tumor with a high incidence rate globally, especially in Asia. Although various causes of gastric cancer have been known, typically, it can be classified into Epstein-Barr virus (EBV)-associated gastric cancer that appears due to EBV infection and gastric cancer cell antigen-related gastric cancer that occurs due to the accumulation of genetic mutations in gastrointestinal cells. Currently, for the treatment of gastric cancer, surgical resection of cancer tissue has been known to be the most effective for a long time, and chemotherapy and radiotherapy are also performed. However, it is a disease that is difficult to be completely cured if gastric cancer cannot be detected at an early stage. In addition, clinical trials have been carried out with various biological agents (antibodies, small molecules), but no therapeutic agent showing good clinical effects has appeared yet.

[0003] Recently, cancer cell-specific targeted treatment methods using patient-derived autologous T cells have been studied at various institutions. As a result of conducting clinical trials on lymphoma using chimeric antigen receptor (CAR) T cells at various institutions, it has attracted attention as a new field for anti-cancer treatment due to good clinical effects and low side effects.

[0004] When patient-derived T cells are used, the induction of immune reactions, which is the most significant side effect of cell therapy agents, is reduced, and the restriction on the donor's HLA type is eliminated. Therefore, it has been regarded as an effective and side effect-free therapeutic agent. So far, the types of cell therapy agents most commonly used in the field of anti-cancer treatment include CD8+ T cells, CD4+ T cells, NK cells, dendritic cells, and CAR T cells. In the case of NK cells, although they have cell-killing efficacy, they have no antigen specificity, so they have various side effects. In the case of dendritic cells, they do not have a function to directly kill cells, but are therapeutic agents based on the vaccine concept that can transfer antigen specificity to T cells in the patient's body and confer cancer cell specificity on T cells with high efficiency. In addition, CD4+ T cells have a role of assisting other cells due to their antigen specificity, and in the case of CD8+ T cells, they are known as cells with the best antigen specificity and cell-killing effect.

[0005] However, most of the cell therapy agents currently in use or under development have limitations, and the current situation is that no clinical effects have appeared. Looking at those limitations, cancer cells either secrete substances that suppress the immune reaction in the human body or cannot present antigens essential for the production of antibodies against cancer cells, thus preventing an appropriate immune reaction from occurring.

[0006] On the one hand, dendritic cells not only play the role of monitors that detect antigens entering from outside the human body or generated inside the body, but also act as specialized antigen-presenting cells that rapidly move to immune organs (secondary lymphoid organs) with the antigens thus recognized and absorbed, and present the antigens to immune cells including T cells that react with the antigens. In the case of anti-cancer immunotherapy vaccines using dendritic cells, they have been developed by various methods, and are roughly divided into ex vivo generated dendritic cell vaccines and in vivo dendritic cell vaccines. In the case of in vivo dendritic cell vaccines, it is a method of directly transmitting antigens to dendritic cells present in the body. In the case of the ex vivo generated dendritic cell vaccine method, dendritic cells are separated from the patient's PBMC, the antigen to be presented to the separated dendritic cells is transmitted, and after the dendritic cells are activated thereby, they are further injected into the patient, and the antigen is transmitted from the injected dendritic cells to T cells. In this case, the method of culturing dendritic cells and the method of antigen transmission in vitro are important. As the currently used antigen presentation methods, the DNA of the antigen to be presented is transfected using a virus or nucleofection, or an antigen is bound to an antibody targeting dendritic cells to transmit the antigen targeting dendritic cells.

[0007] Currently, the most significant problem with dendritic cell vaccines is that it is extremely difficult to activate effective anti-cancer immune cells in the cancer microenvironment where there are severe chronic inflammatory phenomena in the body, the Warburg effect, immunosuppressive cytokines, immunosuppressive T cells, and dendritic cells.

Summary of the Invention

Problems to be Solved by the Invention

[0008] One object of the present invention is to provide an Epstein-Barr virus (EBV)-negative cancer-specific neoepitope and a composition for activating T cells containing the same.

[0009] Another object of the present invention is to provide an antigen-presenting cell loaded with the neoepitope of the present invention, which can activate T cells for cancer treatment.

[0010] Still another object of the present invention is to provide T cells activated by antigen-presenting cells loaded with the neoepitope of the present invention.

[0011] Still another object of the present invention is to provide a method for activating T cells for cancer treatment.

[0012] However, the technical problems that the present invention intends to solve are not limited to the problems described above, and other problems not described will be clearly understood by those with ordinary knowledge in the art from the following description.

Means for Solving the Problems

[0013] According to one embodiment of the present invention, it relates to a cancer-specific epitope.

[0014] In the present invention, the "cancer-specific epitope" is derived from a mutant protein antigen that exists only in cancer cells and not in normal cells. In the present invention, the cancer-specific epitope includes at least one epitope recognized by a T cell receptor, and preferably, it may be a neo-epitope of an auto-cancer antigen that appears due to a mutation of a cancer gene in an Epstein-Barr virus (EBV)-negative cancer.

[0015] In the present invention, the "neo-epitope" refers to an epitope that does not exist in a reference such as normal non-cancer cells or germ cells, but is found in cancer cells. Here, in particular, although the corresponding epitope is found in normal non-cancer cells or germ cells, situations where the sequence of the epitope changes due to one or more mutations in cancer cells, resulting in the generation of a neo-epitope, are also included. In relation to neo-epitopes, neo-epitopes are considered to express random mutations in tumor cells that generate unique tumor-specific antigens. Thus, from another perspective, neo-epitopes can be identified by considering the types (e.g., deletions, insertions, conversions, transpositions, translocations) and effects (e.g., non-sense, missense, frameshift, etc.) of mutations that can act as a first content filter to remove silent and other non-related mutations. Also, the neo-epitope sequence is defined as a sequence extension of relatively short length (e.g., 7-11mer), where it should be understood that such an extension includes a change in the amino acid sequence. Most typically, the changed amino acid should be at or near the central amino acid position. For example, a typical neo-epitope can have a structure of A4-N-A4, or A3-N-A5, or A2-N-A7, or A5-N-A3, or A7-N-A2, where A is a protein-generating amino acid and N is the changed amino acid (relative to the wild type or the matched normal group). For example, the neo-epitope sequences contemplated in the present application include sequence extensions of relatively short length (e.g., 5-30mer, more typically 7-11mer or 12-25mer), where such extensions include a change in the amino acid sequence. Thus, it should be understood that a single amino acid change can be presented in multiple neo-epitope sequences containing the changed amino acid, depending on the position of the changed amino acid. Advantageously, such sequence variability allows for the multiplex selection of neo-epitopes, increasing the number of potentially useful targets that can be selected based on one or more favorable traits (e.g., the highest affinity for the patient HLA-type, the highest structural stability, etc.).Most typically, such neoepitopes are calculated to have a length of 2 to 50 amino acids, more typically 5 to 30 amino acids, and most typically 9 to 15 amino acids, and the changed amino acids are preferably located centrally or, otherwise, in a manner that improves binding to MHC. For example, when the epitope is provided by the MHC-I complex, the typical length of a neoepitope is about 8 to 11 amino acids, and the typical length of a neoepitope presented via the MHC-II complex should have about 13 to 17 amino acids. As can be easily understood, since the position of the changed amino acids in the neoepitope is not central, the actual peptide sequence and the actual phase of the neoepitope can be very different.

