Pharmaceutical composition for preventing and treating lung adenocarcinoma comprising k-ras-specific activated t cells and method for preparing same

Abstract

According to the present invention, a pharmaceutical composition for preventing and treating lung adenocarcinoma, comprising K-ras-specific activated T cells, is used as an antigen composition designed such that a K-ras mutant (G12D, G12V, and G13D) recombinant overlapping peptide is sequentially divided into a total of 12 epitopes (n=1 to 12, and the last epitope (n=12) consists of 23 amino acids) in units of 30 amino acids in the amino acid sequence of K-ras, and 15 amino acid sequences overlap between the epitopes. Thus, the pharmaceutical composition is more effective in recognizing and killing lung adenocarcinoma in which K-ras, K-ras mutant G12V, K-ras mutant G12D, or K-ras mutant G13D is detected. Therefore, according to the present invention, the pharmaceutical composition for preventing and treating lung adenocarcinoma, comprising K-ras-specific activated T cells, can be used to effectively prevent and treat lung adenocarcinoma, particularly lung large cell adenocarcinoma, in which K-ras as well as K-ras mutants are detected.

Description

Pharmaceutical composition for the prevention and treatment of lung adenocarcinoma containing K-RAS-specific activated T cells and method for preparing the same

[0001] The present invention relates to a pharmaceutical composition for the prevention and treatment of lung adenocarcinoma containing K-ras specific activated T cells and a method for preparing the same.

[0002] Conventional chemotherapy has limitations in suppressing cancer recurrence and metastasis, and has the side effect of killing normal cells. To overcome these limitations, various immunotherapy approaches are being studied to treat cancer by enhancing the immune response to tumors.

[0003] Immunotherapy is a treatment method that activates the immune system of cancer patients to increase the effectiveness of cancer treatment. It is a method of killing cancer cells by inducing cancer-specific CTLs (Cytotoxic T Lymphocytes) endogenously or injecting them. Among immunotherapy methods for cancer, a method that utilizes immune cells is lymphokine activated killer (LAK) immunotherapy. The LAK immunotherapy has shown encouraging clinical results in cancer patients who have failed surgery, chemotherapy, and radiation therapy. However, the LAK immunotherapy has the problem of low cancer cell killing efficiency due to the non-specific immune response. In addition, because it uses only interleukin-2 (IL-2) lymphokine during the culture process, immune cell exhaustion occurs, reducing in vivo viability. Therefore, periodic and repeated treatments are required, resulting in clinical limitations.

[0004] To address the above issues, anticancer peptide vaccines are being developed. These peptide vaccines select highly immunogenic portions of cancer antigens, design them into peptides, and then use them to activate immune cells. Therefore, they have the advantage of eliminating concerns about immune cell exhaustion due to the use of IL-2 lymphokine. However, these peptide vaccines have the disadvantage of not only causing immune evasion if the target region of cancer cells undergoes mutations, but also of not receiving CD4 T cell support in the immune response, leading to low immune reactivity. Furthermore, because they are designed as peptides that are loaded onto MHC class I molecules, they are limited in their human leukocyte antigen (HLA) typing.

[0005] To address these issues, overlapping peptide (OLP) vaccines are being developed, which contain the entire antigen and overlap highly immunogenic peptides. Unlike conventional peptide vaccines, these OLP vaccines contain the entire antigen, resulting in a higher immune response with CD4 T cell support, a longer-lasting immune response, and no restrictions on HLA typing. However, these OLP vaccines have the disadvantages of being expensive to manufacture and relatively difficult to immunomodulate.

[0006] In immunocytotherapy, the method of producing antigen-specific T cells using vaccines is also an important factor. Typically, to produce antigen-specific T cells, monocytes are isolated from blood, then maturated to obtain mo dendritic cells. Then, the dendritic cells and T cells are co-cultured in an environment where the antigen has been separately treated, thereby producing antigen-specific T cells. However, the method using moDCs has the disadvantage of requiring additional time, cost, and blood because the antigen-specific T cell induction method is performed separately by performing the DC cell maturation process and the T cell culture process.

[0007] Among various cancer antigens, K-ras mutations are oncogenic mutations found in approximately 20% of solid cancers, mainly in adenocarcinomas of the pancreas and colon, and lung cancer. Therapeutic treatments targeting tumors dependent on K-ras mutant genes have been limited in their effectiveness due to the difficulty in creating antibodies that individually bind to K-ras mutants expressed by the K-ras mutant genes, which is why they are limited to indirect treatments that inhibit or inactivate the function of K-ras.

[0008] Therefore, if an antigen that includes several of the most frequently occurring mutation sites of K-ras but solves the above problems is presented, and endogenous immune cells that can bind to K-ras mutations and cause a direct immune response are selectively amplified and developed into a targeted cancer treatment, it is expected that cancer treatment effects will be maximized and it will be possible to use it as an efficient treatment with a long-lasting anticancer effect because it can efficiently target and kill cancer cells.

[0009] The patent documents and references mentioned in this specification are incorporated herein by reference to the same extent as if each document were individually and specifically identified by reference.

[0010] The purpose of the present invention is to provide a pharmaceutical composition for the prevention and treatment of lung adenocarcinoma, which comprises an antigen for inducing K-ras-specific activated T cells, designed to include the entire amino acid sequence and mutations of K-ras and manufactured using recombinant technology, and which is highly economically efficient, and a method for manufacturing the same.

[0011] Other objects and technical features of the present invention are presented more specifically in the detailed description of the invention, the claims and the drawings below.

[0012] The present invention is characterized by providing a pharmaceutical composition for the prevention and treatment of lung adenocarcinoma, which comprises an antigen composition for inducing K-ras-specific activated T cells and K-ras-specific activated T cells induced using cytokines, and wherein the cancer cells of the lung adenocarcinoma are characterized by detecting K-ras, K-ras mutant G12V, K-ras mutant G12D, or K-ras mutant G13D.

[0013] The antigen composition for inducing the above K-ras specific activated T cell is characterized in that it comprises as an active ingredient a K-ras mutant recombinant overlapping peptide consisting of an amino acid sequence of SEQ ID NO: 1, and the K-ras mutant recombinant overlapping peptide comprises a total of 12 types of epitopes (epitopes (n=1, 2, 3....10, 11, 12); here, n means the order of the epitopes, and the epitopes (n=1 to 11) include a 30 amino acid sequence and the last epitope (n=12) includes a 23 amino acid sequence) in which a 15 amino acid sequence in the N-terminal direction is the same as that in the C-terminal direction of the epitope (n-1) of the immediately preceding order. It is characterized by being designed to overlap with 15 amino acid sequences.

[0014] The above K-ras mutant recombinant overlapping peptide is characterized in that the epitopes (n=1, 2, 3...10, 11, 12) are sequentially located and the epitopes are connected by an LRMK-linker.

[0015] The above epitope (n=1) comprises a K-ras mutant G12V; the N-terminal of the epitope (n=1) is further linked to an epitope (n=1) comprising a K-ras mutant G12D via an LRMK-linker; the C-terminal of the epitope (n=12) is further linked to an epitope (n=1) comprising a K-ras mutant G13D via an LRMK-linker; and the C-terminal of the epitope (n=1) comprising the K-ras mutant G13D is further linked to an epitope (n=1) not comprising a K-ras mutation via an LRMK-linker.

