KR102753588B1 - Pharmaceutical Composition For Preventing Or Treating Lung Adenocarcinoma Comprising K-ras Specific Activated T Cell And Manufacturing Method Thereof - Google Patents

Abstract

The pharmaceutical composition for the prevention and treatment of lung adenocarcinoma comprising K-ras specific activated T cells of the present invention uses, as an antigen composition, K-ras mutant (G12D, G12V, and G13D) recombinant overlapping peptides that 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.
Therefore, the pharmaceutical composition for preventing and treating lung adenocarcinoma containing the K-ras specific activated T cell of the present invention has the advantage of effectively preventing and treating not only K-ras but also lung adenocarcinoma in which a K-ras mutation is detected, particularly lung large cell adenocarcinoma.

Description

Pharmaceutical composition for preventing and treating lung adenocarcinoma comprising K-ras specific activated T cells and manufacturing method thereof {Pharmaceutical Composition For Preventing Or Treating Lung Adenocarcinoma Comprising K-ras Specific Activated T Cell And Manufacturing Method Thereof}

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 producing the same.

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

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. Lymphokine activate killer (LAK) immunotherapy is a method that uses immune cells among immunotherapy methods for cancer. The LAK immunotherapy has shown encouraging clinical results for cancer patients who had failed surgery, chemotherapy, and radiation therapy. However, the LAK immunotherapy has a problem in that the cancer cell killing efficiency is low due to a non-specific immune response. In addition, since only interleukin-2 (IL-2) lymphokine is used in the culture process, the immune cells are exhausted and their viability in the body is reduced, so there is a clinical limitation that periodic and repeated treatment is required.

To solve the above problems, anticancer peptide vaccines are being developed. The peptide vaccine selects a highly immunogenic portion of a cancer antigen, designs it as a peptide, and then uses it to activate immune cells. Therefore, there is an advantage in that there is no concern about immune cell exhaustion due to the use of IL-2 lymphokine. However, the peptide vaccine has the disadvantage that immune evasion may occur if the target portion of the cancer cell mutates, and the immune response may be low because it does not receive help from CD4 T cells in the immune response, and because it is designed as a peptide that is loaded onto an MHC class I molecule, there is a limitation in the human leukocyte antigen (HLA) type.

To solve the above problems, an overlapping peptide (OLP) vaccine is being developed that includes the entire antigen and designs it by overlapping peptides of highly immunogenic parts. Unlike conventional peptide vaccines, the OLP vaccine includes the entire antigen, so it has the advantage of not only having a high immune response with the help of CD4 T cells, but also having a longer immune response period and no restrictions on HLA types. However, the OLP vaccine has the problem of high manufacturing costs and relatively difficult immune regulation.

In immunocytotherapy, the method of producing antigen-specific T cells using vaccines is also an important factor. In general, to produce antigen-specific T cells, monocytes are separated from blood, and then mo dendritic cells are obtained through a maturation process, and then the dendritic cells and T cells are co-cultured in an environment where the antigen is separately processed, thereby producing antigen-specific T cells. The method using moDC has the disadvantage of requiring additional time, cost, and blood because the antigen-specific T cell induction method is performed separately by separating the DC cell maturation process and the T cell culture process.

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

Therefore, if an antigen that includes several of the most frequently occurring mutation sites of K-ras but resolves the above problems is developed to selectively amplify endogenous immune cells that can bind to K-ras mutations and cause a direct immune response, it is expected that it will be possible to efficiently target and kill cancer cells, thereby maximizing the cancer treatment effect and enabling use as an efficient treatment with a high persistence of the anticancer effect.

The patent documents and references mentioned in this specification are incorporated by reference into this specification to the same extent as if each document was individually and specifically designated by reference.

Korean Patent Publication No. 10-2018-0010229 Korean Patent Publication No. 10-2020-0141994

Vaccines. 2014 Jul 2;2(3):515-36. Adv Protein Chem Struct Biol. 2015;99:1-14. Nat Rev Cancer. 2008 May;8(5):351-60).

