Method for t cell activation for cancer treatment

Abstract

To provide cancer-specific epitopes.SOLUTION: A cancer-specific epitope represented by a specific amino acid sequence is provided. Through an antigen-presenting cell loaded with the epitope, that is, a dendritic cell, differentiation and proliferation of cancer antigen-specific T cells, preferably memory T cells, can be rapidly and effectively induced, and the memory T cells thus activated can treat a cancerous or neoplastic condition or prevent recurrence, progression, or metastasis of cancer while avoiding the defense mechanism of cancer cells.SELECTED DRAWING: Figure 1

Description

Technical Field

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

Background Art

[0002] The incidence of gastric cancer is known worldwide as a malignant tumor with a particularly high incidence rate, especially in Asia. Although the causes of gastric cancer are variously known, typically EBV-related gastric cancer caused by infection with Epstein-Barr virus (EBV), and gastric cancer cell antigen-related gastric cancer caused by the accumulation of genetic mutations in gastric cells can be classified. Currently, the treatment strategy for gastric cancer has long been known to be the most effective by incisional surgery of cancer tissue, and chemotherapy and radiotherapy are also performed, but it is known as a disease that is difficult to be completely cured if gastric cancer cannot be detected at an early stage. In addition, clinical trials are being advanced through various biological agents (antibodies, small molecules), but no therapeutic agent with excellent clinical effects has been found yet. · EBV-related gastric cancer caused by infection with Epstein-Barr virus (EBV), and gastric cancer cell antigen-related gastric cancer caused by the accumulation of genetic mutations in gastric cells can be classified. Currently, the treatment strategy for gastric cancer has long been known to be the most effective by incisional surgery of cancer tissue, and chemotherapy and radiotherapy are also performed, but it is known as a disease that is difficult to be completely cured if gastric cancer cannot be detected at an early stage. In addition, clinical trials are being advanced through various biological agents (antibodies, small molecules), but no therapeutic agent with excellent clinical effects has been found yet. · EBV-related gastric cancer caused by infection with Epstein-Barr virus (EBV), and gastric cancer cell antigen-related gastric cancer caused by the accumulation of genetic mutations in gastric cells can be classified. Currently, the treatment strategy for gastric cancer has long been known to be the most effective by incisional surgery of cancer tissue, and chemotherapy and radiotherapy are also performed, but it is known as a disease that is difficult to be completely cured if gastric cancer cannot be detected at an early stage. In addition, clinical trials are being advanced through various biological agents (antibodies, small molecules), but no therapeutic agent with excellent clinical effects has been found yet. Currently, the treatment strategy for gastric cancer has long been known to be the most effective by incisional surgery of cancer tissue, and chemotherapy and radiotherapy are also performed, but it is known as a disease that is difficult to be completely cured if gastric cancer cannot be detected at an early stage. In addition, clinical trials are being advanced through various biological agents (antibodies, small molecules), but no therapeutic agent with excellent clinical effects has been found yet. Currently, the treatment strategy for gastric cancer has long been known to be the most effective by incisional surgery of cancer tissue, and chemotherapy and radiotherapy are also performed, but it is known as a disease that is difficult to be completely cured if gastric cancer cannot be detected at an early stage. In addition, clinical trials are being advanced through various biological agents (antibodies, small molecules), but no therapeutic agent with excellent clinical effects has been found yet. Currently, the treatment strategy for gastric cancer has long been known to be the most effective by incisional surgery of cancer tissue, and chemotherapy and radiotherapy are also performed, but it is known as a disease that is difficult to be completely cured if gastric cancer cannot be detected at an early stage. In addition, clinical trials are being advanced through various biological agents (antibodies, small molecules), but no therapeutic agent with excellent clinical effects has been found yet. Currently, the treatment strategy for gastric cancer has long been known to be the most effective by incisional surgery of cancer tissue, and chemotherapy and radiotherapy are also performed, but it is known as a disease that is difficult to be completely cured if gastric cancer cannot be detected at an early stage. In addition, clinical trials are being advanced through various biological agents (antibodies, small molecules), but no therapeutic agent with excellent clinical effects has been found yet. Currently, the treatment strategy for gastric cancer has long been known to be the most effective by incisional surgery of cancer tissue, and chemotherapy and radiotherapy are also performed, but it is known as a disease that is difficult to be completely cured if gastric cancer cannot be detected at an early stage. In addition, clinical trials are being advanced through various biological agents (antibodies, small molecules), but no therapeutic agent with excellent clinical effects has been found yet.

[0003] Recently, a cancer cell-specific targeted treatment method using patient-derived autologous T cells has been studied at multiple institutions, and clinical trials have been conducted at multiple institutions on lymphoma using chimeric antigen receptor (CAR) T cells. As a result, excellent clinical effects and low side effects have been obtained, and it has come into the spotlight as a new field in anti-cancer treatment. Recently, a cancer cell-specific targeted treatment method using patient-derived autologous T cells has been studied at multiple institutions, and clinical trials have been conducted at multiple institutions on lymphoma using chimeric antigen receptor (CAR) T cells. As a result, excellent clinical effects and low side effects have been obtained, and it has come into the spotlight as a new field in anti-cancer treatment. Recently, a cancer cell-specific targeted treatment method using patient-derived autologous T cells has been studied at multiple institutions, and clinical trials have been conducted at multiple institutions on lymphoma using chimeric antigen receptor (CAR) T cells. As a result, excellent clinical effects and low side effects have been obtained, and it has come into the spotlight as a new field in anti-cancer treatment. Recently, a cancer cell-specific targeted treatment method using patient-derived autologous T cells has been studied at multiple institutions, and clinical trials have been conducted at multiple institutions on lymphoma using chimeric antigen receptor (CAR) T cells. As a result, excellent clinical effects and low side effects have been obtained, and it has come into the spotlight as a new field in anti-cancer treatment. Recently, a cancer cell-specific targeted treatment method using patient-derived autologous T cells has been studied at multiple institutions, and clinical trials have been conducted at multiple institutions on lymphoma using chimeric antigen receptor (CAR) T cells. As a result, excellent clinical effects and low side effects have been obtained, and it has come into the spotlight as a new field in anti-cancer treatment.

[0004] Using patient-derived T cells results in less induction of immune responses, which is the most serious side effect of cell therapy agents. Since there are no restrictions on the donor's HLA type, it has been known as an effective and side effect-free treatment method. To date, the types of cell therapy agents most commonly used in the field of 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 a cell-killing effect, they have no antigen specificity, so they have various side effects. In the case of dendritic cells, they do not have the function of directly killing cells, but instead, they can transmit antigen specificity to T cells in the patient's body and endow T cells with high-efficiency cancer cell specificity as a therapeutic agent based on the vaccine concept. In addition, CD4+ T cells have the role of assisting other cells through antigen specificity. In the case of CD8+ T cells, they are known as the 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 almost reached their limit points, and the actual situation is that no clinical effect has occurred. Specifically, cancer cells either secrete substances that suppress the immune response in the human body or are unable to present the antigens essential for the production of antibodies against cancer cells, resulting in the inability to trigger an appropriate immune response.