[0016] The above K-ras specific activated T cells are characterized by being produced by a method for inducing K-ras specific activated T cells using an antigen composition for inducing K-ras specific activated T cells, comprising: a first step of culturing peripheral blood mononuclear cells (PBMCs) in a medium containing the K-ras specific activated T cell induction antigen composition and a first cytokine to mature dentritic cells and simultaneously induce K-ras specific activated T cells; a second step of adding a second cytokine to the cultured PBMCs and culturing them to further induce K-ras specific activated T cells; and a third step of culturing the PBMCs cultured with the second cytokine added thereto to amplify and then obtain K-ras specific activated T cells.

[0017] The primary cytokines are interleukin-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF); the secondary cytokines are tumor necrosis factor-α, interleukin-1β, and prostaglandin E2.

[0018] The pharmaceutical composition for preventing and treating lung adenocarcinoma comprising K-ras-specific activated T cells of the present invention is designed to use K-ras mutant (G12D, G12V, and G13D) recombinant overlapping peptides as an antigen composition, which are sequentially divided into 12 epitopes (epitopes, n=1 to 12, provided that the last epitope (n=12) is 23 amino acids) in units of 30 amino acids in the amino acid sequence of K-ras, with 15 amino acid sequences overlapping between the epitopes, so that it has the advantage of being more effective in recognizing and killing lung adenocarcinoma in which K-ras, K-ras mutant G12V, K-ras mutant G12D, or K-ras mutant G13D is detected.

[0019] Therefore, the pharmaceutical composition for preventing and treating lung adenocarcinoma containing K-ras-specific activated T cells of the present invention has the advantage of being able to effectively prevent and treat not only K-ras but also lung adenocarcinoma in which K-ras mutations are detected, particularly lung large cell adenocarcinoma.

[0020] Figure 1 shows the amino acid sequence structure of K-ras(M)-ROP of the present invention.

[0021] Figure 2 shows the results of analyzing the reactivity of PBMC to K-ras(M)-ROP of the present invention.

[0022] Figure 3 shows the results of analyzing the specific CD3+ T cell ratio of LP-1 PBMC according to the concentration of K-ras(M)-ROP of the present invention.

[0023] Figure 4 is a K-ras(M)-ROP, K-ras of the present invention. 1-24 Wild-type, and K-ras 1-24Shows the results of analyzing the antigen-specific CD3+ T cell ratio of LP-1 PBMCs for mutations.

[0024] Figure 5 shows the results of comparing the Fast-IVS process and the No-Cytokine process of the present invention.

[0025] Figure 6 shows the results of K-ras mutant epitope screening for ROP-T cells of the present invention.

[0026] Figure 7 shows the results of the HLA-DQ blocking assay of the present invention.

[0027] Figure 8 shows the proportion of CD3+ T cells secreting IFN-γ (IFN-γ+) according to the conditions of the present invention.

[0028] Figure 9 shows the extent to which ROP-T cells and LAK-T cells of the present invention kill cancer cells.

[0029] Figure 10 shows the extent to which the ROP-T cells and LAK-T cells of the present invention inhibit the growth of colon cancer cells (K-ras-G12D) obtained from a colon cancer patient.

[0030] The present invention provides a pharmaceutical composition for the prevention and treatment of lung adenocarcinoma, comprising an antigen composition for inducing K-ras specific activated T cells and K-ras specific activated T cells induced using cytokines. The term "prevention" in the present invention means any act of suppressing or delaying cancer by administering the pharmaceutical composition for the prevention and treatment of lung adenocarcinoma of the present invention, and the term "treatment" in the present invention means any act of improving or beneficially changing the symptoms of cancer by administering the pharmaceutical composition for the prevention and treatment of lung adenocarcinoma.

[0031] The lung adenocarcinoma of the present invention is characterized in that K-ras, K-ras mutant G12V, K-ras mutant G12D, or K-ras mutant G13D are detected in cancer cells. The K-ras gene, KRAS, is a representative proto-oncogene and is involved in cell growth and differentiation in vertebrates. When it is changed into an oncogene by point mutation, chromosomal translocation, gene amplification, etc., the activity of K-ras is abnormally increased, causing cancer. Therefore, the detection of K-ras, K-ras mutant G12V, K-ras mutant G12D, or K-ras mutant G13D in the lung adenocarcinoma cancer cells of the present invention means that KRAS is expressed as an oncogene and the activity of K-ras is abnormally increased. Preferably, the lung adenocarcinoma of the present invention is characterized in that K-ras mutation G12V, K-ras mutation G12D, or K-ras mutation G13D is detected in cancer cells, and more preferably, K-ras mutation G12D is detected.

[0032] The above K-ras mutations G12V, G12D, and G13D are mutations found in lung adenocarcinoma, pancreatic adenocarcinoma, ovarian adenocarcinoma, colorectal adenocarcinoma, and rectal adenocarcinoma. They are known to induce cancer cell proliferation by increasing the cellular level of GTP-bound RAS protein by preventing smooth GTP hydrolysis by GTPase-activating proteins (GAPs), thereby abnormally activating the downstream signaling system.

[0033] The pharmaceutical composition for preventing and treating lung adenocarcinoma comprising K-ras specific activated T cells of the present invention comprises an antigen composition for inducing K-ras specific activated T cells and K-ras specific activated T cells induced using cytokines, and therefore can be used for treating cancers such as lung cancer, colon cancer, breast cancer, and melanoma in which K-ras, K-ras mutant G12V, K-ras mutant G12D, or K-ras mutant G13D are detected, and preferably can be used for treating lung carcinoma in which K-ras, K-ras mutant G12V, K-ras mutant G12D, or K-ras mutant G13D are detected, and more preferably can be used for treating lung large cell adenocarcinoma in which K-ras, K-ras mutant G12V, K-ras mutant G12D, or K-ras mutant G13D are detected, and most preferably can be used for treating lung large cell adenocarcinoma in which K-ras, K-ras mutant G12V, K-ras mutant G12D, or K-ras mutant G13D is detected, and most preferably can be used for treating lung large cell adenocarcinoma in which K-ras, K-ras mutant G12D It can be used to treat lung large cell adenocarcinoma that is detected.

[0034] The “composition” described in the present invention means a combination of the active ingredient with an inert ingredient such as a natural or artificial carrier, label or detector, or an adjuvant, diluent, binder, stabilizer, buffer, salt, lipophilic solvent, preservative, together with the K-ras specific activated T cell according to the present invention as an active ingredient, and includes a pharmaceutically acceptable carrier.

[0035] The carrier may comprise pharmaceutical excipients and additional proteins, peptides, amino acids, lipids, and carbohydrates (e.g., monosaccharides; disaccharides; trisaccharides; tetrasaccharides; oligosaccharides; sugar derivatives such as alditols, aldonic acids, esterified sugars, polysaccharides, or sugar polymers), alone or in combination, in an amount of 1 to 99.99 wt% or vol%.

[0036] Protein excipients may include, but are not limited to, human serum albumin, recombinant human albumin, gelatin, casein, and the like.