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 which comprises K-ras-specific activated T cells, and a method for manufacturing the same.

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

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 is characterized in that cancer cells of the lung adenocarcinoma detect K-ras, K-ras mutant G12V, K-ras mutant G12D, or K-ras mutant G13D.

The antigen composition for inducing the above K-ras specific activated T cell is characterized in that it contains as an active ingredient a K-ras mutant recombinant overlapping peptide consisting of an amino acid sequence of sequence number 1, and the K-ras mutant recombinant overlapping peptide contains 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) whose units are amino acid sequences sequentially listed from any one amino acid in the amino acid sequence of K-ras consisting of sequence number 2, but the epitopes (n=2, 3,...12) excluding the epitope (n=1) have a 15 amino acid sequence in the N-terminal direction that is located in the C-terminal direction of the epitope (n-1) of the previous order. It is characterized by being designed to overlap with 15 amino acid sequences.

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.

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

The above K-ras-specific activated T cells are characterized by being manufactured 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-inducing antigen composition and a first cytokine to mature dentritic cells and 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, which have been cultured with the second cytokine added thereto, to amplify the K-ras-specific activated T cells and then obtain them.

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

The pharmaceutical composition for the prevention and treatment of lung adenocarcinoma comprising K-ras specific activated T cells of the present invention uses, as an antigen composition, K-ras mutant (G12D, G12V, and G13D) recombinant overlapping peptides that 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.

Therefore, the pharmaceutical composition for preventing and treating lung adenocarcinoma containing the K-ras specific activated T cell of the present invention has the advantage of effectively preventing and treating not only K-ras but also lung adenocarcinoma in which a K-ras mutation is detected, particularly lung large cell adenocarcinoma.

Figure 1 shows the amino acid sequence structure of K-ras(M)-ROP of the present invention.
Figure 2 shows the results of analyzing the reactivity of PBMC to K-ras(M)-ROP of the present invention.
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.
Figure 4 shows the results of analyzing the antigen-specific CD3+ T cell ratio of LP-1 PBMCs for K-ras(M)-ROP, K-ras _1-24 Wild-type, and K-ras _1-24 mutant of the present invention.
Figure 5 shows the results of comparing the Fast-IVS process and the No-Cytokine process of the present invention.
Figure 6 shows the results of K-ras mutant epitope screening for ROP-T cells of the present invention.
Figure 7 shows the results of the HLA-DQ blocking assay of the present invention.
Figure 8 shows the ratio of CD3+ T cells secreting IFN-γ (IFN-γ+) according to the conditions of the present invention.
Figure 9 shows the extent to which ROP-T cells and LAK-T cells of the present invention kill cancer cells.
Figure 10 shows the extent to which 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.

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 by which cancer is suppressed or delayed by the administration of 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 by which the symptoms of cancer are improved or beneficially changed by the administration of the pharmaceutical composition for the prevention and treatment of lung adenocarcinoma.

The lung adenocarcinoma of the present invention is characterized in that K-ras, K-ras mutation G12V, K-ras mutation G12D, or K-ras mutation G13D is detected in cancer cells. The K-ras gene, KRAS, is a representative proto-oncogene that is involved in cell growth and differentiation in vertebrates, and when it is changed into an oncogene by point mutation, chromosomal translocation, gene amplification, etc., it abnormally increases the activity of K-ras and causes cancer. Therefore, the detection of K-ras, K-ras mutation G12V, K-ras mutation G12D, or K-ras mutation 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.

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.

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 the treatment of 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 the treatment of 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 the treatment of 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 the treatment of 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 the treatment of lung large cell adenocarcinoma in which K-ras, K-ras mutant G12D It can be used in the treatment of lung large cell adenocarcinoma that is detected.

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 cells according to the present invention as an active ingredient, and includes a pharmaceutically acceptable carrier.

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, and may comprise from 1 to 99.99 wt % or volume %.

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

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.

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.

