WO2023128377A1 - Pharmaceutical composition comprising activated t cells specific to k-ras for preventing and treating lung papillary adenocarcinoma and method for preparing same - Google Patents

Abstract

A pharmaceutical composition comprising activated T cells specific to K-ras for preventing and treating lung papillary adenocarcinoma, according to the present invention, uses, as an antigen composition, a K-ras mutant (G12D, G12V, and G13D) recombinant overlapping peptide, which is designed such that 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 sequentially in the amino acid sequence of K-ras are distinguished, and the sequence of 15 amino acids overlaps between the epitopes. Accordingly, the pharmaceutical composition has an advantageous effect of recognizing and killing lung papillary adenocarcinoma in which K-ras, K-ras mutant G12V, K-ras mutant G12D, or K-ras mutant G13D is detected. Accordingly, the use of the pharmaceutical composition comprising activated T cells specific to K-ras for preventing and treating lung papillary adenocarcinoma of the present invention has the advantage of effectively preventing and treating lung adenocarcinoma, particularly lung papillary adenocarcinoma, in which K-ras as well as K-ras mutants are detected,.

Description

Pharmaceutical composition for preventing and treating lung papillary adenocarcinoma containing K-RAS-specific activated T cells and method for preparing the same

The present invention relates to a pharmaceutical composition for preventing and treating lung papillary adenocarcinoma containing K-ras-specific activated T cells and a method for preparing the same.

Conventional anticancer chemotherapy has limitations in suppressing recurrence and metastasis of cancer and has side effects of killing normal cells. In order to overcome this, various immuno-anticancer treatment methods for treating cancer by enhancing the immune response to tumors have been studied.

Immunotherapy is a method of inducing cancer-specific CTL (Cytotoxic T Lymphocyte) endogenously or injecting it to kill cancer cells. As a method of using immune cells among immuno-anticancer treatments, there is a Lymphokine activate killer (LAK) immune cell therapy.

The LAK immune cell therapy showed encouraging clinical results for cancer patients who had failed surgery, chemotherapy, and radiation therapy. However, the LAK immune cell therapy not only has a problem of low cancer cell killing efficiency due to a non-specific immune response, but also uses only interleukin-2 (IL-2) lymphokine in the culture process, so immune cells are exhausted and survive in vivo There was a clinical limitation that required periodic and repetitive treatment because of poor ability.

In order to solve the above problems, anti-cancer peptide vaccines are being developed. The peptide vaccine selects a part with high immunogenicity among cancer antigens, designs it as a peptide, and uses it to activate immune cells. Therefore, there is an advantage that there is no concern about exhaustion of immune cells due to the use of IL-2 lymphokine. However, the peptide vaccine can not only cause immune evasion when the target site of cancer cells mutates, but also can have low immunoreactivity due to the lack of help from CD4 T cells in the immune response. Design with peptides loaded on MHC class I molecules Because of this, there was a disadvantage in that there was a limitation in the type of human leukocyte antigen (HLA).

In order to solve the above problem, overlapping peptide (OLP) vaccines designed by overlapping peptides containing the entire antigen and having high immunogenicity are being developed. Unlike conventional peptide vaccines, the OLP vaccine contains all antigens, so it has a high immune response with the help of CD4 T cells, a longer immune response period, and no HLA type restrictions. However, the OLP vaccine has a problem in that manufacturing cost is high and immunomodulation is relatively difficult.

In immune cell therapy, a method for producing antigen-specific T cells using a vaccine is also an important factor. In general, in order to produce antigen-specific T cells, after isolating monocytes from blood, mo dendritic cells are obtained through a maturation process, and then the dendritic cells are separately treated in an environment where antigens are separately treated. Ag-specific T cells are produced by co-culture with T cells. The method using the moDC has disadvantages of requiring additional time, cost, and blood because the antigen-specific T cell induction method proceeds by dividing the DC cell maturation process and the T cell culture process.