[0037] Representative amino acid components that can act as a buffer include, but are not limited to, alanine, arginine, glycine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, and aspartame.

[0038] Carbohydrate excipients may include, but are not limited to, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, and sorbose; disaccharides such as lactose, sucrose, trehalose, and cellobiose; polysaccharides such as raffinose, maltodextrin, dextran, and starch; and alditols such as mannitol, xylitol, maltitol, lactitol, sorbitol, and myoinositol.

[0039] The pharmaceutical composition for the prevention and treatment of lung adenocarcinoma containing K-ras specific activated T cells of the present invention can be formulated by a method known to those skilled in the art. The pharmaceutical composition of the present invention can be used parenterally in the form of an injection of a sterile solution or suspension with water or other pharmaceutically acceptable liquid, as needed, and can be formulated by mixing it appropriately with a pharmaceutically acceptable carrier or medium, specifically, sterile water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, excipient, vehicle, preservative, binder, etc., in the form of a unit dosage required for generally recognized pharmaceutical practice. In the above formulation, the amount of the active ingredient means that an appropriate dosage within the indicated range can be obtained.

[0040] When the pharmaceutical composition for the prevention and treatment of lung adenocarcinoma containing the K-ras specific activated T cells of the present invention is formulated as a sterile composition for injection, it can be prescribed according to the usual formulation practice using an excipient such as distilled water for injection. As the aqueous solution for injection, an isotonic solution containing physiological saline, glucose, and other auxiliary drugs, for example, D-sorbitol, D-mannose, D-mannitol, and sodium chloride can be used, and a suitable solubilizing agent, for example, polyalcohol, propylene glycol, and polyethylene glycol as ethanols and polysorbate 80(TM) and HCO-50 as nonionic surfactants can be used in combination. In addition, sesame oil and soybean oil can be used as an oily liquid, and benzyl benzoate and benzyl alcohol can be used in combination as a solubilizing agent.

[0041] Examples of the above injection types include intravenous injection types, intraarterial injection types, selective intraarterial injection types, intramuscular injection types, intraperitoneal injection types, subcutaneous injection types, intraventricular injection types, intracerebral injection types, and intramedullary injection types, and preferably, the above injection types are intravenous injection types.

[0042] The pharmaceutical composition for the prevention and treatment of lung adenocarcinoma comprising K-ras-specific activated T cells of the present invention comprises a pharmaceutically effective amount of K-ras-specific activated T cells. The determination of the effective amount can be readily determined by a person skilled in the art based on the contents disclosed herein.

[0043] In general, the above pharmaceutical effective dose is determined by first administering the active ingredient at a low concentration and then gradually increasing the dose until the desired effect, for example, the effect of reducing or eliminating cancer-related symptoms, is obtained without side effects in the subject. Methods for determining the appropriate dosage or dosing interval of the pharmaceutical composition for preventing and treating lung adenocarcinoma of the present invention are described in detail in Goodman and Gilman's The Pharmacological Basis of Therapeutics, Goodman et al., eds., 11th Edition, McGraw-Hill 2005, and Remington: The Science and Practice of Pharmacy, 20th and 21st Editions, Gennaro and University of the Sciences in Philadelphia, Eds., Lippencott Williams & Wilkins (2003 and 2005).

[0044] The method of administering the pharmaceutical composition for the prevention and treatment of lung adenocarcinoma of the present invention can be determined by taking into consideration various factors such as the type of cancer, the patient's age, weight, sex, medical condition, severity of the disease, administration route, and separately administered drugs. The amount of the pharmaceutical composition for the prevention and treatment of lung adenocarcinoma of the present invention administered to a patient can be determined by many factors such as the administration method, the patient's health condition, weight, and the doctor's prescription, and this is within the scope of knowledge of a person having ordinary skill in the art.

[0045] The pharmaceutical composition for the prevention and treatment of lung adenocarcinoma of the present invention comprises about 1x10 6 cells / ㎖ or more, approximately 2x10 6 cells / ㎖ or more, approximately 3x10 6 cells / ㎖ or more, approximately 4x10 6 cells / ㎖ or more, approximately 5x10 6 cells / ㎖ or more, approximately 6x10 6 cells / ㎖ or more, approximately 7x10 6 cells / ㎖ or more, approximately 8x10 6 cells / ㎖ or more, approximately 9x10 6 cells / ㎖ or more, approximately 1x10 7 cells / ㎖ or more, approximately 2x10 7 cells / ㎖ or more, approximately 3x10 7 cells / ㎖ or more, approximately 4x10 7 cells / ㎖ or more, approximately 5x10 7 cells / ㎖ or more, approximately 6x10 7 cells / ㎖ or more, approximately 7x10 7 cells / ㎖ or more, approximately 8x10 7 cells / ㎖ or more, approximately 9x10 7 cells / ㎖ or more, approximately 1x10 8 cells / ㎖ or more, approximately 2x10 8cells / ㎖ or more, approximately 3x10 8 cells / ㎖ or more, approximately 4x10 8 cells / ㎖ or more, approximately 5x10 8 cells / ㎖ or more, approximately 6x10 8 cells / ㎖ or more, approximately 7x10 8 cells / ㎖ or more, approximately 8x10 8 cells / ㎖ or more, or approximately 9x10 8 cells / ㎖ or more of K-ras specific activated T cells, but a skilled person will be able to adjust the concentration of K-ras specific activated T cells in the composition within a variable range to achieve the same effect.

[0046] The above “about” may be understood as within a range commonly accepted in the art, for example, within the mean standard deviation range, and may be understood as within 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value.

[0047] Additionally, it can be further combined with buffers such as phosphate buffer and sodium acetate buffer, an analgesic such as procaine hydrochloride, a stabilizer such as benzyl alcohol or phenol, and an antioxidant. The above-prepared injection solution is usually filled into a suitable ampoule. Suspensions and emulsions can contain natural gums, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol as a carrier. Suspensions or solutions for intramuscular injection can contain the active compound together with a pharmaceutically acceptable carrier such as sterile water, olive oil, ethyl oleate, glycols, and a suitable amount of lidocaine hydrochloride.

[0048] The pharmaceutical composition for preventing and treating lung adenocarcinoma of the present invention can be administered to a patient by bolus injection or continuous infusion. The pharmaceutical composition for preventing and treating lung adenocarcinoma of the present invention can be administered at least once, at least twice, at least three times, at least four times, or at least five times, continuously, or at regular time intervals, or at time intervals determined by clinical judgment, for 1 hour or less, 1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 8 hours or more, 12 hours or more, 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 2 weeks or more, 3 weeks or more, 4 weeks or more, 1 month or more, 3 months or more, 6 months or more.

[0049] The injection may be formulated in ampoule form or in unit-dose form in multiple-dose containers. However, those skilled in the art will understand that the dosage of the pharmaceutical composition according to the present invention may vary depending on various factors, such as the patient's age, weight, height, sex, general medical condition, and prior treatment history.