The pharmaceutical composition for the prevention and treatment of lung adenocarcinoma containing the K-ras specific activated T cell 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, if necessary, and can be formulated by mixing it appropriately with a pharmaceutically acceptable carrier or medium, specifically, sterile water or 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 active ingredient amount means such that an appropriate dosage within the indicated range can be obtained.

When the pharmaceutical composition for the prevention and treatment of lung adenocarcinoma containing the K-ras specific activated T cell 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, 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.

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, intracerebroventricular injection types, intracerebral injection types, and intramedullary injection types, and preferably, the above injection type is an intravenous injection type.

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 easily determined by a person skilled in the art based on the contents disclosed herein.

In general, the pharmaceutically effective amount 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 administration 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).

The method of administration of the pharmaceutical composition for the prevention and treatment of lung adenocarcinoma of the present invention can be determined by considering 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 the patient can be determined by many factors such as the method of administration, the patient's health condition, weight, and the doctor's prescription, which is within the scope of knowledge of a person having ordinary skill in the art.

The pharmaceutical composition for preventing and treating lung adenocarcinoma of the present invention has an amount of about 1x10 ⁶ cells/㎖ or more, about 2x10 ⁶ cells/㎖ or more, about 3x10 ⁶ cells/㎖ or more, about 4x10 ⁶ cells/㎖ or more, about 5x10 ⁶ cells/㎖ or more, about 6x10 ⁶ cells/㎖ or more, about 7x10 ⁶ cells/㎖ or more, about 8x10 ⁶ cells/㎖ or more, about 9x10 ⁶ cells/㎖ or more, about 1x10 ⁷ cells/㎖ or more, about 2x10 ⁷ cells/㎖ or more, about 3x10 ⁷ cells/㎖ or more, about 4x10 ⁷ cells/㎖ or more, about 5x10 ⁷ cells/㎖ or more, about 6x10 ⁷ cells/㎖ or more, about 7x10 ⁷ cells/㎖ or more, about 8x10 ⁷ cells/㎖ The composition ^comprises K-ras specific activated T cells at a concentration of at least about 9x10 ⁷ cells/㎖, at least about 1x10 ⁸ cells/㎖, at least about 2x10 ⁸ cells/㎖, at least about 3x10 ⁸ cells/㎖, at least about 4x10 ⁸ cells/㎖, at least about 5x10 ⁸ cells/㎖, at least about 6x10 ⁸ cells/㎖, at least about 7x10 ⁸ cells/㎖, at least about 8x10 ⁸ cells/㎖, or at least about 9x10 8 cells/㎖, although one of ordinary skill in the art will be able to adjust the concentration of K-ras specific activated T cells in the composition within a variable range to obtain the same effect.

The above “about” can be understood as within a range commonly accepted in the art, for example, within the mean standard deviation range, and can 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.

In addition, it may further be 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 prepared injection solution is usually filled into a suitable ampoule. Suspensions and emulsions may contain natural gums, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol as a carrier. Suspensions or solutions for intramuscular injection may contain the active compound together with a pharmaceutically acceptable carrier, for example, sterile water, olive oil, ethyl oleate, glycols, and a suitable amount of lidocaine hydrochloride.

The pharmaceutical composition for the prevention and treatment of lung adenocarcinoma of the present invention can be administered to a patient by bolus injection or continuous infusion. The pharmaceutical composition for the prevention and treatment of 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, over 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.

The injection may be formulated in ampoule form or in unit dose form in multiple dose containers. However, those skilled in the art will appreciate 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 previous treatment history.

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 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, in which the second cytokine has been added, to amplify the K-ras-specific activated T cells and then obtain them.

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 separated from blood, and then co-culturing them with T cells in an environment treated with the antigen composition for inducing K-ras-specific activated T cells of the present invention. In contrast, the Fast-IVS of the present invention has a difference in that it performs the maturation process and antigen (antigen composition for inducing K-ras-specific activated T cells) treatment simultaneously on DC cells in PBMCs.