Among various cancer antigens, K-ras mutation is a carcinogenic mutation found in about 20% of solid cancers, and is found mainly in pancreatic and colon adenocarcinomas and lung cancers. Therapies targeting tumors dependent on K-ras mutant genes inhibit or inactivate the function of K-ras because it is difficult to make antibodies that individually bind to K-ras mutants expressed by K-ras mutant genes. The effect was limited because it was limited to an indirect treatment method.

Therefore, by presenting an antigen that solves the above problems, including the most frequently occurring mutation sites of K-ras, it selectively amplifies endogenous immune cells capable of inducing a direct immune response by binding to K-ras mutations, and using them as a target cancer treatment It is expected that it can be used as an effective treatment that maximizes the effect of cancer treatment and has a high durability of anti-cancer effect because it can efficiently target and kill cancer cells.

The patents and references mentioned herein are incorporated herein by reference to the same extent as if each document were individually and expressly specified by reference.

An object of the present invention is to develop a K-ras-specific activated T cell induced with an antigen for inducing K-ras-specific activated T cells, which is designed to include the entire amino acid sequence and mutations of K-ras and is manufactured using recombinant technology and has excellent economic efficiency. It is to provide a pharmaceutical composition for preventing and treating cell-containing lung papillary adenocarcinoma and a method for preparing the same.

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

The present invention relates to an antigen composition for inducing K-ras-specific activated T cells and a medicament for preventing and treating lung papillary adenocarcinoma, including K-ras-specific activated T cells induced using cytokine It is characterized by providing a medical composition, wherein the cancer cells of the lung papillary adenocarcinoma are K-ras, K-ras mutant G12V, K-ras mutant G12D, or K-ras mutant G13D is detected do.

The antigen composition for inducing K-ras-specific activated T cells is characterized by comprising a K-ras mutant recombinant overlapping peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient, wherein the K-ras mutant recombinant overlapping peptide is SEQ ID NO: A total of 12 types of epitopes (epitope (n = 1, 2, 3 .... 10, 11, 12) consisting of amino acid sequences sequentially listed from any one amino acid in the amino acid sequence of K-ras consisting of 2; Here, n means the order of the epitope, and the epitope (n = 1 to 11) includes a 30 amino acid sequence, and the last epitope (n = 12) includes a 23 amino acid sequence), but the epitope (n = For epitopes (n=2, 3,...12) except for 1), the 15 amino acid sequence in the N-terminal direction is designed to overlap with the 15 amino acid sequence in the C-terminal direction of the immediately preceding epitope (n-1). characterized by being

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

The epitope (n=1) includes the K-ras mutation G12V; An epitope (n = 1) containing K-ras mutant G12D is further linked to the N-terminal of the epitope (n = 1) by an LRMK-linker; An epitope (n = 1) containing K-ras mutant G13D is further linked to the C-terminal of the epitope (n = 12) by an LRMK-linker; The C-terminal of the epitope (n = 1) containing the K-ras mutation G13D is characterized in that an epitope (n = 1) not containing the K-ras mutation is further linked by an LRMK-linker.

The K-ras-specific activated T cells are obtained by culturing peripheral blood mononuclear cells (PBMC) in a medium containing the antigen composition for inducing K-ras-specific activated T cells and a primary cytokine. A first step of maturating dentritic cells and inducing K-ras-specific activated T cells; a second step of further inducing K-ras-specific activated T cells by adding and culturing a secondary cytokine to the cultured PBMC; and a third step of amplifying K-ras-specific activated T cells by culturing the PBMCs cultured with the addition of the secondary cytokine. It is characterized in that it is prepared by a method for inducing K-ras-specific activated T cells using the composition.

The primary cytokines are Interleukin-4 and Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF); The secondary cytokines are characterized in that they are tumor necrosis factor-α, interleukin-1β, and prostaglandin E2.