[0050] The present invention is characterized in that the K-ras-specific activated T cells are produced by a method for inducing K-ras-specific activated T cells using an antigen composition for inducing K-ras-specific activated T cells, the method comprising: a first step of culturing peripheral blood mononuclear cells (PBMCs) in a medium containing an antigen composition for inducing K-ras-specific activated T cells and a primary cytokine to mature dentritic cells and simultaneously induce K-ras-specific activated T cells; a second step of adding a secondary cytokine to the cultured PBMCs and culturing them to further induce K-ras-specific activated T cells; and a third step of culturing the PBMCs, which have been cultured with the secondary cytokine added thereto, to amplify and then obtain K-ras-specific activated T cells.

[0051] The method for inducing K-ras-specific activated T cells of the present invention is Fast-IVS (in vitro stimulation), which is distinguished from the conventional IVS. The IVS refers to a method of obtaining monocyte-derived dendritic cells (moDCs) through differentiation and maturation processes from monocytes isolated from blood, and then co-culturing them with T cells in an environment in which the antigen composition for inducing K-ras-specific activated T cells of the present invention has been treated. In contrast, the Fast-IVS of the present invention has a difference in that the maturation process and antigen (antigen composition for inducing K-ras-specific activated T cells) treatment are performed simultaneously on DC cells in PBMCs.

[0052] The antigen composition for inducing K-ras-specific activated T cells used as an antigen in the above T cell induction process may further include cytokines, hormones, and buffers necessary for the maturation and growth of DC cells. Preferably, the cytokines may be interleukin-4, interleukin-1β, Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), and Tumor Necrosis Factor-α (TNF-α), and the hormone may be prostaglandin E2 (PGE2).

[0053] According to one embodiment of the present invention, the interleukin-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) were used as primary cytokines to induce differentiation of monocytes in PBMCs into dendritic cells (DCs), and the tumor necrosis factor-α, interleukin-1β, and prostaglandin E2 were used as secondary cytokines to mature immature DCs.

[0054] According to another embodiment of the present invention, the culture of the first step is performed for 1 day, the culture of the second step is performed for 2 days, and the culture of the third step is performed for 10 days. The culture periods for each step are optimized through the embodiment as a method capable of most efficiently inducing K-ras-specific activated T cells.

[0055] The present invention is characterized in that the antigen composition for inducing K-ras specific activated T cells comprises a K-ras mutant recombinant overlapping peptide consisting of the amino acid sequence of sequence number 1 as an active ingredient.

[0056] The above sequence number 1 is derived from the amino acid sequence (SEQ ID NO: 2) of the K-ras protein consisting of 189 amino acids. The above K-ras mutation means that the 12th amino acid is substituted from glycine (G) to aspartic acid (D), or the 12th amino acid is substituted from glycine (G) to valine (V), or the 13th amino acid is substituted from glycine (G) to aspartic acid (D).

[0057] The above recombination means inserting the genetic information of the designed antigen into a recombinant plasmid DNA, and when the recombinant plasmid DNA is transformed into a microorganism to express the protein and purify it, the antigen for inducing K-ras-specific activated T cells of the present invention is obtained.

[0058] The K-ras mutant recombinant overlapping peptide of the present invention comprises a total of 12 types of epitopes (epitopes (n=1, 2, 3....10, 11, 12); here, n represents the order of the epitopes, and the epitopes (n=1 to 11) include a 30 amino acid sequence and the last epitope (n=12) includes a 23 amino acid sequence) whose units are sequentially listed amino acid sequences from any one amino acid in the amino acid sequence of K-ras consisting of SEQ ID NO: 2, but the epitopes (n=2, 3,...12) excluding the epitope (n=1) are designed such that the 15 amino acid sequences in the N-terminal direction overlap with the 15 amino acid sequences in the C-terminal direction of the epitope (n-1) of the immediately preceding order.

[0059] The above K-ras mutant recombinant overlapping peptide is characterized in that the epitopes (n=1, 2, 3...10, 11, 12) are sequentially positioned and the epitopes are connected by an LRMK-linker. The LRMK-linker is a linker composed of Leucine (L), Arginine (R), Methionine (M), and Lysine (K), and has an advantage in the antigen presentation process (MHC class I pathway) by dendritic cells.

[0060] In detail, the antigen composition for inducing K-ras specific activated T cells of the present invention is designed as follows. The epitope (n=1) includes a K-ras mutant G12V; an epitope (n=1) including a K-ras mutant G12D is further linked to the N-terminal of the epitope (n=1) via an LRMK linker; an epitope (n=1) including a K-ras mutant G13D is further linked to the C-terminal of the epitope (n=12) via an LRMK linker; and an epitope (n=1) not including a K-ras mutation is further linked to the C-terminal of the epitope (n=1) including the K-ras mutant G13D via an LRMK linker.

[0061] By using the antigen composition for inducing K-ras specific activated T cells of the present invention, T cells specific for K-ras mutations (G12D, G12V, G13D) can be induced, and the T cells specific for the K-ras mutations (G12D, G12V, G13D) can be used to treat cancer cells having the K-ras mutations (G12D, G12V, G13D).

[0062] K-ras is a type of ras protein and a small GTPases protein that plays an important role in the signaling system related to cell differentiation, proliferation, and survival. It is known that cancer is induced when the activity of K-ras protein increases abnormally. KRAS, the gene for the ras protein, is well known as an oncogene found as a mutation in various cancers, and 85% of ras-derived cancers are known to be caused by K-ras mutations. Therefore, if T cells that specifically recognize K-ras expressed in cancer cells and its mutations are amplified, cancers in which the activity of K-ras is abnormally increased can be effectively removed and treated. The above cancers are not limited to cancers with increased K-ras activity, and examples thereof include adrenocortical carcinoma (ACC), bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), colon adenocarcinoma (COAD), chronic lymphocytic leukemia (CLL), colorectal cancer (CRC), diffuse large B-cell lymphoma (DLBCL), glioblastoma multiforme (GBM), head and neck squamous cell carcinoma (HNSC), chromophobe kidney (KICH), renal clear cell carcinoma (KIRC), renal papillary cell carcinoma (KIRP), acute myeloid leukemia (LAML), hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), multiple myeloma (MM), ovarian serous cystadenocarcinoma (OV), pancreatic adenocarcinoma (PAAD), prostate adenocarcinoma (PRAD), rectal adenocarcinoma (READ), skin It may be melanoma (SKCM), gastric adenocarcinoma (STAD), testicular germ cell tumor (TGCT), thyroid adenocarcinoma (THCA), uterine corpus endometrial carcinoma (UCEC), or uterine carcinosarcoma (UCS).

[0063] The present invention is described in detail below through examples.

[0064]

[0065] Example

[0066]

[0067] 1. Preparation of K-ras(WT)

[0068] First, the K-ras amino acid sequence (SEQ ID NO: 2) was inserted into the expression vector. K-ras (WT) consists of 189 amino acids, and the amino acid sequence is shown in Table 1 below.