The antigen composition for inducing K-ras specific activated T cells used as an antigen in the above T cell induction process may further contain cytokines, hormones and buffer solutions 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), Tumor Necrosis Factor-α (TNF-α), and the hormone may be prostaglandin E2 (PGE2).

According to one embodiment of the present invention, 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 Tumor Necrosis Factor-α, Interleukin-1β, and Prostaglandin E2 were used as secondary cytokines to mature immature DCs.

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

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

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).

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.

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 30 amino acid sequences and the last epitope (n=12) includes 23 amino acid sequences) 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 sequence in the N-terminal direction overlaps with the 15 amino acid sequence in the C-terminal direction of the epitope (n-1) of the immediately preceding order.

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. 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.

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 mutation G12V; an epitope (n=1) including a K-ras mutation 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 mutation 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 mutation G13D via an LRMK-linker.

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).

K-ras is a type of ras protein and is 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 and its mutations expressed in cancer cells 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), urothelial carcinoma (BLCA), invasive breast 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), corpus endometrial carcinoma (UCEC), or uterine carcinosarcoma (UCS).

The present invention is described in detail below through examples.

Example

1. Production of K-ras(WT)

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 as shown in Table 1 below.

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

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

2. Manufacturing of K-ras(M)-ROP

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).

The above K-ras(M)-ROP is characterized by having a 500 amino acid sequence, and the amino acids are sequentially divided into groups of 30, but each epitope is designed so that 15 amino acid sequences overlap 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.

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

name Amino acid sequence (500aa) K-ras(M)-ROP MTEYKLVVVG ¹⁰ A D GVGKSALT ²⁰ IQLIQNHFVD ³⁰ LRMK ³⁴
MTEYKLVVVG ⁴⁴ A V GVGKSALT ⁵⁴ IQLIQNHFVD ⁶⁴ LRMK ⁶⁸
KSALTIQLIQ ⁷⁸ NHFVDEYDPT ⁸⁸ IEDSYRKQVV ⁹⁸ LRMK ¹⁰²
EYDPTIEDSY ¹¹² RKQVVIDGET ¹²² CLLDILDTAG ¹³² LRMK ¹³⁶
IDGETCLLDI ¹⁴⁶ LDTAGQEEYS ¹⁵⁶ AMRDQYMRTG ¹⁶⁶ LRMK ¹⁷⁰
QEEYSAMRDQ ¹⁸⁰ YMRTGEGGFLC 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 AG D VGKSALT ⁴⁵⁶ IQLIQNHFVD ⁴⁶⁶ LRMK ⁴⁷⁰
MTEYKLVVVG ⁴⁸⁰ AGGVGKSALT ⁴⁹⁰ IQLIQNHFVD ⁵⁰⁰ Epitope name Amino acid sequence
(The sequence number is assigned based on K-rasWT) Epitope 1 (E1, n=1) MTEYKLVVVG ¹⁰ AGGVGKSALT ²⁰ 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 YMRTGEGGFLC 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 ¹⁸⁰ VKIKKCIIM ¹⁸⁹ Epitope 1-G12D (E1-G12D) MTEYKLVVVG ¹⁰ A D GVGKSALT ²⁰ IQLIQNHFVD ³⁰ Epitope 1-G12V (E1-G12V) MTEYKLVVVG ¹⁰ A V GVGKSALT ²⁰ IQLIQNHFVD ³⁰ Epitope 1-G13D (E1-G13D) MTEYKLVVVG ¹⁰ AG D VGKSALT ²⁰ IQLIQNHFVD ³⁰

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

3. K-ras(M)-ROP reactivity screening analysis

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

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

First, ^1x105 cells of PBMC (LP-1 PBMC, LP-4 PBMC, and LP-6 PBMC) obtained from leukocyte apheresis of normal volunteers were seeded and cultured, and then treated with antigen. 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℃, _CO2 5%, 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 the reactivity to K-ras(M)-ROP was the best in LP-1 among normal PBMCs.