A pharmaceutical composition for the prevention and treatment of lung papillary adenocarcinoma containing K-ras-specific activated T cells of the present invention comprises, as an antigen composition, a K-ras mutant (G12D, G12V, and G13D) recombinant overlapping peptide is sequentially divided into a total of 12 epitopes (epitope, n = 1 to 12, but the last epitope (n = 12) is 23 amino acids) in units of 30 amino acids sequentially in the amino acid sequence of K-ras. Since it is designed and used so that 15 amino acid sequences overlap between them, it recognizes lung papillary adenocarcinoma in which K-ras, K-ras mutation G12V, K-ras mutation G12D, or K-ras mutation G13D is detected. It has the advantage of being more effective in killing.

Therefore, when the pharmaceutical composition for preventing and treating lung papillary adenocarcinoma containing K-ras-specific activated T cells of the present invention is used, not only K-ras but also lung adenocarcinoma in which K-ras mutations are detected, especially However, it has the advantage of effectively preventing and treating lung papillary 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.

3 shows the result of analyzing the ratio of specific CD3+ T cells in LP-1 PBMC according to the concentration of K-ras(M)-ROP according to the present invention.

Figure 4 shows the results of analyzing the ratio of antigen-specific CD3+ T cells in LP-1 PBMCs for K-ras(M)-ROP, K-ras1-24Wild-type, and K-ras1-24 mutants of the present invention. .

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

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

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+) for each condition 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.

10 shows the degree 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 relates to an antigen composition for inducing K-ras-specific activated T cells and a medicament for preventing and treating lung papillary adenocarcinoma, including K-ras-specific activated T cells induced using cytokine A scientific composition is provided. "Prevention" of the present invention refers to all activities in which cancer is suppressed or delayed by administration of a pharmaceutical composition for preventing and treating lung papillary adenocarcinoma of the present invention, and "treatment" of the present invention means lung It refers to all activities that improve or beneficially change the symptoms of cancer by administering a pharmaceutical composition for the prevention and treatment of lung papillary adenocarcinoma.

The lung papillary adenocarcinoma of the present invention is characterized in that K-ras, K-ras mutant G12V, K-ras mutant G12D, or K-ras mutant G13D is detected in cancer cells. KRAS, the K-ras gene, is a representative proto-oncogene, which is involved in cell growth and differentiation in vertebrates, and is involved in point mutation, chromosomal translocation, and gene amplification When converted into an oncogene, the activity of K-ras abnormally increases and causes cancer. Therefore, the detection of K-ras, K-ras mutant G12V, K-ras mutant G12D, or K-ras mutant G13D in lung papillary adenocarcinoma cancer cells of the present invention means that KRAS is expressed as an oncogene and K-ras mutant G12V is detected. This means that the activity of ras is abnormally increased. Preferably, the lung papillary 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, more preferably K-ras mutation G13D. It is characterized in that the mutation G12V is detected.

The K-ras mutations G12V, G12D, and G13D are mutations found in lung adenocarcinoma, pancreatic adenocarcinoma, crystalline adenocarcinoma, colorectal adenocarcinoma, and rectal adenocarcinoma, and prevent GTP hydrolysis by GTPase-activating proteins (GAPs) from occurring smoothly, resulting in GTP- It is known to induce the proliferation of cancer cells by increasing the cellular level of bound RAS protein and thereby abnormally activating lower signaling pathways.

The pharmaceutical composition for preventing and treating lung papillary adenocarcinoma containing K-ras-specific activated T cells of the present invention includes an antigen composition for inducing K-ras-specific activated T cells and cytokines. Lung cancer, colorectal cancer, breast cancer, melanoma, etc. in which K-ras, K-ras mutant G12V, K-ras mutant G12D, or K-ras mutant G13D are detected because K-ras-specific activated T cells are included. It can be used for the treatment of cancer, and preferably can be used for the treatment of lung adenocarcinoma in which K-ras, K-ras mutation G12V, K-ras mutation G12D, or K-ras mutation G13D are detected among lung cancers, and more Preferably, it can be used for the treatment of lung papillary adenocarcinoma in which K-ras, K-ras mutation G12V, K-ras mutation G12D, or K-ras mutation G13D is detected, most preferably K-ras It can be used for the treatment of lung papillary adenocarcinoma in which mutant G12V is detected.