[0006] On the other hand, dendritic cells not only play the role of monitors that sense antigens entering from outside the human body or generated inside the body, but also quickly migrate 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. They play the role of specialized antigen-presenting cells. Regarding cancer immunotherapy vaccines using dendritic cells, Thus, it has been developed through various methods and can be broadly 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. Also, in the case of ex vivo generated dendritic cell vaccine methods, dendritic cells are separated from a patient's PBMC (peripheral blood mononuclear cells), antigens to be presented to the separated dendritic cells are transmitted, and after the dendritic cells are activated in this way, they are injected back into the patient so that antigens are transmitted from the injected dendritic cells to T cells. In this case, ex vivo dendritic cell culture methods and antigen transmission methods are important. Currently, among the antigen presentation methods in use, the DNA of the antigen to be presented is transfected using a virus or nucleofection, or antigen transmission targeting dendritic cells is used by binding the antigen to an antibody targeting dendritic cells. Currently, the biggest problem with dendritic cell vaccines is that it is extremely difficult to activate effective anti-cancer immune cells in the microenvironment of cancer where there are profound chronic inflammatory phenomena, the Warburg effect, immunosuppressive cytokines, immunosuppressive T cells, and dendritic cells in the body.

Summary of the Invention

Problems to be Solved by the Invention

[0007]

[0008] ​

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

[0010] Another object of the present invention is to provide an antigen-presenting cell loaded with an epitope for cancer treatment. Another object of the present invention is to provide a method for producing an antigen-presenting cell loaded with an epitope for cancer treatment.

[0011] Another object of the present invention is to provide activated T cells by an antigen-presenting cell loaded with an epitope of the present invention. Another object of the present invention is to provide a method for activating T cells for cancer treatment.

[0012] Another object of the present invention is to provide a method for activating T cells for cancer treatment. Another object of the present invention is to provide a cancer treatment method using an antigen-presenting cell loaded with an epitope.

[0013] Another object of the present invention is to provide a cancer treatment method using T cells activated by an antigen-presenting cell loaded with an epitope. Another object of the present invention is to provide a cancer treatment method using T cells activated by an antigen-presenting cell loaded with an epitope.

[0014] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those having ordinary knowledge in the art from the following description. However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those having ordinary knowledge in the art from the following description.

[0015] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those having ordinary knowledge in the art from the following description. However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those having ordinary knowledge in the art from the following description. However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those having ordinary knowledge in the art from the following description.

Means for Solving the Problems

[0016] According to one embodiment of the present invention, it relates to an epitope specific to cancer (tumor antigen epitope). According to one embodiment of the present invention, it relates to an epitope specific to cancer (tumor antigen epitope).

[0017] In the present invention, the "cancer-specific epitope" is derived from a protein antigen that exists only in cancer cells and does not exist in normal cells. In the present invention, the cancer-specific epitope contains at least one epitope recognized by a T cell receptor, and preferably is an epitope present in Epstein-Barr virus (EBV)-positive cancer cells, which may include the EBV viral epitope or the epitope of cancer cells, and more preferably, is an antigen of Epstein-Barr virus (EBV)-positive cancer cells, Epstein-Barr virus latent membrane protein 2 (Epstein-Barr virus latent membrane protein 2, LMP2a), Epstein-Barr nuclear antigen 1 (Epstein-Barr nuclear antigen 1, EBNA-1), or these epitopes. In the present invention, the "Epstein-Barr virus latent membrane protein 2 (Epstein-Barr virus latent membrane protein 2, LMP2a)" is one of several EBV genes expressed in all type II and type III diseases / malignancies. LMP2a acts as a negative mediator of B cell-receptor signaling and corresponds to a transmembrane protein that promotes cell survival through sequestering of tyrosine kinase. HLA-A2-restricted peptides are epitope-specific cytotoxic T lymphocytes detectable in 60-75% of individuals in vitro.

[0018] In the present invention, the "Epstein-Barr virus latent membrane protein 2 (Epstein -Barr virus latent membrane protein 2, LMP2a)" is one of several EBV genes expressed in all type II and type III diseases / malignancies. LMP2a acts as a negative mediator of B cell-receptor signaling and corresponds to a transmembrane protein that promotes cell survival through sequestering of tyrosine kinase. HLA-A2-restricted peptides are epitope-specific cytotoxic T lymphocytes detectable in 60-75% of individuals in vitro. through sequestering of tyrosine kinase. HLA-A2-restricted peptides are epitope-specific cytotoxic T lymphocytes detectable in 60-75% of individuals in vitro. Yes, it is the LMP epitope that is the most immunodominant in latent diseases. The CLGGLLTMV peptide is preserved in biopsies examined from NPC and HL patients, where the epitope is immunologically vulnerable, similar to other EBV latent epitopes, and thus represents a potential target for the treatment of NPC and HL.

[0019] In the present invention, the "Epstein-Barr nuclear antigen 1 (EBNA-1)" corresponds to a multifunctional, dimeric viral protein associated with Epstein-Barr virus. This corresponds to an EBV protein found only in all EBV associated malignancies. It plays an important role in maintaining the state transformation associated with cell infection with EBV. On the other hand, EBNA-1 corresponds to a glycine-alanine repeat sequence that separates the protein into amino and carboxy terminal domains. Such a sequence stabilizes the protein, prevents proteasome-related degradation (proteasomal degradation), and plays a role in damaging antigen processing and MHC class I-restricted antigen expression. Therefore, the said EBNA-1 suppresses the response of CD8-restricted cytotoxic T cells against virus-infected cells. The said EBNA-1 is expressed by the Qp promoter in all latency programs and corresponds to the only viral protein expressed in latency program I.

[0020] In the present invention, the "epitope" refers to an epitope that does not exist in normal non-cancer cells or references such as germ cells, but is found in cancer cells. ​​​​​​​​​For example, the epitope may be 10mer to 20mer, preferably 15mer, but is not limited thereto. It may be, but is not limited to, these.

[0021] In the present invention, as the epitope, T cells extracted from human blood, preferably, memory T cells can have efficacy, so that HLA-A, HLA-B, HLA- C, HLA-E, HLA-F, HLA-G, β2-microglobulin, HLA-DPA 1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA1, HL A-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DM shows binding affinity to at least one of HLA-DOA and HLA-DOB loci, Among them, it may be an HLA type most highly expressed in Koreans, for example, 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, which shows high binding affinity to at least one of them.