[0016] In the present invention, as the neoepitope, it can show binding affinity to at least one of the HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, β2-microglobulin, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA1, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DM, HLA-DOA, and HLA-DOB gene loci so that T cells extracted from human blood, preferably memory T cells, can have efficacy. Among them, as the HLA types most highly expressed in Koreans, for example, at least one of HLA-A*2402, HLA-A*A0201, HLA-A*3303, HLA-A*1101, HLA-A*0206, HLA-A*3101, HLA-B*5101, HLA-B*4403, HLA-B*5401, HLA-B*5801, and HLA-B*3501, preferably those showing high binding affinity to HLA-A*2402 or HLA-A*A0201 may be used.

[0017] Preferably, in the present invention, the neoepitope has a high binding affinity for HLA-A*2402 and may be a neoepitope represented by any one of SEQ ID NOs: 1 to 49; or has a high binding affinity for HLA-A*0201 and may be a neoepitope represented by any one of SEQ ID NOs: 50 to 214.

[0018] However, in the present invention, as a method for measuring the neoepitope-HLA affinity, NetMHC3.4 (URL: www.cbs.dtu.dk / services / NetMHC-3.4 / ) can be used to predict whether the neoepitope binds to a specific HLA allele, but it is not limited thereto.

[0019] In the present invention, the "HLA" or "human leukocyte antigen" represents a human gene that encodes an MHC (major histocompatibility complex) protein on the surface of cells that cause immune system regulation. "HLA-I" or "HLA class I" represents a human MHC class I gene including the HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, and β2-microglobulin loci. "HLA-II" or "HLA class II" represents a human MHC class II gene including the HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA1, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DM, HLA-DOA, and HLA-DOB loci.

[0020] In the present invention, the cancer includes, without limitation, Epstein-Barr virus (EBV)-negative cancers that express a neoepitope represented by any one of SEQ ID NOs: 1 to 214 of the present invention, and the type thereof is not particularly limited. For example, it may be colorectal cancer, pancreatic cancer, gastric cancer, liver cancer, breast cancer, cervical cancer, thyroid cancer, parathyroid cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, bile duct cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, blood cancer, bladder cancer, kidney cancer, ovarian cancer, melanoma, colon cancer, bone cancer, skin cancer, head cancer, uterine cancer, rectal cancer, brain tumor, cancer near the anus, trumpet duct carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, or pituitary adenoma, and preferably, it may be gastric cancer.

[0021] According to another embodiment of the present invention, it relates to a nucleic acid molecule encoding a neoepitope of an Epstein-Barr virus (EBV)-negative cancer antigen, which is a cancer-specific epitope provided by the present invention, and preferably, is represented by any one of SEQ ID NOs: 1 to 214.

[0022] The nucleic acid molecule of the present invention includes all of the nucleic acid molecules obtained by translating the amino acid sequence of the polypeptide provided by the present invention into a polynucleotide sequence as known to those skilled in the art. Therefore, various polynucleotide sequences based on the open reading frame (ORF) can be produced, and all of these are also included in the nucleic acid molecule of the present invention.

[0023] According to still another embodiment of the present invention, an expression vector into which the isolated nucleic acid molecule provided by the present invention is inserted is provided.

[0024] In the present invention, the "vector" is a nucleic acid molecule capable of transporting another nucleic acid molecule to which a certain nucleic acid molecule is ligated. One type of vector is a "plasmid" which refers to a circular double-stranded DNA to which an additional DNA segment can be ligated. Another type of vector is a phage vector. Still another type of vector is a viral vector, to which an additional DNA segment can be ligated to the viral genome. Some vectors can replicate autonomously in the host cells into which they have flowed (for example, a bacterial vector is an episomal mammalian vector having a bacterial origin of replication). Other vectors (for example, non-episomal mammalian vectors) integrate into the genome of the host cell while flowing into the host cell, and thereby are replicated together with the host genome. In addition, some vectors can direct the expression of genes to which they are operably ligated. Such vectors are named "recombinant expression vectors" or simply "expression vectors" in the present application. Generally, expression vectors useful in recombinant DNA techniques often exist in the form of plasmids. In this specification, "plasmid" and "vector" can be used interchangeably because the plasmid is the most commonly used form of the vector.

[0025] In the present invention, specific examples of the expression vector include the pCDNA vector, F, R1, RP1, Col, pBR322, ToL, Ti vector, which are widely used commercially; cosmids; phages such as lambda, lambdoid, M13, Mu, p1 P22, Qμ, T-even, T2, T3, T7; and plant viruses. However, the present invention is not limited thereto, and all expression vectors known to those skilled in the art as expression vectors can be used in the present invention. When selecting an expression vector, it depends on the properties of the target host cell. When introducing the vector into the host cell, it can be carried out by calcium phosphate transfection, virus infection, DEAE-dextran-mediated transfection, lipofectamine transfection, or electroporation, but is not limited thereto. Those skilled in the art can select and use an introduction method suitable for the expression vector and host cell to be used. Preferably, the vector contains one or more selection markers, but is not limited thereto. It is possible to select by the presence or absence of the production of the product using a vector without a selection marker. The selection of the selection marker is determined by the target host cell, and since methods already known to those skilled in the art are used, the present invention is not limited thereto.

[0026] In order to facilitate the purification of the nucleic acid molecule of the present invention, a tag sequence can be inserted and fused onto the expression vector. Examples of the tag include, but are not limited to, a hexa-histidine tag, a hemagglutinin tag, a myc tag, or a flag tag. All tags known to those skilled in the art that facilitate purification can be used in the present invention.