The “composition” described in the present invention refers to K-ras-specific activated T cells according to the present invention as an active ingredient, along with natural or artificial carriers, inactive ingredients such as labels or detection agents, or adjuvants, diluents, binders, stabilizers, It refers to a combination with active ingredients such as buffers, salts, lipophilic solvents, and preservatives, and includes pharmaceutically acceptable carriers.

Such carriers may include pharmaceutical excipients and additional proteins, peptides, amino acids, lipids, and carbohydrates (e.g., monosaccharides; disaccharides; trisaccharides; tetrasaccharides; oligosaccharides; sugars such as alditols, aldonic acids, esterified sugars); derivatives of, polysaccharides, sugar polymers, etc.) alone or in combination, and may be included in an amount of 1 to 99.99% by weight 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 play a buffering role may include alanine, arginine, glycine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. Not limited.

Carbohydrate excipients include monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose; disaccharides such as lactose, sucrose, trehalose, cellobiose, raffinose, maltodextrin, dextran, polysaccharides such as starch, and alditols such as mannitol, xylitol, maltitol, lactitol, sorbitol, and myoinositol; and the like. can and is not limited thereto.

The pharmaceutical composition for preventing and treating lung papillary adenocarcinoma containing the 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, if necessary, can be used parenterally in the form of an injection of a sterile solution or suspension in water or other pharmaceutically acceptable liquids, and can be used in a pharmaceutically acceptable carrier or medium, specifically As an appropriate combination with sterilized water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, excipient, vehicle, preservative, binder, etc., by mixing in a unit dosage form required for generally recognized pharmaceutical practice can be formulated. In the above formulation, the amount of active ingredient means that an appropriate dose within the indicated range can be obtained.

When the pharmaceutical composition for preventing and treating lung papillary adenocarcinoma containing K-ras-specific activated T cells of the present invention is formulated as a sterile composition for injection, a vehicle such as distilled water for injection is used. It can be prescribed according to the usual pharmaceutical practices. As the aqueous solution for injection, isotonic solutions containing physiological saline, glucose, and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride, can be used. Alcohol, propylene glycol, and polyethylene glycol, polysorbate 80 (TM), and HCO-50, which are nonionic surfactants, may be used in combination. In addition, sesame oil and soybean oil can be used as an oily liquid, and can be used in combination with benzyl benzoate and benzyl alcohol as a dissolution aid.

Examples of the injection formulation include intravenous injection formulation, intraarterial injection formulation, selective intraarterial injection formulation, intramuscular injection formulation, intraperitoneal injection formulation, subcutaneous injection formulation, intraventricular injection formulation, intracerebral injection formulation, and intramedullary injection formulation. And preferably, the injection formulation is an intravenous injection formulation.

The pharmaceutical composition for preventing and treating lung papillary adenocarcinoma containing K-ras-specific activated T cells of the present invention includes a pharmaceutically effective amount of K-ras-specific activated T cells. Determination of the effective amount can be easily determined by those skilled in the art based on the contents disclosed herein.

In general, after the first administration of the active ingredient at a low concentration, the pharmaceutically effective amount is gradually increased until the desired effect, for example, reduction or elimination of cancer-related symptoms, is obtained without side effects in the subject. determined by the method A method for determining an appropriate dosage or administration interval of the pharmaceutical composition for preventing and treating lung papillary adenocarcinoma of the present invention is described 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).

Administration method of the pharmaceutical composition for the prevention and treatment of lung papillary adenocarcinoma of the present invention is the type of cancer, the patient's age, weight, sex, medical condition, severity of disease, administration route, and separately administered It can be determined considering various factors such as drug. The amount of the pharmaceutical composition for the prevention and treatment of lung papillary adenocarcinoma of the present invention administered to a patient may be determined by many factors such as the administration method, the patient's health condition, weight, and the doctor's prescription, which It is within the knowledge of one of ordinary skill in the art.