[0022] Preferably, in the present invention, as the epitope, it has a high binding affinity for HLA-A*2402, and SEQ ID NOs: 1 to 3, SEQ ID NOs: 7 to 9, SEQ ID NOs: 11 ~15, SEQ ID NOs: 19 to 21, SEQ ID NOs: 23 to 39, SEQ ID NOs: 43 to 45, SEQ ID NOs: 47 ~51, SEQ ID NOs: 55 to 57, SEQ ID NOs: 59 to 63, SEQ ID NOs: 67 to 69, SEQ ID NOs: 71 ~75, SEQ ID NOs: 79 to 81, SEQ ID NOs: 83 to 111, SEQ ID NOs: 115 to 117, SEQ ID NO ~75, SEQ ID NOs: 79 to 81, SEQ ID NOs: 83 to 111, SEQ ID NOs: 115 to 117, SEQ ID NO An epitope of the LMP2a antigen, which is a peptide represented by any one of Nos. 119 to 122 may be; or one having a high binding affinity for HLA-A*3101 and is an epitope of the EBNA-1 antigen, which is a peptide represented by any one of SEQ ID NOs: 124 to 127, SEQ ID NOs: 129 to 149, SEQ ID NOs: 151 to SEQ ID NO: 154, SEQ ID NOs: 156 to 158, SEQ ID NOs: 160 to 163, SEQ ID NOs: 165 to 167 , SEQ ID NOs: 168 to 176, SEQ ID NOs: 178 to 181, SEQ ID NOs: 183 to 194, and SEQ ID NOs: 196 to 199 may be.

[0023] However, in the present invention, the method for measuring the epitope-HLA affinity is not limited to using NetMHC3.4 (URL :www.cbs.dtu.dk / services / NetMHC-3.4 / ) to predict whether an epitope binds to a specific HLA allele, but may be other methods although it is not limited to these.

[0024] In the present invention, the "HLA" or "human leukocyte antigen" refers to a human gene encoding an MHC (major histocompatibility complex) protein on the surface of cells that cause immune system regulation . "HLA-I" or "HLA class I" refers to a human MHC class I gene containing the HLA-A, HLA-B, HLA- C, HLA-E, HLA-F, HLA-G, β2-microglobulin locus. "HLA-II" or "HLA class II" refers to HLA- DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-D RA1, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DM, HLA-DOA, and HLA-DOB loci. ​ is referred to as the I gene.

[0025] In the present invention, the cancer may be an Epstein-Barr virus (EBV)-positive cancer, such as EBV-positive gastric cancer, EBV-positive cervical cancer, EBV-positive Burkitt lymphoma, EBV-positive T-cell lymphoma, EBV-positive breast cancer, EBV-positive leiomyosarcomas, EBV-positive smooth muscle tumors, EBV-positive Hodgkin lymphoma, EBV-positive nasopharyngeal cancer or EBV-positive post-transplant lymphoproliferative disorder (PTLD), but preferably EBV-positive gastric cancer.

[0026] According to another embodiment of the present invention, the cancer-specific epitope (tumor antigen epitope) provided by the present invention is preferably a nucleic acid molecule encoding an epitope of LMP2a or EBNA-1, which is an antigen of Epstein-Barr virus (EBV)-positive cancer cells.

[0027] The nucleic acid molecules of the present invention include any nucleic acid molecules translated into a polynucleotide sequence as known to those skilled in the art for the amino acid sequence of the polypeptide provided by the present invention. Therefore, various polynucleotide sequences by ORF (open reading frame) can be produced, and these are also included in the nucleic acid molecules of the present invention.

[0028] According to another embodiment of the present invention, there is provided an expression vector into which the isolated nucleic acid molecule provided by the present invention is inserted.

[0029] In the present invention, the "vector" is the other nucleic acid capable of transporting a certain nucleic acid molecule molecule. One type of vector is the "plasmid" which refers to circular double-stranded DNA that can ligate additional DNA segments. Another type of vector is the phage vector. Another type of vector is the viral vector wherein an additional DNA segment can be ligated to the viral genome . A certain vector can autonomously replicate in the host cell into which they flow (for example, a bacterial vector is an episomal mammalian vector having a bacterial origin of replication). Other vectors (for example, non-episomal mammalian vectors) can flow into the host cell and be integrated into the genome of the host cell, and then replicated together with the host genome. Not only that, a certain vector can direct the expression of the genes ligated for them to operate . Such a vector is named "recombinant expression vector" or simply "expression vector" in the present application. Generally, expression vectors useful in recombinant DNA techniques sometimes 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 among vectors . . .

[0030] In the present invention, specific examples of the expression vector include the commercially widely used pCDNA A vector, F, R1, RP1, Col, pBR322, ToL, Ti vector; cosmid ; phages such as lambda, lambdoid, M13, Mu, p1 P22, Qμ, T -even, T2, T3, T7; selected from the group consisting of plant viruses​​​​​​ is also acceptable, but not limited thereto, and is known to those skilled in the art as an expression vector All expression vectors can be used in the present invention. When selecting an expression vector, it is necessary to consider the purpose and follow the properties of the host cell to be used. When introducing the vector into the host cell, calcium phosphate transfection , viral infection, DEAE-dextran-mediated transfection, lipo fectamine transfection, or electroporation can be used, but 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 not limited thereto. It is also possible to select whether to produce the product using a vector without a selection marker . The selection of the selection marker is determined by the host cell to be used, and since methods already known to those skilled in the art are used, the present invention does not limit this . The present invention does not limit this .

[0031] For the nucleic acid molecule of the present invention, in order to facilitate purification, a tag sequence can be inserted and fused onto the expression vector . Examples of the tag include, but are not limited to, a hexahistidine tag, a hemagglutinin tag , a myc tag, or a flag tag. Any tag known to those skilled in the art that facilitates purification can be used in the present invention .

[0032] According to another embodiment of the present invention, there is provided a host cell transfected with the expression vector provided by the present invention .

[0033] In the present invention, the "host cell" includes a vector for incorporating a polypeptide insert include a recipient such as (etc.) or an individual cell or cell culture that was a recipient Host cells include the progeny of a single host cell, and said progeny may not necessarily be identical (either morphologically or genomically) to the original mother cell due to natural, accidental, or intentional mutations. Host cells include cells that have been transfected in vivo with the polypeptide (etc.) of the present application. DNA complement).

[0034] In the present invention, the host cells may include cells of mammalian, plant, insect, fungal or cellular origin, for example, bacteria such as Escherichia coli, Streptomyces, Salmonella typhimurium, etc. cells; fungal cells such as yeast cells, Pichia pastoris, etc.; insect cells such as Drosophila, Spodoptera tera Sf9 cells, etc.; CHO (Chinese hamster ovary cells, Chinese h amster ovary cells), SP2 / 0 (mouse myeloma), human lymphoblastoid, COS, NSO (mouse myeloma ), 293T, Bowes melanoma cells, HT-1080, BHK (Baby Hamster Kidney cells, Baby Hamster Kidney cells), HEK (Human Embryonic Kidney cells, Human Embryonic Kidney cells), or PERC.6 (human retinal cells); or plant cells, but are not limited thereto, and any cells that can be used as host cells known to those skilled in the art are available (human retinal cells); or may be plant cells, but are not limited thereto, and any cells that can be used as host cells known to those skilled in the art are available

[0035] According to another embodiment of the present invention, a cancer-specific epitope (tumor antigen epitope) provided by the present invention, a nucleic acid molecule encoding the same, and an expression vector into which the nucleic acid molecule is inserted ​​​​​​​or a composition for activating T cells, comprising a host cell transformed with the expression vector. This is what we do.