[0069] Name Amino acid sequence (189aa) K-ras (WT) MTEYKLVVVG 10 AGGVGKSALT 20 IQLIQNHFVD 30 EYDPTIEDSY 40 RKQVVIDGET 50 CLLDILDTAG 60 QEEYSAMRDQ 70 YMRTGEGFLC 80 VFAINNTKSF 90 EDIHHYREQI 100 KRVKDSEDVP 110 MVLVGNKCDL 120 PSRTVDTKQA 130 QDLARSYGIP 140 FIETSAKTRQ 150 RVEDAFYTLV 160 REIRQYRLKK 170 ISKEEKTPGC 180 VKIKKCIIM 189

[0070] The above K-ras-WT was treated with a restriction enzyme and inserted into the pET30a vector to produce an expression vector, and the expression vector was transformed into E. coli to express the protein.

[0071]

[0072] 2. Preparation of K-ras(M)-ROP

[0073] The antigen of K-ras Mutant Recombinant Overlapping Peptide (K-ras(M)-ROP) was designed and an expression vector was prepared in the same manner as K-ras(WT). The K-ras(M)-ROP includes G12D, in which G(Glycine) at the 12th position of the K-ras amino acid is mutated to D(Asapartic acid), G12V, in which G(Glycine) at the 12th position of the K-ras amino acid is mutated to V(Valine), or G13D, in which G(Glycine) at the 13th position of the K-ras amino acid is mutated to D(Asapartic acid).

[0074] The above K-ras(M)-ROP is characterized by having a 500 amino acid sequence, and the amino acids are sequentially separated into groups of 30, but each epitope is designed so that 15 amino acid sequences overlap with each other. Table 2 shows the amino acid sequence of K-ras(M)-ROP (SEQ ID NO: 1) and the amino acid sequence of the K-ras(M)-ROP epitope.

[0075] Figure 1 shows the epitope structure of K-ras(M)-ROP of the present invention.

[0076] Name Amino acid sequence (500aa) K-ras (M) - ROPMTEYKLVVVG 10 ADGVGKSALT 20 IQLIQNHFVD 30 LRMK 34 MTEYKLVVVG 44 AVGVGKSALT 54 IQLIQNHFVD 64 LRMK 68 KSALTIQLIQ 78 NHFVDEYDPT 88 IEDSYRKQVV 98 LRMK 102 EYDPTIEDSY 112 RKQVVIDGET 122 CLLDILDTAG 132 LRMK 136 IDGETCLLDI146 LDTAGQEEYS 156 AMRDQYMRTG 166 LRMK 170 QEEYSAMRDQ 180 YMRTGEGFLC 190 VFAINNTKSF 200 LRMK 204 EGFLCVFAIN 214 NTKSFEDIHH 224 YREQIKRVKD 234 LRMK 238 EDIHHYREQI 248 KRVKDSEDVP 258 MVLVGNKCDL 268 LRMK 272 SEDVPMVLVG 282 NKCDLPSRTV 292 DTKQAQDLAR 302 LRMK 306 PSRTVDTKQA 316 QDLARSYGIP 326 FIETSAKTRQ 336 LRMK 340 SYGIPFIETS 350 AKTRQRVEDA 360 FYTLVREIRQ 370 LRMK 374 RVEDAFYTLV 384 REIRQYRLKK 394 ISKEEKTPGC 404 LRMK 408 YRLKKISKEE 418 KTPGCVKIKK 428 CIIM 432 LRMK 436 MTEYKLVVVG 446 AGDVGKSALT 456 IQLIQNHFVD 466 LRMK 470 MTEYKLVVVG 480 AGGVGKSALT 490 IQLIQNHFVD 500 Epitope name Amino acid sequence (sequence number assigned based on K-rasWT) Epitope 1 (E1, n=1) MTEYKLVVVG 10 AGGVGKSALT20 IQLIQNHFVD 30 Epitope 2 (E2, n=2) KSALT 20 IQLIQNHFVD 30 EYDPTIEDSY 40 RKQVV 45 Epitope 3 (E3, n=3)EYDPTIEDSY 40 RKQVVIDGET 50 CLLDILDTAG 60 Epitope 4 (E4, n=4) IDGET 50 CLLDILDTAG 60 QEEYSAMRDQ 70 YMRTG 75 Epitope 5 (E5, n=5)QEEYSAMRDQ 70 YMRTGEGFLC 80 VFAINNTKSF 90 Epitope 6 (E6, n=6) EGFLC 80 VFAINNTKSF 90 EDIHHYREQI 100 KRVKD 105 Epitope 7 (E7, n=7) EDIHHYREQI 100 KRVKDSEDVP 110 MVLVGNKCDL 120 Epitope 8 (E8, n=8)SEDVP 110 MVLVGNKCDL 120 PSRTVDTKQA 130 QDLAR 135 Epitope 9 (E9, n=8)PSRTVDTKQA 130 QDLARSYGIP 140 FIETSAKTRQ 150 Epitope 10 (E10, n=10) SYGIP 140 FIETSAKTRQ 150 RVEDAFYTLV 160 REIRQ 165 Epitope 11 (E11, n=11)RVEDAFYTLV 160 REIRQYRLKK 170 ISKEEKTPGC 180 Epitope 12 (E12, n=12)YRLKK 170 ISKEEKTPGC 180VKIKKCIIM 189 Epitope 1-G12D (E1-G12D)MTEYKLVVVG 10 ADGVGKSALT 20 IQLIQNHFVD 30 Epitope 1-G12V (E1-G12V)MTEYKLVVVG 10 AVGVGKSALT 20 IQLIQNHFVD 30 Epitope 1-G13D (E1-G13D)MTEYKLVVVG 10 AGDVGKSALT 20 IQLIQNHFVD 30

[0077] The above K-ras(M)-ROP was synthesized (Genescript Co. Ltd.) and cloned into the pET30a vector, and expressed by transformation into E. coli. The expressed protein was cleaved using APC (Activated protein C) to produce K-ras(M)-ROP.

[0078]

[0079] 3. K-ras(M)-ROP reactivity screening assay

[0080] The reactivity of K-ras(M)-ROP was screened in peripheral blood mononuclear cells (PBMCs) from normal individuals. The screening was performed using the enzyme-linked immune absorbent spot assay (ELISpot assay).

[0081] Figure 2 shows the results of analyzing the reactivity of PBMCs to K-ras(M)-ROP of the present invention. Panel A shows an SFC image of an ELISpot(IFN-γ) assay, and Panel B shows an SFC graph of the ELISpot(IFN-γ) assay.

[0082] First, 1x10 PBMCs (LP-1 PBMCs, LP-4 PBMCs, and LP-6 PBMCs) obtained from leukocyte apheresis of normal volunteers 5 Cells were seeded, cultured, and then treated with antigens. K-ras(M)-ROP 5㎍ / ㎖, 1.0㎍ / ㎖, and 0.1㎍ / ㎖ were used as the antigen, and anti-CD3 was used as a positive control. Cell culture was performed under the conditions of 37℃, CO25%, overnight (O / N), and the cultured cells were stained with IFN-γ and analyzed by reading SFC (Spot Forming Cell). As a result of the experiment, it was confirmed that among normal PBMCs, LP-1 had the best reactivity to K-ras(M)-ROP.

[0083]

[0084] 4. Analysis of specific CD3+ T cell ratios by K-ras(M)-ROP concentration

[0085] The proportion of K-ras(M)-ROP-specific CD3+ T cells according to the concentration of K-ras(M)-ROP in the above LP-1 PBMCs was analyzed. To this end, LP-1 PBMCs were treated with antigen, and then IFN-γ capture staining was performed and analyzed.