4. Analysis of specific CD3+ T cell ratio by K-ras(M)-ROP concentration

The K-ras(M)-ROP-specific CD3+ T cell ratio according to the K-ras(M)-ROP concentration was analyzed for the above LP-1 PBMC. To this end, LP-1 PBMC was treated with antigen, and then IFN-γ capture staining was performed and analyzed.

Figure 3 shows the results of analyzing the K-ras(M)-ROP-specific CD3+ T cell ratio of LP-1 PBMC according to the K-ras(M)-ROP concentration 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 ratio (%) of IFN-γ secreting CD3+ T cells.

First, LP-1 PBMC 1x10 ⁶ cells were seeded and cultured, and then treated with antigens at different concentrations (K-ras(M)-ROP 5 ㎍/㎖, K-ras(M)-ROP 1.0 ㎍/㎖, K-ras(M)-ROP 0.1 ㎍/㎖). In addition, LP-1 PBMC 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℃, _CO2 5%, overnight (O/N).

IFN-γ capture staining was performed by treating LP-1 PBMCs treated with the ^1st capture antibody, incubating at 37℃ for 45 minutes, and then treating with ^2nd detection antibodies and CD3, CD4, CD8, and CD137. LP-1 PBMCs subjected to the IFN-γ capture staining were analyzed for their cell characteristics using a fluorescence activated cell sorter (FACS).

As a result of the experiment, it was confirmed that when LP-1 PBMCs were treated with antigens according to their concentration, the proportion of K-ras(M)-ROP-specific CD2+ T cells increased, which was well consistent with the above ELISpot (IFN-γ) results. Therefore, it is judged that the reactivity of LP-1 PBMCs increases in a concentration-dependent manner to K-ras(M)-ROP. In addition, as a result of quantitatively evaluating the reactivity of LP-1 PBMCs to K-ras(M)-ROP, it was confirmed that the proportion of K-ras(M)-ROP-specific CD3+ T cells was about 2.4% when 5㎍/㎖ of K-ras(M)-ROP was treated.

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

After treating LP-1 PBMCs with K-ras(M)-ROP(500aa), K-ras _1-24 Wild-type (Peptide Wt, 24aa), or K-ras _1-24 mutant (24aa), the proportion of antigen-specific CD3+ T cells was compared and analyzed.

Figure 4 shows the results of analyzing the antigen-specific CD3+ T-cell ratio of LP-1 PBMC for K-ras(M)-ROP, K-ras _1-24 Wild-type, and K-ras _1-24 mutant of the present invention. Panel A shows the results of IFN-γ capture FACS analysis of LP-1 PBMC treated with K-ras(M)-ROP, K-ras _1-24 Wild-type, and K-ras _1-24 mutant. Panel B shows a graph of the percentage of IFN-γ secreting CD3+ T cells, and Panel C shows a table summarizing the graph of the percentage of antigen-specific CD3+ T cells. No Ag means that only the effector was used, and @CD3 means that anti-CD3 was used as a positive control.

The above K-ras _1-24 mutations refer to the 12th amino acid G being substituted with D or V in K-ras _1-24 Wild-type, or the 12th amino acid G being replaced with 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.

As a result of the experiment, CD+ T cells that reacted with peptide Wt were not confirmed, and CD3+ T cells that reacted with Pep.G12D, Pep.G12V, and Pep.G13D were also significantly low at 0.31% (Pep.G12D), 0.11% (Pep.G12V), and 0.25% (Pep.G13D). Considering that the ratio 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 an epitope consisting of a peptide composed of 24 amino acids.

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

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

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 characteristic analysis process 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.

The above Fast-IVS process is characterized by simultaneously performing the processes of inducing antigen-specific CD3+ T cells using an antigen and performing cell expansion by treating 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.

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).

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

Fast-IVS process No-Cytokine Process Day-0 LP-1 PBMC Seeding with Ag, IL-4, and GM-CSF LP-1 PBMC Seeding Without Cytokine Day-1 Adding TNF-α, IL-1β and PGE2 No Cytokine Adding Day 3 Expansion Expansion Day 5, 7, 9, 11, 12 Media Adding Media Adding Day 13 Harvest Harvest

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.