[0036] As used herein, "activation of T cells" refers to activation of T cells by the activation of at least one tumor antigen. A single clone (e.g., one that encodes the same TCR) with a T cell receptor that recognizes the peptide or polyclonal (e.g., having clones encoding different TCRs). Activated T cells are classified into cytotoxic T cells, helper T cells, and natural T cells. Selected from the group consisting of ral killer T cells, γδ T cells, regulatory T cells and memory T cells. The present invention may contain one or more subtypes of T cells, including, but not limited to, one or more of the subtypes of T cells that are However, preferably, the T cells may be memory T cells.

[0037] In the present invention, the activated T cells evade the defense mechanisms of cancer cells and inhibit the proliferation of cancer or tumor cells. The condition may be treated or the recurrence, progression or metastasis of cancer may be prevented.

[0038] According to another embodiment of the present invention, the cancer-specific epitope (tumor Antigen presenting cells loaded with tumor antigen epitopes g cells, APCs).

[0039] In the present invention, the antigen-presenting cells are dendritic cells (DCs). The cells may include one or more of B cells and macrophages, but preferably include dendritic cells. may be also possible.

[0040] In the present invention, the antigen-presenting cells may be isolated from an individual.

[0041] In the present invention, the "dendritic cells" are members of various populations of cell types found in lymphoid or non-lymphoid tissues. Such cells are characterized by these unique morphologies and high expression levels of surface class I and class II MHC molecules, which are proteins that present antigenic peptides to T cells. DCs, other APCs, and T cells can be readily isolated from peripheral blood, such as peripheral blood mononuclear cells (PBMCs) derived from peripheral blood, and from a number of tissue sources, or can be derived (e.g., differentiated) therefrom. DCs, other APCs, and T cells can be readily isolated from peripheral blood, such as peripheral blood mononuclear cells (PBMCs) derived from peripheral blood, and from a number of tissue sources, or can be derived (e.g., differentiated) therefrom. In the present invention, the antigen-presenting cells can induce the differentiation and proliferation of T cells specific for cancer antigens, preferably memory T

[0042] cells, avoid the defense mechanisms of cancer cells, and treat cancer or a neoplastic condition or prevent cancer recurrence, progression, or metastasis. or prevent cancer recurrence, progression, or metastasis.

[0043] According to another embodiment of the present invention, it relates to a fusion protein comprising a cancer-specific epitope (tumor antigen epitope) provided by the present invention; and an antibody specific for dendritic cells, or a fragment thereof. According to another embodiment of the present invention, it relates to a fusion protein comprising a cancer-specific epitope (tumor antigen epitope) provided by the present invention; and an antibody specific for dendritic cells, or a fragment thereof. The fusion protein provided by the present invention enables dendritic cells to be loaded with the cancer-specific epitopes provided by the present invention.

[0044] The fusion protein provided by the present invention enables dendritic cells to be loaded with the cancer-specific epitopes provided by the present invention. The fusion protein provided by the present invention enables dendritic cells to be loaded with the cancer-specific epitopes provided by the present invention.

[0045] In the present invention, examples of the antibody specific for dendritic cells include DCIR, MHC class I, MHC class II, CD1, CD2, CD3, CD4, CD8, CD11b, CD1 4, CD15, CD16, CD19, CD20, CD29, CD31, CD40, CD4 3, CD44, CD45, CD54, CD56, CD57, CD58, CD83, CD8 6, CMRF-44, CMRF-56, DCIR, DC-ASPGR, CLEC-6, C D40, 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 an antibody specific for ASPGR, but is not limited thereto.

[0046] In the fusion protein of the present invention, the cancer-specific epitope may be conjugated to an antibody specific for the dendritic cell or a fragment thereof. Here, the “conjugate” refers to any substance formed by covalently bonding two moieties together. Representative conjugates according to the present invention include those formed by conjugating 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 Vander Waals bonding force. The step of linking an antigen to an antibody can also be performed by direct chemical linkage through a non-covalent association such as dockerin-cohesin association [(U.S. Patent Publication No. US20100135994 by Banchereau et al., related to the portion incorporated by reference herein) , or by forming a peptide or chemical bond. The step of linking an antigen to an antibody can also be performed by direct chemical linkage through a non-covalent association such as dockerin-cohesin association [(U.S. Patent Publication No. US20100135994 by Banchereau et al., related to the portion incorporated by reference herein) , or by forming a peptide or chemical bond. The step of linking an antigen to an antibody can also be performed by direct chemical linkage through a non-covalent association such as dockerin-cohesin association [(U.S. Patent Publication No. US20100135994 by Banchereau et al., related to the portion incorporated by reference herein) , or by forming a peptide or chemical bond. , or by forming a peptide or chemical bond. linkage.

[0047] According to another embodiment of the present invention, it relates to a method for producing antigen-presenting cells loaded with an epitope specific to cancer (tumor antigen epitope) provided by the present invention.

[0048] In the present invention, the antigen-presenting cells may include one or more of dendritic cells, B cells, and macrophages, and preferably may be dendritic cells.

[0049] In the present invention, the dendritic cells (for example, immature dendritic cells) can be obtained from various sources including an autologous source, that is, a source derived from the subject individual, and preferably can also be obtained from peripheral blood mononuclear cells (Peripheral blood mononuclear cell; PBMC) derived from peripheral blood, and more preferably can be obtained by separating monocytes from PBMC derived from an individual and contacting them with a plurality of cytokines. Here, the types of cytokines that induce the differentiation of the monocytes into dendritic cells are not particularly limited, and for example, may include one or more of GM-CSF and IL-4.

[0050] In the present invention, the "subject individual" means an individual who has cancer or is highly likely to develop cancer.

[0051] In the present invention, when the antigen-presenting cells are prepared as described above, the antigen-presenting cells can be loaded with an epitope specific to cancer of the present invention. Generally, immature dendritic cells capture antigens through phagocytosis or receptor-mediated endocytosis, process the antigens through a series of intracellular processes, and then load antigen peptides onto MHC and present them to T lymphocytes. As the process of processing antigens, dendritic cells further mature, along with phagocytosis ​ Lose the receptors used for intracellular uptake, and increase the expression of MHC class I, II, costimulatory molecules, and cell adhesion molecules, express new chemokine receptors, and migrate to areas rich in peripheral lymph node T lymphocytes, and present antigens to T lymphocytes, thereby inducing an immune response in T lymphocytes.