[0086] Figure 3 shows the results of analyzing the K-ras(M)-ROP-specific CD3+ T cell ratio of LP-1 PBMC according to the concentration of K-ras(M)-ROP of the present invention. Panel A shows the SFC image of the ELISpot(IFN-γ) assay according to the conditions, and Panel B shows the SFC graph of the ELISpot(IFN-γ) assay according to the conditions. Panel C shows the principle and method of IFN-γ capture staining, and Panel D shows the result of IFN-γ capture FACS analysis. Panel E shows a graph of the percentage (%) of IFN-γ-secreting CD3+ T cells.

[0087] First, LP-1 PBMC 1x10 6 After seeding and culturing the cells, antigens (K-ras(M)-ROP 5 ㎍ / ㎖, K-ras(M)-ROP 1.0 ㎍ / ㎖, K-ras(M)-ROP 0.1 ㎍ / ㎖) were treated at different concentrations. In addition, LP-1 PBMCs treated with 5 ㎍ / ㎖ and 1.0 ㎍ / ㎖ of tetanus toxoid vaccine (TTX) and anti-CD3 were used as positive controls. Cell culture was performed at 37°C, CO25%, overnight (O / N).

[0088] IFN-γ capture staining was performed by incubating antigen-treated LP-1 PBMCs with primary capture antibody (1 st After treatment with capture antibody, the cells were incubated at 37°C for 45 minutes and then treated with secondary detection antibody (2 nd The method was performed by processing IFN-γ capture and antigen (antibody) and CD3, CD4, CD8, and CD137. The LP-1 PBMCs on which the IFN-γ capture staining was performed were analyzed for cell characteristics using a fluorescence activated cell sorter (FACS).

[0089] The experimental results showed that when LP-1 PBMCs were treated with antigens at various concentrations, the proportion of K-ras(M)-ROP-specific CD2+ T cells increased, which was well consistent with the ELISpot (IFN-γ) results above. Therefore, it is believed that the reactivity of LP-1 PBMCs increases in a concentration-dependent manner to K-ras(M)-ROP. In addition, when the reactivity of LP-1 PBMCs to K-ras(M)-ROP was quantitatively evaluated, it was confirmed that when 5㎍ / ㎖ of K-ras(M)-ROP was treated, the proportion of K-ras(M)-ROP-specific CD3+ T cells was around 2.4%.

[0090]

[0091] 5. Comparative analysis of antigen-specific CD3+ T cell ratios by antigen type

[0092] K-ras(M)-ROP(500aa), K-ras on LP-1 PBMC 1-24 Wild-type (Peptide Wt, 24aa), or K-ras 1-24 After treatment with the mutation (24aa), the proportion of antigen-specific CD3+ T cells was compared and analyzed.

[0093] Figure 4 is a K-ras(M)-ROP, K-ras of the present invention. 1-24 Wild-type, and K-ras 1-24 Analysis of the antigen-specific CD3+ T-cell ratio in LP-1 PBMCs for mutations is shown. Panel A shows K-ras(M)-ROP, K-ras 1-24 Wild-type, and K-ras 1-24 IFN-γ capture FACS analysis of mutant-treated LP-1 PBMCs. Panel B shows a graph of the percentage of IFN-γ-secreting CD3+ T cells, and Panel C shows a tabular summary of the percentage of antigen-specific CD3+ T cells. No Ag indicates that only effector was used, and @CD3 indicates that anti-CD3 was used as a positive control.

[0094] The above K-ras 1-24 The mutation is K-ras 1-24 It refers to the 12th amino acid G being substituted with D or V in the wild type, or the 12th amino acid G being D (Pep.G12D, Pep.G12V, Pep.G13D). The antigen-specific CD3+ T-cell ratio was analyzed using the same method as above, and K-ras(M)-ROP (500aa), Peptide Wt, Pep.G12D, Pep.G12V, and Pep.G13D were used as antigens.

[0095] As a result of the experiment, CD+ T cells that responded to peptide Wt were not confirmed, and CD3+ T cells that responded to Pep.G12D, Pep.G12V, and Pep.G13D were also confirmed to be significantly low at 0.31% (Pep.G12D), 0.11% (Pep.G12V), and 0.25% (Pep.G13D). Considering that the proportion of CD3+ T cells induced in response to K-ras(M)-ROP was 2.41%, the above results imply that the induction of antigen-specific CD3+ T cells is minimal with only the epitope, which is a peptide composed of 24 amino acids.

[0096]

[0097] 6. Production of ROP-T cells using K-ras(M)-ROP and Fast-IVS

[0098] Based on the above experimental results, CD3+ T cells (ROP-T cells) that respond to K-ras(M)-ROP were produced. In the present invention, ROP-T cells were produced by applying Fast-IVS (Fast-In vitro Stimulation).

[0099] Figure 5 shows the results of comparing the Fast-IVS process and the No-Cytokine process of the present invention. Panel A shows the manufacturing process and evaluation process of ROP-T cells using Fast-IVS, and the process of analyzing the characteristics of ROP-T cells. Panel B shows the results of comparing the IFN-γ+ CD3+ T cell ratios of T cells amplified under Fast-IVS process conditions and No-Cytokine process conditions.

[0100] The Fast-IVS process is characterized by simultaneously performing the steps of inducing antigen-specific CD3+ T cells using an antigen and performing cell expansion by treating the cells with cytokines. In contrast, the No-Cytokine process is characterized by performing cell expansion on antigen-specific CD3+ T cells induced by the antigen without treating them with cytokines.

[0101] The cytokines used for cell amplification in the above Fast-IVS process are interleukin-4 (IL-4), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and prostaglandin E2 (PGE2).

[0102] Table 3 below shows the Fast-IVS process and the No-Cytokine process of the present invention.

[0103] Fast-IVS ProcessNo-Cytokine ProcessDay-0LP-1 PBMC Seeding with Ag, IL-4, and GM-CSFLP-1 PBMC Seeding Without CytokineDay-1Adding TNF-α, IL-1β and PGE2No Cytokine AddingDay 3ExpansionExpansionDay 5, 7, 9, 11, 12Media AddingMedia AddingDay 13HarvestHarvest

[0104] The analysis results confirmed that the proportion of antigen-specific CD3+ T cells secreting IFN-γ was amplified approximately four times more in the Fast-IVS process than in the No-Cytokine process.

[0105]

[0106] 7. Optimization of the Fast-IVS Process for ROP-T Cell Production

[0107] The Fast-IVS process for ROP-T cell production was optimized by changing the treatment concentration of K-ras(M)-ROP and the Fast-IVS process conditions. Table 4 below shows examples for optimizing the Fast-IVS process.