7. Optimization of Fast-IVS process for ROP-T cell production

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 optimization of the Fast-IVS process.

Example 1 Example 2 Example 3 Experimental conditions Scale PBMCs 10M@24well 10M@24well 10M@24well Cytokine manufacturing company JW Creagene JW Creagene JW Creagene K-ras(M)-ROP ㎍/㎖ 5.0 1.0 1.0 Fast-IVS (Badge AIM-V) period Days 5 5 7 Expansion period Days 10 10 10 Experimental Results Expansion Fold No Ag-T 45 34 55 ROP-T 55 49 65 Helper T cell No Ag-T 54.7 35.8 21.1 ROP-T 64.4 75.3 60.4 K-ras(M)-ROP specific T cell(IFN-γ+, CD3+)(%) No Ag-T 4.6 1.1 1.2 ROP-T 19.7 18.6 52.9

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/㎖ of K-ras(M)-ROP and a Fast-IVS period of 7 days.

8. Analysis of ROP-T cell characteristics

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 re-stimulation IFN-γ secretion T frequency (%).

Figure 6 shows the results of K-ras mutant epitope screening for 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 ratio (%) of the conditionally restimulated IFN-γ secreting T cell ratio.

First, autologous DCs were cultured for 4 days and sensitized to antigen to prepare Ag pulsed DCs. The Ag pulsed DCs were dispensed into 96-well at ^5x103 cells/100㎕. K-ras(M)-ROP-specific CD3+ T cells were dispensed into the 96-well at ^1x105 cells/100㎕, with a cell ratio of 1:20 between the Ag pulsed DCs and K-ras(M)-ROP-specific CD3+ T cells. The medium containing Ag pulsed DCs 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 FACS. The antigens used in the production of the above 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 G13D mutant peptide (G13D_DC). In addition, DC using only the effector without the antigen (Ag) (NoAg_DC) was used for comparison.

The experimental results showed that when restimulated using ROP_DC, the ratios (%) of K-ras(M)-ROP-specific CD3+/CD4+ T cells and K-ras(M)-ROP-specific CD3+/CD8+ T cells in CD3+ ROP-T cells were 10% and 5%, respectively. When restimulated using G12D_DC, the ratios (%) of K-ras(M)-ROP-specific CD3+/CD4+ T cells and K-ras(M)-ROP-specific CD3+/CD8+ T cells in CD3+ ROP-T cells were 1.5% and 0.5%, respectively. When restimulated with G13D_DC, the percentages (%) 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 with 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.

9. HLA restriction analysis of ROP-T cells

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.

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

First, antigen-sensitized DC (Ag pulsed DC) was prepared. The antigens used in the preparation 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 G13D mutant peptide (G13D_DC). HLA-DQ blocking was performed on the prepared Ag pulsed DC by treating it with HLA-DQ antibody for 1 hour. After re-stimulation of ROP-T using the HLA-DQ blocked Ag pulsed DC, IFN-γ+, CD3+, and CD4+ were analyzed through FACS.

The results of the analysis showed that the proportion of CD3+CD4+ T cells secreting IFN-γ was minimal for antigen-nonspecific T cells (NoAg-T), regardless of the type of DC used for restimulation and 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.

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

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

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

Figure 8 shows the ratio 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, T cells were induced using the Fast-IVS process using a mixture of K- _{ras 1-24} wild type peptide, K-ras _1-24 G12D peptide, K-ras 1-24 G12V peptide, and K-ras _1-24 G13D peptide as antigens. As a control, T cells were induced using the Fast-IVS process using only an executor without an antigen. The induced T cells were restimulated using ROP_DC, and the ratio of IFN-γ+ CD3+ T cells was analyzed using FACS.