[0052] As an example of the present invention, to load the cancer-specific epitope onto the antigen-presenting cell the antigen-presenting cell may be contacted with the cancer-specific epitope of the present invention, or preferably, the antigen-presenting cell (e.g., immature dendritic cell), or an antigen-presenting cell (e.g., dendritic cell) contained in or derived from PBMC (e.g., differentiated) may be pulsed with the cancer-specific epitope of the present invention. As is known in the art, pulsing involves mixing a cell (e.g., dendritic cell ) with a solution containing the cancer-specific epitope peptide of the present invention, and subsequently, optionally removing the cancer-specific epitope from the mixture. In the present invention, when contacting the immature dendritic cell with the cancer-specific epitope, Toll-like receptor agonists can be used to further 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.

[0053] As another example of the present invention, to load the cancer-specific epitope onto the antigen-presenting cell the antigen-presenting cell may be nucleofected with an expression vector, preferably a plasmid, into which a nucleic acid molecule encoding the cancer-specific epitope of the present invention has been inserted. Yes. Here, at the time of the nucleofection, for example, it can be generated by any useful means in this field including the Amaxa (trademark) nucleofection system or the InVitrogen (trademark) nucleofection system.

[0054] As another example of the present invention, in order to load the cancer-specific epitope onto the antigen-presenting cell the cancer-specific epitope provided by the present invention; and a fusion protein containing an antibody specific for dendritic cells or a fragment thereof may be used.

[0055] According to another embodiment of the present invention, it relates to T cells activated by the antigen-presenting cells provided by the present invention.

[0056] In the present invention, the T cells may be those separated from an individual.

[0057] In the present invention, the T cells refer to a single clone (for example, encoding the same TCR) or a polyclonal (for example, having clones encoding different T CRs) T cell population having a T cell receptor that recognizes a tumor antigen peptide, and include one or more selected from the group consisting of cytotoxic T cells, helper T cells, natural killer T cells, γδT cells, regulatory T cells, and memory T cells, and may contain one or more subtypes of T cells not limited thereto, but preferably may be memory T cells. In the present invention, the "memory T cells" are T cells differentiated from T cells that have previously encountered and reacted with these specific

[0058] antigens or activated T cells. ​​​​​ exist. Tumor-specific memory T cells constitute a small portion of the total T cell population, and these perform an important function in monitoring tumor cells throughout an individual's 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 long-term tumor antigen-specific response of T cells. The activated T cells, preferably activated memory T cells, in the present invention specifically recognize the antigens of cancer cells, evade the defense mechanisms of cancer cells, and can treat cancer or a neoplastic condition, or prevent the recurrence, progression, or metastasis of cancer.

[0059] According to another embodiment of the present invention, it relates to a method for activating T cells by antigen-presenting cells (APC) provided by the present invention.

[0060] In the present invention, for the activation of the T cells, the T cells may be co-cultured with antigen-presenting cells loaded with the cancer-specific epitopes of the present invention.

[0061] In the present invention, the T cells may be obtained from various sources including self-sources, i.e., from the intended individual, and preferably may be obtained from peripheral blood mononuclear cells (PBMC) derived from peripheral blood. More preferably, they may be obtained from the non-adherent portion of the peripheral blood mononuclear cells. As an example of the present invention, the non-adherent portion of the PBMC is obtained by density gradient centrifugation of a peripheral blood sample. ​ It may be obtained, and may be obtained by culturing with at least one cytokine (for example, IL-2) with or without the presence of an anti-CD3 antibody (for example, OKT3).

[0062] In the present invention, the T cells refer to a single clone (for example, encoding the same TCR) or a polyclonal (for example, having clones encoding different TCRs) T cell population having a T cell receptor that recognizes a tumor antigen peptide, and cytotoxic T cells, helper T cells, natural killer T cells, γδ T cells, regulatory T cells, and memory T cells. It may contain one or more selected from the group consisting of, but is not limited to, one or more subtypes of T cells, and preferably may be memory T cells.

[0063] Also, in the present invention, the T cells and the antigen-presenting cells may be derived from the same individual, for example, an individual suffering from cancer, preferably an EBV-positive cancer (for example, low-grade to intermediate-grade cancer), but are not limited thereto.

[0064] In the present invention, for the activation of the T cells, the T cells may be 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 1 day to 21 days, 1 day to 14 days, 2 days to 10 days, 2 days to 5 days, 2 days to 5 days, 3 days, 5 days, 7 days, 10 days, 14 days, 16 days, 18 days or 21 days, but is not limited thereto.

[0065] In the present invention, during the co-culture of the T cells and the antigen-presenting cells of the present invention, one or more cytokines Adding in can promote the activation, maturation and / or proliferation of 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.

[0066] 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 a constant region of an immunoglobulin heavy chain is 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), IL-18, tumor necrosis factor (TNF), or granulocyte macrophage colony-stimulating factor (GMCSF), but is not limited thereto. Further, the constant region of the immunoglobulin heavy chain may be a constant region of an immunoglobulin heavy chain 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. Further, the constant region of the immunoglobulin heavy chain may be IgA (Igα), IgD (Igδ), IgE (Igε), IgG (Igγ) and Ig ​​​​​​​​​​​​​​​​M(Igμ), which may be derived from any of the five immunoglobulin classes known in the art, preferably the constant region of an immunoglobulin heavy chain derived from the IgG class. It may be the constant region of an immunoglobulin heavy chain derived from the IgG class.

[0067] In another embodiment of the present invention, during the co-culture of the T cells and 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 the constant region of an immunoglobulin heavy chain 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. Also, the constant region of the immunoglobulin heavy chain may be IgA (Igα), IgD (Igδ), IgE (Igε), IgG (Igγ), and IgM (Igμ), which may be derived from any of the five immunoglobulin classes known in the art, preferably the constant region of an immunoglobulin heavy chain derived from the IgG class.

[0068] According to another embodiment of the present invention, the cancer-specific epitope provided by the present invention is ​​​​​​​​​​​​​​It relates to an immunotherapeutic agent containing, as an active ingredient, pulsed antigen-presenting cells. In the present invention The immunotherapeutic agent according to the present invention can increase the immune response or selectively enhance a part of the immune response favorable for the treatment or prevention of a specific disease, for example, cancer.

[0069] According to another embodiment of the present invention, a cancer vaccine containing, as an active ingredient, antigen-presenting cells pulsed with a cancer-specific epitope provided by the present invention; and / or activated T cells, or a pharmaceutical composition for the prevention or treatment of cancer is provided. In the present invention, the above-mentioned "cancer" refers to or indicates a physiological state characterized by the growth of cells that cannot typically be regulated in mammals. In the present invention, the cancer to be the subject of prevention, improvement or treatment

[0070] is an Epstein-Barr virus (EBV )-positive cancer, which may be EBV-positive gastric cancer, EBV-positive cervical cancer, EBV-positive Burkitt lymphoma, EBV-positive T cell lymphomas, EBV-positive breast cancer, EBV-positive leiomyosarcomas, EBV-positive smooth muscle tumors, EBV-positive Hodgkin lymphoma, EBV -positive nasopharyngeal cancer or EBV-positive post-transplant lymphoproliferative disorder (PTLD), preferably EBV-positive gastric cancer, but is not limited thereto. The antigen-presenting cells provided by the present invention are T cells specific to EBV-positive cancer antigens, preferably

[0071] ​​​​​​​Alternatively, it can induce the differentiation and proliferation of memory T cells, and the thus-activated me mory T cells can avoid the cancer cell defense mechanism and treat cancer or a neoplastic condition, or prevent cancer recurrence, progression, or metastasis.