[0108] Example 1 Example 2 Example 3 Experimental Conditions Scale PBMCs 10M@24well 10M@24well 10M@24well Cytokine Manufacturer JW Creagene JW Creagene JW Creagene K-ras(M)-ROP ㎍ / ㎖ 5.0 1.0 1.0 Fast-IVS (medium AIM-V) Period Days 5 5 7 Expansion Period Days 10 10 10 Experimental Results Expansion Fold No Ag-T4 5 3 4 5 5 ROP-T5 5 4 9 6 5 Helper T cell No Ag-T5 4.7 3 5.8 2 1.1 ROP-T6 4.4 7 5.3 6 0.4 K-ras(M)-ROP specific T cell(IFN-γ+, CD3+)(%) No Ag-T4 6 1.1 1.2 ROP-T19 ​​7 18 6 5 2.9

[0109] The experimental results confirmed that ROP-T cells were amplified in all examples, and the optimal Fast-IVS process was confirmed to be a treatment concentration of 1.0 μg / ml of K-ras(M)-ROP and a Fast-IVS period of 7 days.

[0110]

[0111] 8. Analysis of ROP-T cell characteristics

[0112] The characteristics of amplified ROP-T cells were analyzed by performing K-ras mutant epitope screening. To this end, autologous dendritic cells (ADCs) were induced from PBMCs, and antigen-sensitized ADCs were prepared to stimulate antigen-specific T cells, thereby confirming the reactivity of ROP-T cells. The reactivity was analyzed by calculating the restimulation IFN-γ secretion T cell frequency (%).

[0113] Figure 6 shows the results of screening for K-ras mutant epitopes in ROP-T cells of the present invention. Panel A shows the results of FACS analysis for IFN-γ+, CD3+, and CD4+. Panel B shows the results of analyzing the cell percentage (%) of the restimulated IFN-γ-secreting T cells by condition.

[0114] First, autologous DCs were cultured for 4 days and sensitized to antigen to prepare Ag-pulsed DCs. The Ag-pulsed DCs were seeded in 96-wells at a density of 5x10 3 cells / 100㎕. K-ras(M)-ROP specific CD3+ T cells were seeded in the 96-well at a density of 1x10 5The cells were divided into 100 μl and the cell numbers of Ag pulsed DC and K-ras(M)-ROP specific CD3+ T cells were in a ratio of 1:20. The medium containing Ag pulsed DC and K-ras(M)-ROP specific CD3+ T cells was cultured for 4 hours. The ratios (%) of CD3+, CD4+, CD137+, IFN-γ cap, and IFN-γ secreting T cells of the cultured cells were analyzed using the FACS. The antigens used in the production of the Ag pulsed DC were K-ras(M)-ROP(500aa)(ROP_DC), K-ras 1-24 wild type peptide (WT_DC), K-ras 1-24 G12D mutant peptide (G12D_DC), K-ras 1-24 G12V mutant peptide (G12V_DC), and K-ras 1-24 It was G13D mutant peptide (G13D_DC). In addition, DC using only effector without antigen (Ag) (NoAg_DC) was used for comparison.

[0115] The experimental results showed that when restimulated using ROP_DC, the proportions (%) of K-ras(M)-ROP-specific CD3+ / CD4+ T cells and K-ras(M)-ROP-specific CD3+ / CD8+ T cells present in CD3+ ROP-T cells were 10% and 5%, respectively. When restimulated using G12D_DC, the proportions (%) of K-ras(M)-ROP-specific CD3+ / CD4+ T cells and K-ras(M)-ROP-specific CD3+ / CD8+ T cells present in CD3+ ROP-T cells were 1.5% and 0.5%, respectively. When restimulated using G13D_DC, the proportions (%) of K-ras(M)-ROP-specific CD3+ / CD4+ T cells and K-ras(M)-ROP-specific CD3+ / CD8+ T cells present in CD3+ ROP-T cells were confirmed to be 3.0% and 1.5%, respectively. In contrast, when restimulated using Wt_DC and G12V_DC, K-ras(M)-ROP-specific CD3+ / CD4+ T cells and K-ras(M)-ROP-specific CD3+ / CD8+ T cells were hardly detected.

[0116]

[0117] 9. HLA restriction analysis of ROP-T cells

[0118] To confirm HLA restriction of K-ras G13D mutant-specific T cells among induced ROP-T, human leukocyte antigen DQ (HLA-DQ) assay was performed.

[0119] Figure 7 shows the results of the HLA-DQ blocking assay of the present invention.

[0120] First, antigen-sensitized DC (Ag pulsed DC) was prepared. The antigen used in the preparation of the Ag pulsed DC was K-ras(M)-ROP(500aa)(ROP_DC), K-ras 1-24 wild type peptide (WT_DC), K-ras1-24 G12D mutant peptide (G12D_DC), K-ras 1-24 G12V mutant peptide (G12V_DC), and K-ras 1-24 It was G13D mutant peptide (G13D_DC). HLA-DQ blocking was performed on the manufactured Ag pulsed DC by treating it with HLA-DQ antibody for 1 hour. After restimulation of ROP-T using HLA-DQ-blocked Ag pulsed DC, IFN-γ+, CD3+, and CD4+ were analyzed by FACS.

[0121] The analysis results showed that antigen-nonspecific T cells (NoAg-T) had a minimal proportion of CD3+CD4+ T cells secreting IFN-γ, regardless of the type of DC used for restimulation or the presence or absence of HLA-DQ blocking on the DC. In contrast, when ROP-T cells were restimulated using ROP_DC, the proportion of CD3+CD4+ T cells secreting IFN-γ increased to more than 15%, regardless of the presence or absence of HLA-DQ blocking on the DC. In addition, when ROP-T cells were restimulated using HLA-DQ blocked G13D_DC, the proportion of CD3+CD4+ T cells secreting IFN-γ was confirmed to decrease by approximately 6% (7% → 1%) compared to when restimulated using HLA-DQ unblocked G13D_DC.

[0122] As a result, it is judged that the ROP-T cells of the present invention induce the expansion of CD4+ T cells that are specific for the ROP antigen, restricted to HLA-DQ, and specific for the G13D mutation.

[0123]

[0124] 10. Comparison of control native K-ras-T cells and peptide mix-T cells

[0125] We verified the specific response induction of K-ras(M)-ROP to K-ras mutants.

[0126] Figure 8 shows the percentage of CD3+ T cells secreting IFN-γ (IFN-γ+) according to the conditions of the present invention. First, T cells were induced using the Fast-IVS process using K-ras(M)-ROP or native K-ras(189aa) as an antigen. In addition, K-ras 1-24 wild type peptide, K-ras 1-24 G12D peptide, K-ras 1-24 G12V peptide and K-ras 1-24 T cells were induced using the Fast-IVS process using a G13D peptide mixture as an antigen. As a control, T cells were induced using the Fast-IVS process using only the executor without antigen. The induced T cells were restimulated using ROP_DC, and the proportion of IFN-γ+ CD3+ T cells was analyzed using FACS.

[0127] Table 5 below shows the specific response-inducing verification experiment method for K-ras mutants of K-ras(M)-ROP. In Table 5 below, K-ras epitope wild type is Native K-ras 1-24 (24aa); K-ras epitope G12D is K-ras 1-24 G12D(24aa) refers to K-ras epitope G12V refers to K-ras 1-24 G12V(24aa) refers to K-ras epitope G13D; K-ras 1-24 It means G13D(24aa).