[0027] According to still another embodiment of the present invention, there is provided a host cell transfected with the expression vector provided by the present invention.

[0028] In the present invention, the "host cell" includes a recipient of a vector for incorporation of a polypeptide insert, or an individual cell or cell culture that was a recipient. Host cells include the progeny of a single host cell, and such progeny may not necessarily be identical (either morphologically or in genomic DNA complement) to the original parent cell due to natural, accidental or deliberate mutations. Host cells include cells into which the polypeptide of the present application has been transfected in vivo.

[0029] In the present invention, the host cell can include cells of mammalian, plant, insect, fungal, or cellular origin, for example, bacterial cells such as Escherichia coli, Streptomyces, Salmonella typhimurium; fungal cells such as yeast cells, Pichia pastoris; insect cells such as Drosophila, Spodoptera Sf9 cells; animal cells such as CHO (Chinese hamster ovary cells), SP2 / 0 (mouse myeloma), Human lymphoblastoid, COS, NSO (mouse myeloma), 293T, BOW melanoma cells, HT-1080, BHK (Baby Hamster Kidney cells), HEK (Human Embryonic Kidney cells), or PERC.6 (human retinal cells); or plant cells, but is not limited thereto, and all cells that can be used as host cells known to those skilled in the art are available.

[0030] According to still another embodiment of the present invention, it relates to a composition for activating T cells, which comprises a cancer-specific epitope provided by the present invention, a nucleic acid molecule encoding the same, an expression vector into which the nucleic acid molecule has been inserted, or a host cell transformed with the expression vector.

[0031] As used herein, "activation of T cells" refers to a monoclonal (e.g., encoding the same TCR) or polyclonal (e.g., having clones encoding different TCRs) T cell population having a T cell receptor that recognizes at least one tumor antigen peptide. The activated T cells can contain one or more subtypes of T cells including, but not limited to, one or more selected from the group consisting of cytotoxic T cells, helper T cells, natural killer T cells, γδ T cells, regulatory T cells, and memory T cells, and preferably may be memory T cells.

[0032] In the present invention, the activated T cells can avoid the defense mechanism of cancer cells and treat cancer or tumor states, or prevent cancer recurrence, progression or metastasis.

[0033] According to still other embodiments of the present invention, the cancer-specific epitope provided by the present invention may be an antigen-presenting cell (APC) loaded with a tumor antigen epitope.

[0034] In the present invention, the antigen-presenting cells can include one or more of dendritic cells (DC), B cells, and macrophages, and preferably may be dendritic cells.

[0035] In the present invention, the "dendritic cells" are members of a diverse population of morphologically similar cell types found in lymphoid or non-lymphoid tissues. Such cells are characterized by their unique morphology and high expression levels of surface class I and class II MHC molecules, which are proteins that present antigen peptides to T cells. DCs, other APCs, and T cells can conveniently be isolated from peripheral blood and from a number of tissue sources, or can be derived (e.g., differentiated), such as from peripheral blood mononuclear cells (PBMCs) derived from peripheral blood.

[0036] In the present invention, the antigen-presenting cell induces the differentiation and proliferation of cancer antigen-specific T cells, preferably memory T cells, to avoid the cancer cell defense mechanism and treat cancer or tumor states, or prevent cancer recurrence, progression, or metastasis.

[0037] According to still another embodiment of the present invention, it relates to a cancer-specific epitope provided by the present invention; and a fusion protein containing a dendritic cell-specific antibody or a fragment thereof.

[0038] The fusion protein provided by the present invention enables the cancer-specific epitope provided by the present invention to be loaded onto dendritic cells.

[0039] In the present invention, the dendritic cell-specific antibody may be an antibody specific for DCIR, MHC class I, MHC class II, CD1, CD2, CD3, CD4, CD8, CD11b, CD14, CD15, CD16, CD19, CD20, CD29, CD31, CD40, CD43, CD44, CD45, CD54, CD56, CD57, CD58, CD83, CD86, CMRF-44, CMRF-56, DCIR, DC-ASPGR, CLEC-6, CD40, BDCA-2, MARCO, DEC-205, Clec9A, 33D1, mannose receptor, Langerin, DECTIN-1, B7-1, B7-2, IFN-γ receptor, IL-2 receptor, ICAM-1, Fcγ receptor, LOX-1, or ASPGR of dendritic cells, but is not limited thereto.

[0040] In the fusion protein of the present invention, the cancer-specific epitope may be conjugated to the dendritic cell-specific antibody or a fragment thereof. Here, the "conjugate" refers to any substance formed by binding two parts together. Representative conjugates according to the present invention include those formed by binding an antigen, an antibody, and a TLR agonist together. The term "conjugation" refers to the process of forming a conjugate, and generally refers to physical coupling, such as covalent bonding, simultaneous-coordination covalent bonding, or a secondary binding force, such as Van der Waals bonding force. The step of linking an antigen to an antibody is further carried out by non-covalent association such as dockerin-cohesin association [(related to the part cited as a reference in U.S. Patent Publication No. 20100135994 by (Banchereau) et al.)], or by direct chemical linkage by forming a peptide or chemical bond.

[0041] According to still another embodiment of the present invention, there is provided a method for producing antigen-presenting cells (APCs) loaded with the cancer-specific epitope provided by the present invention.

[0042] In the present invention, the antigen-presenting cells can include one or more of dendritic cells, B cells, and macrophages, but preferably may be dendritic cells.

[0043] In the present invention, the dendritic cells (e.g., immature dendritic cells) can be obtained from various sources including self-sources, i.e., from the individual of interest. Preferably, they are obtained from peripheral blood mononuclear cells (PBMC) derived from peripheral blood. More preferably, monocytes are separated from the individual-derived PBMC and contacted with a plurality of cytokines to obtain them. Here, the types of cytokines that induce the differentiation of the monocytes into dendritic cells are not particularly limited, and for example, they can include one or more of GM-CSF and IL-4.

[0044] In the present invention, the "individual of interest" means an individual who has developed cancer or has a high likelihood of developing cancer.