[0072] The anti-cancer vaccine according to the present invention can include any of an immunization method performed by a single administration and an immunization method performed by continuous administration.

[0073] In the present invention, "prevention" can include, without limitation, any act of blocking cancer symptoms or suppressing or delaying the symptoms using the pharmaceutical composition of the present invention.

[0074] Also, in the present invention, "treatment" can include, without limitation, any act in which the cancer symptoms improve or become advantageous using the pharmaceutical composition of the present invention.

[0075] In the present invention, the pharmaceutical composition can be characterized by being in the form of capsules, tablets, granules, injections, ointments, powders, or beverages, and the pharmaceutical composition can be characterized by being targeted at humans.

[0076] In the present invention, the pharmaceutical composition, although not limited thereto, can be formulated into oral dosage forms such as powders, granules, capsules, tablets, aqueous suspensions, etc., external preparations, suppositories, and sterile injection solutions by ordinary methods respectively and used. The pharmaceutical composition of the present invention can include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers can include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, flavors, etc. for oral administration, and buffers, preservatives, soothing agents, solubles for injections. ​ It can be used by mixing with a solubilizing agent, an isotonic agent, a stabilizing agent, etc. In the case of topical administration, a base, an excipient, a lubricant, a preservative, etc. can be used. The dosage form of the pharmaceutical composition of the present invention can be variously manufactured by mixing with a pharmaceutically acceptable carrier as described above. For example, for oral administration, it can be manufactured in the form of tablets, troches, capsules, elixirs, sus pensions, syrups, wafers, etc., and in the case of injections, it can be manufactured in unit-dose ampoules or multiple-dose forms. In addition, it can be formulated into solutions, sus pensions, tablets, capsules, sustained-release preparations, etc.

[0077] On the other hand, examples of carriers, excipients, and diluents suitable for formulation include lactose, dex trose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate tricalcium, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvi nylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate , talc, magnesium stearate, or mineral oil, etc. can be used. Further, it can further contain a filler, an anticoagulant, a lubricant, a wetting agent, a fragrance, an emulsifier, a preservative, etc.

[0078] In the present invention, the administration route of the pharmaceutical composition is not limited to these, but includes oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, local, sublingual, or rectal. Oral or parenteral administration is preferred.

[0079] In the present invention, the term "parenteral" refers to subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, or intrathoracic administration. The pharmaceutical compositions of the present invention include intraosseous, intrathecal, intralesional and intracranial injection or infusion techniques. It may also be administered in the form of suppositories for rectal administration.

[0080] The pharmaceutical compositions of the present invention may be prepared in a variety of ways, depending on the activity of the particular compound used, age, weight, general health, etc. Health, sex, meal, administration time, administration route, excretion rate, drug combination and characteristics to be prevented or treated The amount of the drug that can be administered may vary depending on various factors, including the severity of a particular disease. The dosage varies depending on the patient's condition, weight, severity of illness, dosage form, route of administration and duration. However, this can be appropriately selected by those skilled in the art, and the daily dose is 0.0001 to 50 mg / kg. The dosage may be once a day, or 0.001 to 50 mg / kg. The dosage may be administered in one dose or in several divided doses. The pharmaceutical composition according to the present invention may be in the form of a pill, a sugar-coated tablet, a capsule, a liquid, or the like. The composition can be formulated into a liquid formulation, a gel, a syrup, a slurry, or a suspension.

[0081] According to another embodiment of the present invention, in order to prevent or treat cancer, a subject is administered Antigen-presenting cells loaded with the cancer-specific epitopes provided by the present invention; and / or The present invention relates to a method for preventing or treating cancer, comprising administering activated T cells. It is.

[0082] In the present invention, the cancer is caused by Epstein-Barr virus. EBV) positive cancers, including EBV positive gastric cancer, EBV positive cervical cancer, EBV-positive Burkitt’s lymphoma, EBV-positive T cell lymphomas, EBV-positive breast cancer, EBV-positive leiomyosarcomas, EBV-positive smooth muscle tumors, EBV-positive muscle tumors, EBV-positive Hodgkin lymphoma, EBV-positive nasopharyngeal cancer or EBV-positive post-transplant lymphoproliferative disorder (PTLD), although preferably, it may be EBV-positive gastric cancer, but is not limited to these .

[0083] The dosage, schedule and route of administration of antigen-presenting cells or activated T cells loaded with cancer-specific epitopes provided by the present invention depend on the size and condition of the individual and are determined by standard pharmaceutical practices. Exemplary routes of administration include intravenous, intraarterial , intraperitoneal, intralung, intravascular, intramuscular, intratracheal, subcutaneous, intraocular, intrathecal or transdermal.

[0084] The volume of cells administered to an individual can vary, for example, depending on the specific type of cells administered, the route of administration and the specific type and stage of cancer being treated. The amount should be sufficient to produce the desired response, such as a therapeutic response to cancer, without serious toxicity or adverse events. 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 based on the size of the corresponding tumor, the number of cancer cells or the tumor in the same individual before treatment compared to its growth rate or to the corresponding activity in other untreated individuals such that it reduces the tumor size by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% , 90%, 95% or 100%, or reduces the number of cancer cells or is an amount sufficient to reduce the tumor growth rate. The scale of the effect can be measured using standard methods such as in vitro assays, cell-based assays, animal models or human experiments using the purified enzyme. In one embodiment of the invention, the antigen-presenting cells (e.g., dendritic cells) loaded with the cancer-specific epitopes of the invention are 1×10 ~5×10 , 5×10

[0085] ~1×10 , 1×10 5 ~2×10 5 , 2×10 5 ~3×10 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 , 1×10 6 ~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 or 5× 10 6 ~1×10 7 Any one of these cells / individuals may be administered at a dosage, but it is not limited thereto.

[0086] In another embodiment of the present invention, antigen-presenting cells (e.g., dendritic cells) loaded with cancer-specific epitopes of the present invention are 1×10 ~5×10 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 、6×1 0 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 or 1×10 5 ~1×10 7 Any one of these cells / kg may be administered at a dosage, but it is not limited thereto.

[0087] Also, in one embodiment of the present invention, the activated T cells of the present invention are 1×10 8 ~5× 10 8 、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 Any of the pieces It may be administered in a dose of one cell / individual, but is not limited thereto.

[0088] In another embodiment of the invention, the activated T cells of the invention are administered at a concentration of 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 , 1×10 9 ~2×10 9 , 2×10 9 ~ 4×10 9 , 4×10 9 ~1×10 10 , 2×10 8 ~6×10 8 , 6×10 8 ~1×1 0 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 Individual Any one of these may be administered at a dose of 100 cells / kg, but is not limited thereto. do not have.