Table 5 below shows the specific response induction verification experimental method for K-ras mutations of K-ras(M)-ROP. In Table 5 below, K-ras epitope wild type means Native K-ras _1-24 (24aa); K-ras epitope G12D means K-ras _1-24 G12D(24aa); K-ras epitope G12V means K-ras _1-24 G12V(24aa); K-ras epitope G13D means K-ras _1-24 G13D(24aa).

conditions Fast-IVS Expansion Antigen Cytokine Days Media Days No Ag-T - D0:IL-4, GM-CSF
D+1:TNF-a, IL-1b, PGE2 7 Days Alys+IL-2+SR3% 10 Days ROP-T K-ras(M)-ROP(8.5μM=5㎍/㎖) Pep.-T K-ras epitope (24mer) 4 types mixture (wild type, G12D, G12V, G13D) (8.5 μM) WT-T Native K-ras(8.5μM=2㎍/㎖)

The experimental results showed that the proportion of IFN-γ+ CD3+ T cells was approximately 8% in the case of T cells induced through Fast-IVS without using an antigen (No Ag-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 K-ras(M)-ROP as an antigen (ROP-T). The proportion of IFN-γ+ CD3+ T cells was approximately 18.4% in the case of T cells induced through the Fast-IVS process using Native K-ras as an antigen (WT-T). In the case of T cells (Pep_T) induced through the Fast-IVS process using a mixture of K-ras _1-24 wild type peptide, K-ras _1-24 G12D peptide, K-ras _1-24 G12V peptide, and K-ras _1-24 G13D peptide as antigens, the ratio of IFN-γ+ CD3+ T cells was confirmed to be 19.0%.

In summary, it was determined that the K-ras(M)-ROP antigen had a 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.

11. Confirmation of cancer cell toxicity of ROP-T cells

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 (ROP-T) induced using the Fast-IVS process using ROP as an antigen or T cells (LAK-T) induced using the Fast-IVS process without using an antigen, and the extent to which each T cell lysed each cancer cell was confirmed.

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

Cell line name carcinoma K-ras mutation HLA type HLA-A HLA-DR(B1) HLA-DQ(B1) MCF7 breast cancer
(breast adenocarcinoma) doesn't exist 02:01, 02:01 15:01, 15:01 06:02, 06:02 526mel Melanoma
(melanoma) doesn't exist 02:01, 03 - - MDA MB231 breast cancer
(breast adenocarcinoma) G13D 02:17, 02:01 13:05, 07:01 03:04, 03:04 SW480 Colon cancer
(colorectal adenocarcinoma) G12V 24:02, 02:01 13:27, 15:01 06:03, 05:01 NCI-H441 Pulmonary papillary adenocarcinoma
(lung papillary adenocarcinoma) G12V 03:01, 02:01 13:23, 14:10 06:07, 06:07 T3M-10 pulmonary large cell carcinoma
(lung large cell carcinoma) G12D 24:02, 11:01 11:01, 08:03 06:01, 03:04

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

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

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 have not undergone ROP antigen treatment.

After obtaining cancer tissue from a patient with colon cancer, tests for HLA-typing and K-ras mutation were performed.

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

carcinoma K-ras mutation HLA type HLA-A HLA-DR(B1) HLA-DQ(B1) Colorectal cancer G12D 02:01, 24:02 04:06, 12:01 03:01, 03:02

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.

Figure 10 shows the results of analyzing the growth curve of the ROP-T cells of the present invention against cancer cells of colon cancer patients. As a result of the experiment, it was confirmed that the ROP-T cells of the present invention inhibited the growth of colon cancer cells by about 10% compared to LAK-T cells.

The specific embodiments described in this specification are meant to represent preferred embodiments or examples of the present invention, and the scope of the present invention is not limited thereby. It will be apparent to those skilled in the art that modifications and other uses of the present invention do not depart from the scope of the invention described in the claims of this specification.

Attach electronic file of sequence list

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 cytokines;
The above K-ras specific activated T cell inducing antigen composition 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, and 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 a first step of culturing peripheral blood mononuclear cells (PBMCs) in a medium containing the above K-ras specific activated T cell inducing antigen composition and a first cytokine to mature dentritic cells and induce K-ras specific activated T cells;
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 above K-ras specific activated T cell inducing antigen composition 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.