[0128] conditionsFast-IVSExpansionAntigenCytokineDaysMediaDaysNo Ag-T-D0:IL-4, GM-CSFD+1:TNF-a, IL-1b, PGE27 DaysAlys+IL-2+SR3%10 DaysROP-TK-ras(M)-ROP(8.5μM=5μg / ml)Pep.-TK-ras Epitope (24mer) mixture of 4 types (wild type, G12D, G12V, G13D) (8.5μM) WT-TNative K-ras (8.5μM=2μg / ml)

[0129] The experimental results showed that the proportion of IFN-γ+ CD3+ T cells was approximately 8% in T cells induced through Fast-IVS without using an antigen (No Ag-T). In the case of T cells induced through the Fast-IVS process using K-ras(M)-ROP as an antigen (ROP-T), the proportion of IFN-γ+ CD3+ T cells was approximately 37.4%. In the case of T cells induced through the Fast-IVS process using Native K-ras as an antigen (WT-T), the proportion of IFN-γ+ CD3+ T cells was approximately 18.4%. K-ras 1-24 wild type peptide, K-ras 1-24 G12D peptide, K-ras 1-24 G12V peptide and K-ras 1-24 In the case of T cells (Pep_T) induced through the Fast-IVS process using the G13D peptide mixture as an antigen, the proportion of IFN-γ+ CD3+ T cells was confirmed to be 19.0%.

[0130] In summary, it was determined that the K-ras(M)-ROP antigen had an IFN-γ+ CD3+ T cell induction effect that was approximately twice as superior to that using an epitope containing native K-ras or a K-ras mutation.

[0131]

[0132] 11. Confirmation of the cancer cell toxicity effect of ROP-T cells

[0133] The cancer cell killing effect of T cells (ROP-T) induced through the Fast-IVS process was confirmed. To this end, cell lines of breast adenocarcinoma, melanoma, colorectal adenocarcinoma, lung papillary adenocarcinoma, or lung large cell carcinoma were co-cultured with T cells induced using the Fast-IVS process using ROP as an antigen (ROP-T) or T cells induced using the Fast-IVS process without using an antigen (LAK-T), and the extent to which each T cell lysed each cancer cell was confirmed.

[0134] Table 6 below shows the analysis information for cell lines of each cancer type.

[0135] Cell lineCarcinomaK-ras mutationHLA-typeHLA-AHLA-DR(B1)HLA-DQ(B1)MCF7breast adenocarcinomaNone02:01, 02:0115:01, 15:0106:02, 06:02526melmelanomaNone02:01, 03--MDA MB231breast adenocarcinomaG13D02:17, 02:0113:05, 07:0103:04, 03:04SW480colorectal adenocarcinomaG12V24:02, 02:0113:27, 15:0106:03, 05:01 NCI-H441 Lung papillary adenocarcinoma G12V 03:01, 02:01 13:23, 14:10 06:07, 06:07 T3M-10 Lung large cell carcinoma G12D 24:02, 11:01 11:01, 08:03 06:01, 03:04

[0136] As a result of the analysis, it was confirmed that the breast carcinoma cell line MCF7 and the melanoma cell line 526mel contained K-ras but no mutations; the breast adenocarcinoma cell line MDA MB231 contained K-ras G13D mutations; the colorectal adenocarcinoma cell line SW480 and the lung papillary adenocarcinoma cell line NCI-H441 contained K-ras G12V mutations; and the lung large cell carcinoma cell line T3M-10 contained K-ras G12D mutations.

[0137] Figure 9 shows the results of a toxicity test on cancer cells of the ROP-T cells of the present invention.

[0138] As a result of the experiment, it was confirmed that the ROP-T cells of the present invention have significantly higher toxicity against breast adenocarcinoma cells, melanoma cells, colon adenocarcinoma cells, lung papillary adenocarcinoma cells, and lung large cell carcinoma cells compared to LAK-T cells that did not undergo ROP antigen treatment.

[0139] After obtaining cancer tissue from a colon cancer patient, HLA-typing and K-ras mutation testing were performed.

[0140] Table 7 below shows the test results for cancer cells (primary culture) obtained from colon cancer patients.

[0141] Carcinoma K-ras mutation HLA-type HLA-AHLA-DR(B1) HLA-DQ(B1) Colorectal cancer G12D 02:01, 24:02 04:06, 12:01 03:01, 03:02

[0142]

[0143] Colon cancer cells were obtained by performing primary culture from the cancer tissue of the above colon cancer patient, and T cells induced using the Fast-IVS process using ROP as an antigen (ROP-T) or T cells induced using the Fast-IVS process not using an antigen (LAK-T) were cultured together to determine how much each T cell suppresses the growth of colon cancer cells.

[0144] Figure 10 shows the results of analyzing the growth curve of ROP-T cells of the present invention against cancer cells of colon cancer patients. The experimental results confirmed that the ROP-T cells of the present invention inhibited the growth of colon cancer cells by approximately 10% compared to LAK-T cells.

[0145]

[0146] The specific embodiments described herein are intended to represent preferred embodiments or examples of the present invention and are not intended to limit the scope of the present invention. It will be apparent to those skilled in the art that variations and other uses of the present invention do not depart from the scope of the invention described in the claims of this specification.

[0147]

[0148] The pharmaceutical composition for preventing and treating lung adenocarcinoma comprising the K-ras specific activated T cell of the present invention has the advantage of being able to effectively prevent and treat not only K-ras but also lung adenocarcinoma in which a K-ras mutation is detected, particularly lung large cell adenocarcinoma.

Claims

1. A pharmaceutical composition for preventing or treating lung adenocarcinoma, comprising K-ras specific activated T cells induced in a medium containing an antigen composition for inducing K-ras specific activated T cells and a cytokine; The antigen composition for inducing the above K-ras specific activated T cells comprises a K-ras mutant recombinant overlapping peptide consisting of the amino acid sequence of sequence number 1 as an active ingredient; The above cytokines include interleukin-4, granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor-α, interleukin-1β, or prostaglandin E2; The above lung adenocarcinoma is a pharmaceutical composition for the prevention or treatment of lung adenocarcinoma, characterized in that it is lung large cell carcinoma in which K-ras mutation G12D is detected. The above K-ras specific activated T cells are produced by culturing peripheral blood mononuclear cells (PBMCs) in a medium containing the antigen composition for inducing the K-ras specific activated T cells and a primary cytokine, thereby maturing dentritic cells and simultaneously inducing K-ras specific activated T cells in the first step; A second step of adding secondary cytokines to the above cultured PBMCs and culturing them to further induce K-ras-specific activated T cells; and A third step of obtaining PBMCs by adding the above secondary cytokine and culturing them to amplify K-ras specific activated T cells; The antigen composition for inducing the above K-ras specific activated T cells comprises a K-ras mutant recombinant overlapping peptide consisting of the amino acid sequence of sequence number 1 as an active ingredient; The primary cytokines are interleukin-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF); A pharmaceutical composition for the prevention or treatment of lung adenocarcinoma, characterized in that the secondary cytokines are tumor necrosis factor-α, interleukin-1β, and prostaglandin E2, and is manufactured by a method for inducing K-ras specific activated T cells using an antigen composition for inducing K-ras specific activated T cells.

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