[0089] In the present invention, the cancer-specific epitope-loaded antigen-presenting cells (e.g., dendritic Upon administration of the activated T cells, a stabilizing agent such as human serum albumin may be used. An agent or excipient may be used together.

[0090] In the present invention, the cancer-specific epitope-loaded antigen-presenting cells (e.g., dendritic The dosage and administration schedule of the administered mAb and / or activated T cells will depend on the therapeutic agent being administered. The dosage may be adjusted over the course of treatment based on the practitioner's judgment. The activated T cells then target the cancer-specific epitopes loaded on antigen-presenting cells. Administered on approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11 days On the 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st It may be administered 1 month later or simultaneously with the antigen-presenting cells, but is not limited to these It is not limited thereto.

[0091] 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 can be administered 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. Furthermore people at high risk of developing a proliferative disease can receive treatment to suppress and / or delay the onset of the disease

Advantages of the Invention

[0092] The antigen-presenting cells loaded with the Epstein-Barr virus (EBV)-positive cancer-specific epitope provided by the present invention, that is, dendritic cells, can rapidly and effectively induce the differentiation and proliferation of T cells specific to the cancer antigen preferably memory T cells. The memory T cells thus activated can avoid the cancer cell defense mechanism and treat Epstein-Barr virus (EBV)-positive cancer or a neoplastic state or prevent the recurrence, progression or metastasis of cancer.

[0093] Hitherto, in adoptive T cell therapy, it has taken 3 to 6 months to generate a large amount of T cells for the treatment of cancer patients, and there have been major problems in the cell production process in immunotherapy. However, according to the present invention, 10 to be used for the treatment of patients ​​​​​​​ 9 Its autologous memory T cells can be produced within three weeks, reducing costs and minimizing the risk factors of external contamination sources and can minimize the risk of infection against external contamination sources. Therefore, according to the present invention, a quicker treatment approach is possible for more solid cancer patients and can also be applied to terminal cancer patients, which is a technology that is applicable.

Brief Description of the Drawings

[0094]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

BEST MODE FOR CARRYING OUT THE INVENTION

[0095] According to one embodiment of the present invention, SEQ ID NOs: 1 to 3, SEQ ID NOs: 7 to 9, SEQ ID NOs: 11 to 15, SEQ ID NOs: 19 to 21, SEQ ID NOs: 23 to 39, SEQ ID NOs: 43 to 45, SEQ ID NOs: 47 to 51, SEQ ID NOs: 55 to 57, SEQ ID NOs: 59 to 63, SEQ ID NOs: 67 to 69, SEQ ID NOs: 71 to 75, SEQ ID NOs: 79 to 81, SEQ ID NOs: 83 to 111, SEQ ID NOs: 115 to 117, SEQ ID NO: 119 ~122, SEQ ID NOs: 124 to 127, SEQ ID NOs: 129 to 149, SEQ ID NOs: 151 to SEQ ID NO No. 154, SEQ ID NOs: 156 to 158, SEQ ID NOs: 160 to 163, SEQ ID NOs: 165 to 167 、SEQ ID NOs: 168 to 176, SEQ ID NOs: 178 to 181, SEQ ID NOs: 183 to 194 and SEQ It relates to an epitope specific to Epstein-Barr virus (EBV) positive cancer represented by any one of ID NOs: 196 to 199. stein barr virus; EBV) positive cancer-specific epitope (epit ope).

[0096] According to another embodiment of the present invention, the cancer-specific epitope provided by the present invention is low related to loaded antigen-presenting cells (APCs ).

[0097] According to another embodiment of the present invention, it relates to T cells activated by the antigen-presenting cells provided by the present invention .

[0098] According to another embodiment of the present invention, a cancer vaccine containing, as an active ingredient, the antigen-presenting cells loaded with the cancer-specific epitope provided by the present invention; and / or activated T cells, or a pharmaceutical composition for preventing or treating cancer is provided . According to another embodiment of the present invention, it relates to a method for preventing or treating cancer, which includes the step of administering an effective amount of the antigen-presenting cells loaded with the cancer-specific epitope provided by the present invention; and / or activated T cells to a subject

[0099] .

Example

[0100] Hereinafter, the present invention will be described in more detail through examples. These examples are only for more specifically explaining the present invention, and it is self-evident to those with ordinary knowledge in the art that the scope of the present invention is not limited by these examples

[0101] [Example 1] Method for preparing an epitope of an EBV-positive gastric cancer cell antigen The prediction of the epitope was carried out as shown in Tables 1 and 2, predicting to have the number of 15-mer amino acids in the entire sequence of LMP2a or EBNA-1, and regarding the peptide preparation process​​​​​ By the usual method of connection, the epitopes of the sequence listing described in Table 1 (LNP2a) and Table 2 (EBNA-1) below were prepared. were prepared.

[0102] [Table 1] JPEG2025106402000003.jpg233139JPEG2025106402000004.jpg233137JPEG2025106402000005.jpg56143

[0103] [Table 2] JPEG2025106402000007.jpg251133JPEG2025106402000008.jpg26148

[0104] [Example 2] ELISPOT results of T cells activated by dendritic cells loaded with the selected LMP2a epitope PBMC extracted from the blood of three healthy humans (N5, N9, or N15) were separated into monocytes and leukocytes through autologous cell sorting. The monocytes were cultured in a culture solution containing GM-CSF and IL-4 cytokines for 4 days to differentiate into dendritic cells. The monocytes that had differentiated into dendritic cells were placed in a culture solution containing 10 or 11 epitope peptides as described in Table 3 together with poly(I:C), transferred to the dendritic cells, and matured for 1 day. In the case of leukocytes, they were cultured for 3 days in a culture solution containing anti-CD3 / CD28 antibodies and IL-2 cytokines.

[0105] [Table 3]

[0106] Next, CD8-expressing T cells were isolated from leukocytes cultured in a culture solution containing anti-CD3 / CD28 antibodies and IL-2 cytokine by autologous activation cell sorting method, and co-cultured with mature dendritic cells at a ratio of 1:10 (dendritic cells:CD8-expressing T cells). During the co-culture, a cytokine cocktail containing IL-2 and IL-7, which are cytokines that help the survival and immune response of T cells, was mixed and cultured. After 18 hours, the degree of IFN-γ secretion of the T cells activated in this way was measured by ELISPOT and shown in Figures 1 to 4. However, as the peptide of the positive control group, a peptide consisting of the amino acid sequence from the 58th to the 66th of the M1 protein, which is one of the proteins expressed by influenza virus, was adopted. As can be seen from Figures 1 and 2, in the case of N5 (N5-1 and N5-2; using the blood of the same person and repeating the experiment twice by the same method), it was confirmed that the expression of IFN-γ in T cells increased by dendritic cells in which the epitopes of the 3rd, 7th, 9th, 11th, 12th, 15th, 18th, or 21st group were transformed. As can be seen from Figure 3, in the case of N9, it was confirmed that the expression of IFN-γ in T cells increased by dendritic cells in which the epitopes of the 1st, 2nd, 3rd, 7th, 8th, 10th, 15th, 16th, 19th, or 20th group were transformed. As can be seen from Figure 4, in the case of N15, it was confirmed that the expression of IFN-γ in T cells increased by dendritic cells in which the epitopes of the 1st, 2nd, 3rd, 5th, 6th, 7th, 8th, 9th, 11th, 15th, 21st, or 22nd group were transformed. As can be seen from Figures 1 and 2, in the case of N5 (N5-1 and N5-2; using the blood of the same person and repeating the experiment twice by the same method), it was confirmed that the expression of IFN-γ in T cells increased by dendritic cells in which the epitopes of the 3rd, 7th, 9th, 11th, 12th, 15th, 18th, or 21st group were transformed. As can be seen from Figure 3, in the case of N9, it was confirmed that the expression of IFN-γ in T cells increased by dendritic cells in which the epitopes of the 1st, 2nd, 3rd, 7th, 8th, 10th, 15th, 16th, 19th, or 20th group were transformed.