[0045] In the present invention, when the antigen-presenting cells are prepared as described above, the cancer-specific epitopes of the present invention can be loaded onto the antigen-presenting cells. Generally, immature dendritic cells capture antigens by phagocytosis or receptor-mediated endocytosis, process the antigens through a series of intracellular processes, load antigen peptides onto MHC, and present them to T lymphocytes. Along with the process of processing antigens, dendritic cells gradually mature, lose the receptors used for phagocytosis and endocytosis, increase the expression of MHC class I, II, co-stimulatory molecules, and cell adhesion molecules, express new chemokine receptors, and migrate to areas rich in T lymphocytes in the peripheral lymph nodes to present antigens to T lymphocytes, thereby triggering a T lymphocyte immune response.

[0046] As an example of the present invention, in order to load the cancer-specific epitope onto the antigen-presenting cell, the antigen-presenting cell can be brought into contact with the cancer-specific epitope of the present invention. Preferably, as the antigen-presenting cell, for example, immature dendritic cells, or antigen-presenting cells (e.g., dendritic cells) contained in or derived from (e.g., differentiated) PBMC can be pulsed with the cancer-specific epitope of the present invention. As is known in the art, pulsing refers to the process of mixing a cell, e.g., a dendritic cell, with a solution containing an antigen peptide and then optionally removing the antigen peptide from the mixture. In the present invention, when the immature dendritic cells are brought into contact with the cancer-specific epitope, a Toll-like receptor agonist can be used to additionally induce the maturation of the population of immature dendritic cells. At this time, exemplary TLR agonists include, but are not limited to, Poly IC, MALP, and R848.

[0047] As another example of the present invention, in order to load the cancer-specific epitope onto the antigen-presenting cell, the antigen-presenting cell can be nucleofected with an expression vector, preferably a plasmid, into which a nucleic acid molecule encoding the cancer-specific epitope has been inserted. Here, the nucleofection can be carried out by any useful means in the art, including, for example, the Amaxa® nucleofection system or the InVitrogen® nucleofection system.

[0048] As yet another example of the present invention, loading the cancer-specific epitope onto the antigen-presenting cell is performed using the cancer-specific epitope provided by the present invention; and a fusion protein comprising a dendritic cell-specific antibody or a fragment thereof.

[0049] According to still other embodiments of the present invention, it relates to T cells activated by the antigen-presenting cells provided by the present invention.

[0050] In the present invention, the T cells refer to a monoclonal (e.g., encoding the same TCR) or polyclonal (e.g., having clones encoding different TCRs) T cell population having a T cell receptor that recognizes tumor antigen peptides, and include one or more selected from the group consisting of cytotoxic T cells, helper T cells, natural killer T cells, γδ T cells, regulatory T cells, and memory T cells, and may contain one or more subtypes of T cells, but are not limited thereto. Preferably, they may be memory T cells.

[0051] In the present invention, the "memory T cells" are T cells differentiated from T cells that have encountered and reacted with these specific antigens before or activated T cells. Tumor-specific memory T cells constitute a small part of the total T cell population, but they perform an important function in monitoring tumor cells during an individual's entire life. When tumor-specific memory T cells encounter tumor cells expressing these specific tumor antigens, the memory T cells are immediately activated and clonally expanded. The activated and proliferated T cells differentiate into effector T cells and kill tumor cells with high efficiency. Memory T cells are important for establishing and maintaining the organ tumor antigen-specific response of T cells. In the present invention, activated T cells, preferably activated memory T cells, can specifically recognize the antigens of cancer cells and treat cancer or tumor states by avoiding the defense mechanism of cancer cells, or prevent cancer recurrence, progression, or metastasis.

[0052] According to still another embodiment of the present invention, it relates to a method for activating T cells by the antigen-presenting cells provided by the present invention.

[0053] In the present invention, for the activation of the T cells, the T cells can be co-cultured with antigen-presenting cells loaded with the cancer-specific epitopes of the present invention.

[0054] In the present invention, the T cells can be obtained from a variety of sources, including self-sources, i.e., from the individual of interest. Preferably, they are obtained from peripheral blood mononuclear cells (PBMCs) derived from peripheral blood, and more preferably, from the non-adherent portion of the PBMCs. As an example of the present invention, the non-adherent portion of the PBMCs can be obtained by density gradient centrifugation of a peripheral blood sample, and this can be obtained by culturing it with at least one cytokine (e.g., IL-2) in the presence or absence of an anti-CD3 antibody (e.g., OKT3).

[0055] In the present invention, the T cells refer to a monoclonal (e.g., encoding the same TCR) or polyclonal (e.g., having clones encoding different TCRs) T cell population having a T cell receptor that recognizes a tumor antigen peptide, and can include one or more selected from the group consisting of cytotoxic T cells, helper T cells, natural killer T cells, γδ T cells, regulatory T cells, and memory T cells, and can contain one or more subtypes of T cells, but are not limited thereto. Preferably, they may be memory T cells.

[0056] Also, in the present invention, the T cells and the antigen-presenting cells can be derived from the same individual, for example, an individual suffering from cancer (e.g., low-grade to intermediate-grade cancer), but are not limited thereto.

[0057] In the present invention, for the activation of the T cells, the T cells are co-cultured with the antigen-presenting cells of the present invention for any one or more of 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 days, preferably for 1 to 21 days, 1 to 14 days, 2 to 10 days, 2 to 5 days, 2 to 5 days, 3 days, 5 days, 7 days, 10 days, 14 days, 16 days, 18 days, or 21 days, but are not limited thereto.

[0058] In the present invention, when co-culturing the T cells with the antigen-presenting cells of the present invention, one or more cytokines can be added to promote the activation, maturation and / or proliferation of the T cells and prime the T cells for subsequent differentiation into memory T cells. Exemplary cytokines that can be used in such steps include, but are not limited to, interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-7 (IL-7), interleukin-15 (IL-15), interleukin-21 (IL-21), or combinations thereof.

[0059] Also, in the present invention, when co-culturing the T cells with the antigen-presenting cells of the present invention, a fusion protein containing a cytokine and an immunoglobulin heavy chain constant region can be added to promote the activation, maturation and / or proliferation of the T cells and prime the T cells for subsequent differentiation into memory T cells. Here, the cytokine may be interferon-γ (IFN-γ), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-12 (IL-12), interleukin-18 (IL-18), tumor necrosis factor (TNF), or granulocyte macrophage colony-stimulating factor (GMCSF), but is not limited thereto. Further, the immunoglobulin heavy chain constant region may be an immunoglobulin heavy chain constant region selected from the group consisting of an immunoglobulin hinge region and optionally a CH2 domain, a CH3 domain, and a CH4 domain or a combination thereof, but is not limited thereto. Furthermore, the immunoglobulin heavy chain constant region can be derived from an immunoglobulin belonging to any of the five immunoglobulin classes known in the art, namely IgA (Igα), IgD (Igδ), IgE (Igε), IgG (Igγ), and IgM (Igμ), and preferably may be an immunoglobulin heavy chain constant region derived from an immunoglobulin of the IgG class.