The pharmaceutical composition for preventing and treating lung papillary adenocarcinoma of the present invention contains about 1x10 ⁶ cells/mL or more, about 2x10 ⁶ cells/mL or more, about 3x10 ⁶ cells/mL or more, about 4x10 ⁶ cells/mL or more. ㎖ or more, about 5x10 ⁶ cells/mL or more, about 6x10 ⁶ cells/mL or more, about 7x10 ⁶ cells/mL or more, about 8x10 ⁶ cells/mL or more, about 9x10 ⁶ cells/mL or more, about 1x10 ⁷ cells/mL or more , about 2x10 ⁷ cells/mL or more, about 3x10 ⁷ cells/mL or more, about 4x10 ⁷ cells/mL or more, about 5x10 ⁷ cells/mL or more, about 6x10 ⁷ cells/mL or more, about 7x10 ⁷ cells/mL or more, about 8x10 ⁷ cells/mL or more, about 9x10 ⁷ cells/mL or more, about 1x10 ⁸ cells/mL or more, about 2x10 ⁸ 108 cells/mL or more, about 3x10 ⁸ cells/mL or more, about 4x10 ⁸ cells/mL or more, about 5x10 ⁸ cells/mL or more, about 6x10 ⁸ cells/mL or more, about 7x10 ⁸ cells/mL or more, about 8x10 ⁸ cells/mL or more, or about 9x10 ⁸ cells/mL or more K-ras-specific activated T cells, but usually A person skilled in the art will be able to adjust the concentration of K-ras-specific activated T cells in the composition within a modifiable range to obtain the same effect.

The "about" can be understood within a range commonly accepted in the art, for example, within the average standard deviation range, and is 50%, 45%, 40%, 35%, 30%, 25%, Understand to within 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% It can be.

In addition, buffering agents such as phosphate buffer and sodium acetate buffer, analgesic agents such as procaine hydrochloride, stabilizers such as benzyl alcohol or phenol, and antioxidants may be further combined. The prepared injection solution is usually filled into an appropriate ampoule.

Suspensions and emulsions may contain, as carriers, natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. Suspensions or solutions for intramuscular injection can contain the active compound together with pharmaceutically acceptable carriers such as sterile water, olive oil, ethyl oleate, glycols and suitable amounts of lidocaine hydrochloride.

The pharmaceutical composition for preventing and treating lung papillary adenocarcinoma of the present invention may be administered to patients by bolus injection or continuous infusion. The pharmaceutical composition for preventing and treating lung papillary adenocarcinoma of the present invention is 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 hour or less More than 1 day, more than 2 days, more than 3 days, more than 4 days, more than 5 days, more than 6 days, more than 7 days, more than 2 weeks, more than 3 weeks, more than 4 weeks, more than 1 month, more than 3 months, more than 6 months It may 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.

The injection may be formulated in the form of an ampoule or a unit dosage form in a multi-dose container. 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 previous treatment history.

In the present invention, the K-ras-specific activated T cells are isolated from peripheral blood mononuclear cells (PBMC) in a medium containing an antigen composition for inducing K-ras-specific activated T cells and a primary cytokine. A first step of culturing to mature dentritic cells and inducing K-ras-specific activated T cells; a second step of further inducing K-ras-specific activated T cells by adding and culturing a secondary cytokine to the cultured PBMC; and a third step of amplifying K-ras-specific activated T cells by culturing the PBMCs cultured with the addition of the secondary cytokine. It is characterized in that it is prepared by a method for inducing K-ras-specific activated T cells using the composition.