[0107] As can be seen from Figures 1 and 2, in the case of N5 (N5-1 and N5-2; using the blood of the same person and repeating the experiment twice by the same method), it was confirmed that the expression of IFN-γ in T cells increased by dendritic cells in which the epitopes of the 3rd, 7th, 9th, 11th, 12th, 15th, 18th, or 21st group were transformed. As can be seen from Figure 3, in the case of N9, it was confirmed that the expression of IFN-γ in T cells increased by dendritic cells in which the epitopes of the 1st, 2nd, 3rd, 7th, 8th, 10th, 15th, 16th, 19th, or 20th group were transformed. As can be seen from Figure 4, in the case of N15, it was confirmed that the expression of IFN-γ in T cells increased by dendritic cells in which the epitopes of the 1st, 2nd, 3rd, 5th, 6th, 7th, 8th, 9th, 11th, 15th, 21st, or 22nd group were transformed. As can be seen from Figures 1 and 2, in the case of N5 (N5-1 and N5-2; using the blood of the same person and repeating the experiment twice by the same method), it was confirmed that the expression of IFN-γ in T cells increased by dendritic cells in which the epitopes of the 3rd, 7th, 9th, 11th, 12th, 15th, 18th, or 21st group were transformed.

[0108] As can be seen from Figure 3, in the case of N9, it was confirmed that the expression of IFN-γ in T cells increased by dendritic cells in which the epitopes of the 1st, 2nd, 3rd, 7th, 8th, 10th, 15th, 16th, 19th, or 20th group were transformed. As can be seen from Figure 4, in the case of N15, it was confirmed that the expression of IFN-γ in T cells increased by dendritic cells in which the epitopes of the 1st, 2nd, 3rd, 5th, 6th, 7th, 8th, 9th, 11th, 15th, 21st, or 22nd group were transformed. As can be seen from Figure 3, in the case of N9, it was confirmed that the expression of IFN-γ in T cells increased by dendritic cells in which the epitopes of the 1st, 2nd, 3rd, 7th, 8th, 10th, 15th, 16th, 19th, or 20th group were transformed.

[0109] Also, as can be seen from Figure 4, in the case of N15, it was confirmed that the expression of IFN-γ in T cells increased by dendritic cells in which the epitopes of the 1st, 2nd, 3rd, 5th, 6th, 7th, 8th, 9th, 11th, 15th, 21st, or 22nd group were transformed. As can be seen from Figure 4, in the case of N15, it was confirmed that the expression of IFN-γ in T cells increased by dendritic cells in which the epitopes of the 1st, 2nd, 3rd, 5th, 6th, 7th, 8th, 9th, 11th, 15th, 21st, or 22nd group were transformed. It was confirmed that the expression of IFN-γ in T cells was increased by dendritic cells.

[0110] Thus, in the present invention, cytotoxic T lymphocytes (CTLs) can be activated by dendritic cells loaded with epitopes selected from among the epitopes described in Tables 1 and 2 above, and it was found that the T cells activated in this way have antigen specificity capable of recognizing an epitope, which is a new antigen.

[0111] [Example 3] ELISPOT results of T cells activated by dendritic cells loaded with the selected EBNA-1 epitopes Using PBMC extracted from the blood of three healthy humans (N2, N15, or N19), the degree of T cell activation was confirmed in the same manner as in Example 2 above, and the results are shown in FIGS. 5 to 8. However, monocytes differentiated into dendritic cells were transformed with a group containing 10 or 11 epitope peptides as described in Table 4 below.

[0112] [Table 4]

[0113] As can be seen from FIG. 5, in the case of N2, it was confirmed that cytotoxic T lymphocytes were activated by dendritic cells transformed with the epitopes of the 6′, 7′, 8′, and 15′ groups.

[0114] Also, as can be seen from FIGS. 6 and 7, in the case of N15 (N15-1 and N15-2; the same person's blood was used and the experiment was repeated twice in the same manner), in some partial groups ​​​​​​​​​​​Cytotoxic T lymphocytes were activated by dendritic cells in which the epitopes of all groups except were transformed.

[0115] Also, as can be seen from FIG. 8, in the case of N19, the epitopes of the 3′, 4′, 5′, 9′, 12 ′ and 15′ groups were transformed by dendritic cells, and cytotoxic T lymphocytes were confirmed to be activated.

[0116] From the results of Examples 2 and 3 above, it was found that the epitopes according to the present invention can very effectively activate cytotoxic T lymphocytes.

[0117] Having described the specific parts of the present invention in detail above, for those with ordinary knowledge in the art such specific descriptions are merely preferred embodiments, and it is clear that the scope of the present invention is not limited thereby. Therefore, it can be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

Industrial Applicability

[0118] The present invention relates to an epitope specific to cancer, an antigen-presenting cell loaded with the epitope, and a method for activating T cells for cancer treatment by the antigen-presenting cell. ​

Claims

【Claim 1】 An epitope specific to cancer, represented by any one of SEQ ID NO: 1 to 3, SEQ ID NO: 7 to 9, SEQ ID NO: 11 to 15, SEQ ID NO: 19 to 21, SEQ ID NO: 23 to 39, SEQ ID NO: 43 to 45, SEQ ID NO: 47 to 51, SEQ ID NO: 55 to 57, SEQ ID NO: 59 to 63, SEQ ID NO: 67 to 69, SEQ ID NO: 71 to 75, SEQ ID NO: 79 to 81, SEQ ID NO: 83 to 111, SEQ ID NO: 115 to 117, SEQ ID NO: 119 to 122, SEQ ID NO: 124 to 127, SEQ ID NO: 129 to 149, SEQ ID NO: 151 to SEQ ID NO: 154, SEQ ID NO: 156 to 158, SEQ ID NO: 160 to 163, SEQ ID NO: 165 to 167, SEQ ID NO: 168 to 176, SEQ ID NO: 178 to 181, SEQ ID NO: 183 to 194, and SEQ ID NO: 196 to 199.

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