[0060] In addition, in the present invention, when co-culturing the T cells with the antigen-presenting cells of the present invention, a ligand that binds to a cell surface protein highly expressed in memory T cells; and a fusion protein containing an immunoglobulin heavy chain constant region are added to promote the activation, maturation, and / or proliferation of T cells, and prime the T cells for subsequent differentiation into memory T cells. Here, the cell surface protein highly expressed in the memory T cells may be CD27, CXCR3, or CD62L. The ligand that can bind to CD27 may be CD70, the ligand that can bind to CXCR3 may be CXCR9 or CXCR10, and the ligand that can bind to CD62L may be GlyCAM-1, CD34, MadCAM-1, or PSGL-1, but is not limited thereto. Furthermore, the immunoglobulin heavy chain constant region is derived from an immunoglobulin belonging to any five immunoglobulin classes known in the art, such as IgA (Igα), IgD (Igδ), IgE (Igε), IgG (Igγ), and IgM (Igμ), and preferably may be an immunoglobulin heavy chain constant region derived from an immunoglobulin of the IgG class.

[0061] According to still another embodiment of the present invention, it relates to an immunotherapeutic agent containing, as an active ingredient, antigen-presenting cells loaded with the cancer-specific epitope provided by the present invention. The immunotherapeutic agent according to the present invention can increase the immune response or selectively increase a part of the immune response favorable for the treatment or prevention of a specific disease, for example, cancer.

[0062] According to still another embodiment of the present invention, it relates to an anti-cancer vaccine and a pharmaceutical composition for the prevention or treatment of cancer, containing, as an active ingredient, antigen-presenting cells loaded with the cancer-specific epitope provided by the present invention; and / or activated T cells.

[0063] The antigen-presenting cell provided by the present invention can induce the differentiation and proliferation of cancer antigen-specific T cells, preferably memory T cells. The memory T cells activated in this way can avoid the cancer cell defense mechanism and treat cancer or tumor states, or prevent cancer recurrence, progression or metastasis.

[0064] In the present invention, the "cancer" refers to or indicates a physiological state characterized by unregulated cell growth typically in mammals. In the present invention, cancers to be targeted for prevention, improvement or treatment are Epstein-Barr virus (EBV)-negative cancers, and are inclusively and unrestrictedly cancers expressing neoepitopes represented by any one of SEQ ID NOs: 1 to 214 of the present invention, and the types thereof are not particularly limited. For example, they may be colon cancer, pancreatic cancer, gastric cancer, liver cancer, breast cancer, cervical cancer, thyroid cancer, parathyroid cancer, non-pharyngeal cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, bile duct cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, blood cancer, bladder cancer, kidney cancer, ovarian cancer, melanoma, colon cancer, bone cancer, skin cancer, head cancer, uterine cancer, rectal cancer, brain tumor, cancer near the anus, fallopian tube cancer, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer, renal cell carcinoma, renal pelvis cancer, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, or pituitary adenoma, etc., and preferably may be gastric cancer, but is not limited thereto.

[0065] In the present invention, "prevention" can inclusively and unrestrictedly include any act of blocking cancer symptoms, or suppressing or delaying those symptoms using the pharmaceutical composition of the present invention.

[0066] Also, in the present invention, "treatment" can inclusively and unrestrictedly include any act in which cancer symptoms improve or become advantageous using the pharmaceutical composition of the present invention.

[0067] In the present invention, the pharmaceutical composition is characterized by being in the form of a capsule, tablet, granule, injection, ointment, powder, or beverage, and the pharmaceutical composition can be characterized by being targeted at humans.

[0068] In the present invention, the pharmaceutical composition can be formulated into various dosage forms such as powders, granules, capsules, tablets, oral dosage forms such as aqueous suspensions, external preparations, suppositories, and sterile injection solutions by conventional methods, although not limited thereto. The pharmaceutical composition of the present invention can contain a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers can include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, dyes, flavors, etc. for oral administration, and buffers, preservatives, soothing agents, solubilizers, isotonic agents, stabilizers, etc. can be mixed and used for injections, and bases, excipients, lubricants, preservatives, etc. can be used for topical administration. The dosage forms of the pharmaceutical composition of the present invention can be manufactured in various ways by mixing with pharmaceutically acceptable carriers as described above. For example, for oral administration, it can be manufactured in the form of tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc., and for injections, it can be manufactured in the form of unit-dose ampoules or multiple-dose forms. Additionally, it can be formulated into solutions, suspensions, tablets, capsules, sustained-release preparations, etc.

[0069] On the other hand, examples of carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, amorphous cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil, etc. Additionally, fillers, anticoagulants, lubricants, wetting agents, flavors, emulsifiers, preservatives, etc. can be further included.

[0070] In the present invention, the administration route of the pharmaceutical composition is not limited thereto, and includes oral, intravenous, intramuscular, intra-arterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal. Oral or parenteral administration is preferred.

[0071] In the present invention, the term "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intradural, intralesional, and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention is further administered in the form of a suppository for rectal administration.

[0072] The pharmaceutical composition of the present invention can vary widely depending on various factors including the activity of the specific compound used, age, body weight, general health, sex, formulation, administration time, administration route, excretion rate, drug combination, and the severity of the specific disease to be prevented or treated. The dosage of the pharmaceutical composition varies depending on the patient's condition, body weight, degree of disease, drug form, administration route, and duration, but can be appropriately selected by those skilled in the art and can be administered at 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg per day. Administration may be once a day or divided into several times. The dosage does not limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention is formulated into tablets, dragees, capsules, solutions, gels, syrups, slurries, suspensions.

[0073] According to another embodiment of the present invention, there is provided a method for preventing or treating cancer, comprising the step of administering to a subject in need thereof antigen-presenting cells loaded with a cancer-specific epitope provided by the present invention; and / or activated T cells.

[0074] The dosage, schedule, and administration route of the antigen-presenting cells loaded with the cancer-specific epitope provided by the present invention or activated T cells can be determined depending on the size and condition of the subject and according to standard pharmaceutical practice. Exemplary administration routes include intravenous, intra-arterial, intraperitoneal, intralung, intravascular, intramuscular, intra-organ, subcutaneous, intraocular, intrathecal, or transdermal.