The method of inducing K-ras-specific activated T cells according to the present invention is Fast-IVS (in vitro stimulation), which is different from conventional IVS. The IVS induces K-ras-specific activated T cells of the present invention after obtaining monocyte-derived dendritic cells (moDC) through a differentiation and maturation process from monocytes isolated from blood. It refers to a method of co-culture with T cells in an environment treated with an antigen composition for use. In contrast, the Fast-IVS of the present invention has a difference in that it simultaneously performs the maturation process and antigen (antigen composition for inducing K-ras-specific activated T cells) treatment of DC cells in PBMC.

The antigen composition for inducing K-ras-specific activated T cells used as an antigen in the T cell induction process may further include cytokines, hormones, and buffers required for DC cell maturation and growth. Preferably, the cytokine is interleukin-4, interleukin-1β, granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor-α (Tumor Necrosis Factor). -α, TNF-α), and the hormone may be prostaglandin E2 (PGE2).

According to one embodiment of the present invention, the interleukin-4 and the granulocyte-macrophage colony-stimulating factor (GM-CSF) are primary cytokines, which induce monocytes in PBMC into DC It was used to induce differentiation into dendritic cells, and the tumor necrosis factor-α, interleukin-1β, and prostaglandin E2 are secondary cytokines, immature DC ) was used to mature.

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 step-by-step culturing period was optimized through the examples to most efficiently induce K-ras-specific activated T cells.

The present invention is characterized in that the antigen composition for inducing K-ras-specific activated T cells includes the K-ras mutant recombinant overlapping peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

SEQ ID NO: 1 is derived from the amino acid sequence (SEQ ID NO: 2) of K-ras protein. In the K-ras mutation, 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), It means that the 13th amino acid is substituted from glycine (G) to aspartic acid (D).

The recombinant means inserting into a recombinant plasmid DNA containing the genetic information of the designed antigen, and the recombinant plasmid DNA is transformed into a microorganism to express a protein and purify the K-ras-specific An antigen for inducing activated T cells is obtained.

The K-ras mutant recombinant overlapping peptide of the present invention has a total of 12 types of epitopes (epitope (n = 1, 2 , 3....10, 11, 12); where n means the sequence of the epitope, the epitope (n = 1 to 11) contains a sequence of 30 amino acids and the last epitope (n = 12) is 23 amino acids Sequence.), but epitope (n = 2, 3, ... 12) except for epitope (n = 1) is the sequence of 15 amino acids in the N-terminal direction of the immediately preceding epitope (n-1) It is designed to overlap with the 15 amino acid sequence in the C-terminal direction.

The K-ras mutant recombinant overlapping peptide is characterized in that the epitopes (n = 1, 2, 3 ... 10, 11, 12) are located in sequence, and the epitopes are linked by an LRMK-linker, and the LRMK- The linker is a linker composed of Leucine (L), Arginine (R), Methionine (M), and Lysine (K), and has an advantage in the MHC class I pathway by dendritic cells. there is.

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 the K-ras mutation G12V; An epitope (n = 1) containing K-ras mutant G12D is further linked to the N-terminal of the epitope (n = 1) by an LRMK-linker; An epitope (n = 1) containing K-ras mutant G13D is further linked to the C-terminal of the epitope (n = 12) by an LRMK-linker; An epitope (n=1) not containing the K-ras mutation is further linked to the C-terminal of the epitope (n=1) containing the K-ras mutation G13D by an LRMK-linker.

When the antigen composition for inducing K-ras-specific activated T cells of the present invention is used, T cells specific for K-ras mutations (G12D, G12V, G13D) can be induced, and the K-ras mutations (G12D, G12V, G13D) can be used to treat cancer cells with K-ras mutations (G12D, G12V, G13D).