[0075] The dosage of cells administered to an individual varies, for example, depending on the specific type of cells administered, the route of administration, and the specific type and stage of the cancer being treated. The amount must be sufficient to bring about a desired response, such as a therapeutic response to cancer, without severe toxicity or adverse cases. In some embodiments, the amount of activated T cells or antigen-presenting cells (e.g., dendritic cells) administered is a therapeutically effective amount. In some embodiments, the amount of cells (e.g., dendritic cells or activated T cells loaded with cancer-specific epitopes) is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% less in terms of tumor size, number of cancer cells, or tumor growth rate compared to the corresponding tumor size, number of cancer cells, or tumor growth rate in the same individual before treatment, or compared to the corresponding activity in other untreated individuals. The magnitude of the effect can be measured using standard methods such as in vitro assays using purified enzymes, cell-based assays, animal models, or human experiments.

[0076] In one embodiment of the present invention, the antigen-presenting cells (e.g., dendritic cells) loaded with the cancer-specific epitopes of the present invention are 1×10 5 ~5×10 5 、5×10 5 ~1×10 6 、1×10 6 ~2×10 6 、2×10 6 ~3×10 6 、3×10 6 ~4×10 6 、4×10 6 ~5×10 6 、5×10 6 ~6×10 6 、6×10 6 ~7×10 6 、7×10 6 ~8×10 6 、8×10 6 ~1×10 8 、1×106 ~3×10 6 、3×10 6 ~5×10 6 、5×10 6 ~7×10 6 、2×10 6 ~4×10 6 、1×10 6 ~5×10 6 、または5×10 6 ~1×10 7 administered at a dose of any one of the cells / individual, but not limited thereto.

[0077] In another embodiment of the present invention, the antigen-presenting cells (e.g., dendritic cells) loaded with the cancer-specific epitope of the present invention are 1×10 4 ~5×10 4 、5×10 4 ~1×10 5 、1×10 5 ~2×10 5 、2×10 5 ~4×10 5 、4×10 5 ~6×10 5 、6×10 5 ~8×10 5 、8×10 5 ~1×10 6 、1×10 6 ~2×10 6 、2×10 6 ~1×10 7 、1×10 4 ~1×10 5 、1×10 5 ~1×10 6 、1×10 6 ~1×10 7 、1×10 4 ~1×10 6 、または1×10 5 ~1×10 7 administered at a dose of any one of the cells / kg, but not limited thereto.

[0078] Also, in one embodiment of the present invention, the activated T cells of the present invention are 1×10 8 ~5×108 , 5×10 8 ~9×10 8 , 9×10 8 ~1×10 9 , 1×10 9 ~2×10 9 , 2×10 9 ~3×10 9 , 3×10 9 ~4×10 9 , 4×10 9 ~5×10 9 , 5×10 9 ~6×10 9 , 6×10 9 ~1×10 10 , 1×10 9 ~3×10 9 , 3×10 9 ~5×10 9 , 5×10 9 ~7×10 9 , 7×10 9 ~1×10 10 , 1×10 9 ~5×10 9 , 5×10 9 ~1×10 10 , 3×10 9 ~7×10 9 , 1×10 10 ~1.5×10 10 , 1×10 10 ~2×10 10 , or 1×10 9 ~1×10 10 are administered at a dose of any one of the cells / individuals, but are not limited thereto.

[0079] In other embodiments of the present invention, the activated T cells of the present invention are 1×10 7 ~1×10 8 , 1×10 8 ~2×10 8 , 2×10 8 ~4×10 8 , 4×10 8 ~6×10 8 , 6×10 8 ~8×10 8 , 8×10 8 ~1×10 9 , 1×109 ~2×10 9 、 2×10 9 ~4×10 9 、 4×10 9 ~1×10 10 、 2×10 8 ~6×10 8 、 6×10 8 ~1×10 9 、 1×10 8 ~2×10 8 、 2×10 8 ~2×10 9 、 1×10 7 ~1×10 8 、 1×10 8 ~1×10 9 、 1×10 9 ~1×10 10 、 or 1×10 7 ~1×10 9 administered at a dose of any one of the cells / kg, but not limited thereto.

[0080] In the present invention, when administering antigen-presenting cells (e.g., dendritic cells) loaded with the cancer-specific epitope and / or activated T cells, stabilizers or excipients such as human albumin can be used together.

[0081] In the present invention, the dosage and administration schedule of antigen-presenting cells (e.g., dendritic cells) loaded with the cancer-specific epitope and / or activated T cells can be adjusted over the course of treatment based on the judgment of the administering physician. In some embodiments, the activated T cells are administered about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, or 1 month after the administration of the antigen-presenting cells loaded with the cancer-specific epitope, or simultaneously with the antigen-presenting cells, but not limited thereto.

[0082] In the present invention, when administering antigen-presenting cells (e.g., dendritic cells) loaded with the cancer-specific epitope and / or activated T cells, it is carried out alone or together with other therapies such as surgery, radiotherapy, gene therapy, immunotherapy, bone marrow transplantation, stem cell transplantation, hormone therapy, targeted therapy, cryotherapy, ultrasonic therapy, photodynamic therapy, chemotherapy, etc. Additionally, those at a greater risk of developing a proliferative disease can receive treatment to suppress and / or delay the onset of the disease.

Advantages of the Invention

[0083] The antigen-presenting cells loaded with the cancer-specific epitope provided by the present invention, that is, dendritic cells, can rapidly and effectively induce the differentiation and proliferation of cancer antigen-specific T cells, preferably memory T cells. The memory T cells thus activated can treat cancer or tumor states by avoiding the cancer cell defense mechanism, or prevent cancer recurrence, progression, or metastasis.

[0084] Previously, in adoptive T cell therapy, it took 3 to 6 months to produce a large number of T cells for the treatment of cancer patients, and there were major problems in the cell production process in immunocyte therapy. However, in the case of the present invention, 10 9 autologous memory T cells that must be used for the treatment of patients can be produced within 3 weeks, and cost savings and the risk factor of infection against external contamination sources can be minimized. Therefore, in the case of the present invention, it is a technology that enables rapid therapeutic access to more solid cancer patients and is also applicable to terminal cancer patients.

Brief Description of the Drawings

[0085]

Figure 1

Figure 2

Figure 3

Figure 4

Mode for Carrying Out the Invention

[0086] According to one embodiment of the present invention, it relates to an Epstein-Barr virus (EBV)-negative cancer-specific neo-epitope represented by any one of SEQ ID NOs: 1 to 214.

[0087] According to another embodiment of the present invention, it relates to antigen presenting cells (APCs) loaded with the cancer-specific neo-epitope provided by the present invention.

[0088] According to still another embodiment of the present invention, it relates to T cells activated by the antigen presenting cells provided by the present invention.

[0089] According to still another embodiment of the present invention, it relates to an anti-cancer vaccine or a pharmaceutical composition for the prevention or treatment of cancer, which contains, as an active ingredient, antigen presenting cells loaded with the cancer-specific epitope provided by the present invention; and / or activated T cells.

Example

[0090] Hereinafter, the present invention will be described in more detail through examples. These examples are merely for explaining the present invention more specifically, and it will be apparent to those with ordinary knowledge in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention.

[0091] [Example 1] Method for preparing autologous memory T cells specific to EBV-negative gastric cancer cells and clinical application 1. Selection of EBV-negative gastric cancer cell antigen neo-epitopes An algorithm for predicting the most main sequences in which gene mutations accumulate in gastric cancer cells and predicting the epitopes that bind to the HLA of T cells was developed using Neopepsee. At this time, in the neo-epitope prediction algorithm, peptide sequences expected to have a high binding affinity with the HLA types (HLA-A2402, A0201) most expressed in Koreans were discovered. For this purpose, currently, missense mutations expressed in mRNA were predicted by whole exome sequencing and RNAseq data analysis of all EBV-negative gastric cancer patients existing in TCGA. The binding affinity between each HLA type and the discovered neo-epitope was IC obtained from NetMHC50 Taking into account all the values (in nM) and the rank-based predictive values of MHC-peptide binding obtained from NetCTLpan, and additionally considering protein cleavage, the hydrophobicity of amino acids at TCR contact residues, the polarity and charge values of amino acids, as well as the molecular size and entropy of the peptides, the final score of Neopepsee was calculated. As a result, the possibility of cancer-specific neo-antigens was ultimately predicted to discover neo-epitopes.

[0092] The following Tables 1-6 show the RNAseq data of patients expressing HLA-A2402 and HLA-A0201, which are known to be the most highly expressed in Koreans. They show the sequences predicted to be neo-epitopes, the genes expressing these sequences, and the normal sequences, predict the binding affinity between these sequences and HLA, and predict the types of neo-epitopes that can be used when actually producing cell therapy agents.

[0093]

Table 1

[0094]

Table 2

[0095]

Table 3

[0096]

Table 4

[0097]

Table 5

[0098]

Table 6

[0099] As is clear from Tables 1 to 3 above, by in silico prediction, as neoepitopes having high binding affinity for HLA-A2402, AYNSISSEVI (SEQ ID NO: 45) (IC 50 = 274 nM), TYQNDNKPEF (SEQ ID NO: 42) (IC 50 = 187 nM), FFYPCHPDVF (SEQ ID NO: 49) (IC 50 = 436 nM), VYNMPSTPSF (SEQ ID NO: 32) (IC 50 = 38 nM), RYLGPTDWQL (SEQ ID NO: 41) (IC 50 = 159 nM), LMVIGIPFFF (SEQ ID NO: 48) (IC 50 = 404 nM) were selected.

[0100] Also, as is clear from Tables 4 to 6, as neoepitopes having high binding affinity for HLA-A0201, MLIDVLLIGV (SEQ ID NO: 122) (IC50 = 4 nM), MMDRQMLPPV (SEQ ID NO: 123) (IC 50 = 6 nM), LMWVCALGHL (SEQ ID NO: 173) (IC 50 = 120 nM), GIVDIFLSFL (SEQ ID NO: 174) (IC 50 = 125 nM), AVFVICWTPI (SEQ ID NO: 213) (IC 50 = 448 nM), TLRVLLIVGV (SEQ ID NO: 214) (IC 50 = 457 nM) were selected.

[0101] For the following experiments, the neoepitopes selected as described above were synthesized with the prepared MHC-peptide multimers (8, 9, 10 mers). Using this, cells capable of recognizing this among patient-derived T cells were extracted, and thereafter, EBV-negative gastric cancer cell antigen-specific autologous memory T cells were prepared.

[0102] 2. ELISPOT results of T cells activated by dendritic cells loaded with the selected neoepitopes PBMCs were isolated from the blood of healthy humans into monocytes and leukocytes by flow cytometry. For monocytes to differentiate into dendritic cells, GM-CSF and IL-4 cytokines were added to the culture medium and cultured for 2 days. Also, in the case of leukocytes, they were cultured for 3 days with anti-CD3 / CD28 antibodies, and then cultured in a culture medium containing IL-2 cytokine. The neoepitope peptide selected as described above was introduced into the dendritic cells differentiated from monocytes by electroporation. After that, they were cultured for 5 days, and after confirming the expression of the neoepitope on the surface of the dendritic cells, they were co-cultured with leukocytes cultured in a culture medium containing anti-CD3 / CD28 antibodies at a ratio of 1:20 (dendritic cells: leukocytes). During co-culture, a cytokine cocktail containing IL-4, a cytokine that increases the efficacy of the antigen-presenting function of dendritic cells, and IL-2 and IL-7, cytokines that assist in the conversion of T cells into memory cells, was mixed and cultured. After 16 hours, the degree of IFN-γ expression in the T cells activated in this way was measured by ELISPOT, and the results are shown in Figures 1 to 4. After 72 hours of co-culture, to select memory T cells presented with antigens via dendritic cells, EBV antigen-specific memory T cells were extracted using a cell extractor (MACS) with a magnet that can extract T cells secreting the IFNr cytokine. The extracted memory T cells were cultured in a culture medium mixed with IL-2, IL-7, and IL-15 cytokines to maintain their memory function and increase the number of cells until the number of cells injectable into mice was reached. However, as a control group, cells introduced with non-stimulated EBV-positive gastric cancer peptide (HLA-A3101) were used.

[0103] As a result, it was confirmed that in T cells cultured with dendritic cells loaded with the neoepitope peptide predicted by Neopepsee, regardless of the binding affinity of the peptide to HLA, the secretion of IFNr was much higher than that of the control group.