K-ras, a type of ras protein, is a small GTPases protein that plays an important role in the signal transduction system related to cell differentiation, proliferation, and survival. It is known that cancer is induced when the activity of K-ras protein abnormally increases. KRAS, a gene of the ras protein, is well known as an oncogene found as a mutation in various carcinomas, and 85% of ras-derived carcinomas are known to be caused by K-ras mutation. Therefore, when T cells specifically recognizing K-ras expressed in cancer cells and its mutations are amplified, cancer with abnormally increased activity of K-ras can be effectively removed and treated. The cancer is not limited as long as the cancer has increased K-ras activity, and examples thereof include adrenocortical carcinoma (ACC), bladder urothelial carcinoma (BLCA), invasive breast carcinoma (BRCA), cervical squamous cell carcinoma and intracervical 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 myelogenous 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 melanoma (SKCM), gastric adenocarcinoma (STAD), testicular germ cell tumor (TGCT) , thyroid adenocarcinoma (THCA), cervical endometrial carcinoma (UCEC) or uterine carcinosarcoma (UCS).

In the following, the present invention will be described in detail through examples.

Example

1. Preparation 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 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

After treating the K-ras-WT with a restriction enzyme, an expression vector was prepared by inserting it into a pET30a vector, and the expression vector was transformed into E. coli to express the protein.

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

An 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 for K-ras(WT). The K-ras(M)-ROP is G12D in which G (Glycine) at the 12th position of K-ras amino acid is mutated to D (Asapartic acid), and G (Glycine) at the 12th position of K-ras amino acid is V (Valine) It includes G12V mutated to , or G13D in which G (Glycine) at the 13th position of K-ras amino acid is mutated to D (Asapartic acid).

The K-ras(M)-ROP is characterized by having a sequence of 500 amino acids, and the amino acids are sequentially divided into 30 units, and one epitope is designed such 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 K-ras(M)-ROP epitope.

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 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 IDGETCLLDI 146 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 numbers were 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 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 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 K-ras(M)-ROP was synthesized (Genescript Co. Ltd.), cloned into a pET30a vector, and then transformed into E.coli and expressed. The expressed protein was cleaved using activated protein C (APC) to prepare K-ras(M)-ROP.

3. K-ras(M)-ROP Reactivity Screening Assay

The reactivity of K-ras(M)-ROP was screened in peripheral blood mononuclear cells (PBMC) of normal subjects. The screening was performed using 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, 1x10 ⁵ cells of PBMC (LP-1 PBMC, LP-4 PBMC, and LP-6 PBMC) obtained from leukocyte division of normal volunteers were seeded, cultured, and treated with antigen. As the antigen, 5 μg/ml, 1.0 μg/ml, and 0.1 μg/ml of K-ras(M)-ROP were used, and anti-CD3 was used as a positive control. Cell culture was performed under conditions of 37°C, 5% CO ₂ , 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 ratio of K-ras(M)-ROP-specific CD3+ T cells according to the K-ras(M)-ROP concentration was analyzed for the LP-1 PBMCs. To this end, IFN-γ capture staining was performed after antigen treatment on LP-1 PBMCs, and this was analyzed.

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

First, LP-1 PBMC 1x10 ⁶ cells were seeded and cultured, and then antigens (K-ras(M)-ROP 5μg/ml, K-ras(M)-ROP 1.0μg/ml, K-ras(M)-ROP 0.1 μg/ml) was treated. In addition, LP-1 PBMC treated with tetanus toxoid vaccine (TTX) at 5 μg/ml and 1.0 μg/ml and anti-CD3 were used as positive controls. Cell culture was performed under conditions of 37°C, 5% CO ₂ , and overnight (O/N).

For IFN-γ capture staining, antigen-treated LP-1 PBMC was treated with ^1st capture antibody, then incubated at 37°C for 45 minutes, and secondary detection antibody ( ^2nd detection andtibody) and CD3, CD4 , CD8, and CD137. Cell characteristics of the LP-1 PBMCs subjected to the IFN-γ capture staining were analyzed using a Fluorescence activated cell sorter (FACS).