[0104] Accordingly, in the present invention, cytotoxic T lymphocytes (CTLs) can be activated by dendritic cells loaded with neoepitopes having high binding affinity for HLA-A2402 or HLA-A0201 in Tables 1 to 6 above. It has been found that T cells activated in this way have antigen specificity that can recognize neoepitopes, which are neoantigens.

[0105] As described above, specific parts of the present invention have been described in detail. However, for those with ordinary knowledge in the art, such specific descriptions are merely preferred embodiments, and thus it is obvious that the scope of the present invention is not limited. Therefore, the substantial scope of the present invention should be defined by the appended claims and their equivalents.

Industrial Applicability

[0106] The present invention relates to cancer-specific neoepitopes, antigen-presenting cells loaded with the neoepitopes, and a method for activating T cells for cancer treatment by the antigen-presenting cells.

Claims

1. An Epstein - Barr virus (EBV) - negative cancer - specific neo - epitope for T - cell activation, represented by any one of SEQ ID NOs: 41, 48, 49, 122, 123, 173, 174, 213, and 214.

2. The Epstein - Barr virus (EBV) - negative cancer - specific neo - epitope according to claim 1, which shows binding affinity to at least one of HLA - A*2402 and HLA - A*0201.

3. The Epstein - Barr virus (EBV) - negative cancer - specific neo - epitope according to claim 1, wherein the cancer expresses a cancer - specific neo - epitope represented by any one of SEQ ID NOs: 41, 48, 49, 122, 123, 173, 174, 213, and 214.

4. The Epstein - Barr virus (EBV) - negative cancer - specific neo - epitope according to claim 3, wherein the cancer is colorectal cancer, pancreatic cancer, gastric cancer, liver cancer, breast cancer, cervical cancer, thyroid cancer, parathyroid cancer, lung cancer, non - small - cell lung cancer, prostate cancer, gallbladder cancer, bile duct cancer, non - Hodgkin lymphoma, Hodgkin lymphoma, blood cancer, bladder cancer, kidney cancer, ovarian cancer, melanoma, colon cancer, bone cancer, skin cancer, head cancer, uterine cancer, rectal cancer, brain tumor, cancer near the anus, fallopian tube cancer, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer, renal cell carcinoma, renal pelvis cancer, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, or pituitary adenoma.

5. A nucleic acid molecule encoding the Epstein - Barr virus (EBV) - negative cancer - specific neo - epitope according to any one of claims 1 to 4.

6. An expression vector into which the nucleic acid molecule according to claim 5 is inserted.

7. A host cell transfected with the expression vector according to claim 6.

8. A composition for activating T cells for cancer prevention or treatment, comprising an Epstein-Barr virus (EBV)-negative cancer-specific neoepitope represented by any one of SEQ ID NOs: 41, 48, 49, 122, 123, 173, 174, 213, and 214.

9. The composition for activating T cells according to claim 8, wherein the T cells comprise one or more selected from the group consisting of cytotoxic T cells, helper T cells, natural killer T cells, γδ T cells, regulatory T cells, and memory T cells.

10. An antigen-presenting cell loaded with an Epstein-Barr virus (EBV)-negative cancer-specific neoepitope represented by any one of SEQ ID NOs: 41, 48, 49, 122, 123, 173, 174, 213, and 214.

11. The antigen-presenting cell according to claim 10, comprising one or more of dendritic cells, B cells, and macrophages.

12. The antigen-presenting cell according to claim 10, which promotes the proliferation or differentiation of T cells.

13. A method for producing an antigen-presenting cell, comprising the step of loading an Epstein-Barr virus (EBV)-negative cancer-specific neoepitope represented by any one of SEQ ID NOs: 41, 48, 49, 122, 123, 173, 174, 213, and 214 onto the antigen-presenting cell (except when the step is performed in a human body).

14. The method according to claim 13, wherein the antigen-presenting cell comprises one or more of dendritic cells, B cells, and macrophages.

15. The method according to claim 13, wherein the antigen-presenting cell is obtained from peripheral blood mononuclear cells (PBMCs) derived from the peripheral blood of the subject individual.

16. The loading is performed by contacting the antigen-presenting cell with the Epstein-Barr virus (EBV)-negative cancer-specific neoepitope, according to the method of claim 13.

17. The loading is performed by pulsing the antigen-presenting cell with the Epstein-Barr virus (EBV)-negative cancer-specific neoepitope, according to the method of claim 13.

18. The loading is performed by nucleofecting the antigen-presenting cell with an expression vector into which a nucleic acid molecule encoding the Epstein-Barr virus (EBV)-negative cancer-specific neoepitope has been inserted, according to the method of claim 13.

19. The loading is performed using a fusion protein comprising the Epstein-Barr virus (EBV)-negative cancer-specific neoepitope and a dendritic cell-specific antibody or a fragment thereof, according to the method of claim 13.

20. A T cell activated by the antigen-presenting cell according to any one of claims 10 to 12.

21. A method for activating a T cell by the antigen-presenting cell according to any one of claims 10 to 12 (except when the activation is performed in a human body).

22. The method according to claim 21, wherein the activation is performed by co-culturing the T cell with the antigen-presenting cell.

23. The method according to claim 21, wherein the T cell is obtained from peripheral blood mononuclear cells (PBMCs) of a subject.

24. The method according to claim 21, wherein the T cell comprises one or more selected from the group consisting of cytotoxic T cells, helper T cells, natural killer T cells, γδ T cells, regulatory T cells, and memory T cells.

25. The method according to claim 22, wherein the co-culture is carried out by adding interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-7 (IL-7), interleukin-15 (IL-15), interleukin-21 (IL-21), or a combination thereof.

26. The method according to claim 22, wherein the co-culture is carried out by adding a fusion protein comprising a cytokine and an immunoglobulin heavy chain constant region.

27. The method according to claim 26, wherein the cytokine is interferon-γ (IFN-γ), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-12 (IL-12), IL-18, tumor necrosis factor (TNF), or granulocyte macrophage colony-stimulating factor (GM-CSF).

28. The co-culture is The method according to claim 22, wherein the co-culture is carried out by adding a fusion protein comprising a ligand of CD27, CXCR3, or CD62L, and an immunoglobulin heavy chain constant region.

29. A pharmaceutical composition for preventing or treating Epstein-Barr virus (EBV)-negative cancer, comprising, as an active ingredient, the antigen-presenting cell according to any one of claims 10 to 12.

30. A pharmaceutical composition for preventing or treating Epstein-Barr virus (EBV)-negative cancer, comprising, as an active ingredient, the activated T cell according to claim 20.

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