As a result of the experiment, it was confirmed that the ratio of K-ras(M)-ROP-specific CD2+ T cells increased when LP-1 PBMCs were treated with the antigen according to the concentration, which was in good agreement with the ELISpot (IFN-γ) result. Therefore, it is believed that the reactivity of LP-1 PBMC increases in a concentration-dependent manner to K-ras(M)-ROP. In addition, as a result of quantitatively evaluating the reactivity of LP-1 PBMC to K-ras(M)-ROP, K-ras(M)-ROP-specific CD3+ T It was confirmed that the percentage of cells was 2.4%.

5. Comparative analysis of the ratio of antigen-specific CD3+ T cells according to the type of antigen

After treating LP-1 PBMC with K-ras(M)-ROP (500aa), K-ras _1-24 Wild-type (Peptide Wt, 24aa), or K-ras _1-24 mutant (24aa), antigen-specific The ratio of red CD3+ T cells was comparatively analyzed.

Figure 4 analyzes the ratio of antigen-specific CD3+ T-cells of LP-1 PBMC to K-ras(M)-ROP, K-ras _1-24 Wild-type, and K-ras _1-24 mutants of the present invention show one result. Panel A shows the results of IFN-γ capture FACS analysis of LP-1 PBMCs treated with K-ras(M)-ROP, K-ras _1-24 Wild-type, and K-ras _1-24 mutants. Panel B shows a graph of IFN-γ-secreting CD3+ T cell ratio (%), and panel C shows the graph of antigen-specific CD3+ T cell ratio (%) tabulated. No Ag means that only the effector was used, and @CD3 means that anti-CD3 was used as a positive control.

In the K-ras _1-24 mutation, the 12th amino acid G in K-ras _1-24 Wild-type is substituted with D or V, or the 12th amino acid G is replaced with D (Pep.G12D, Pep.G12V, Pep.G13D). The ratio of antigen-specific CD3+ T-cells was analyzed in the same manner 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 responded to Peptide Wt were not confirmed, and CD3+ T cells that responded to Pep.G12D, Pep.G12V, and Pep.G13D were also 0.31% (Pep.G12D), 0.11% (Pep.G12V) and It was confirmed that it was remarkably low at 0.25% (Pep.G13D). Considering that the ratio of CD3+ T cells induced in response to K-ras(M)-ROP is 2.41%, the above results indicate that the induction of antigen-specific CD3+ T cells is insignificant with only the epitope, which is a peptide composed of 24 amino acids.

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

Based on the above experimental results, CD3+ T cells (ROP-T cells) responding to K-ras(M)-ROP were prepared. In the present invention, ROP-T cells were prepared 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 characterization process of ROP-T cells. Panel B shows the result of comparing the ratio of IFN-γ+ CD3+ T cells in T cells expanded under the Fast-IVS process condition and the No-Cytokine process condition.

The Fast-IVS process is characterized in that a process of inducing antigen-specific CD3+ T cells using an antigen and a process of performing cell expansion by treating cytokines are performed at the same time. In contrast, the No-Cytokine process is characterized in that cell amplification is performed without treating antigen-specific CD3+ T cells with cytokines.

The cytokines used for cell amplification in the Fast-IVS process include Interleukin-4 (IL-4), Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), and tumor necrosis. They are 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
Days 5, 7, 9, 11, 12	Media Adding	Media Adding
Day 13	Harvest	Harvest

As a result of the analysis, it was confirmed that the ratio of antigen-specific CD3+ T cells secreting IFN-γ was amplified about 4 times more in the Fast-IVS process than in the No-Cytokine process.

7. Optimization of the Fast-IVS process for manufacturing ROP-T cells

The Fast-IVS process for preparing ROP-T cells 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
Experiment conditions	Scale	PBMCs	10M@24well	10M@24well	10M@24well
	Cytokine	manufacturing company	JW Creagene	JW Creagene	JW Creagene
	K-ras(M)-ROP	μg/mL	5.0	1.0	1.0
	Fast-IVS (Badge AIM-V) Period		Days	5	5	7
	Expansion period		Days	10	10	10
Experiment result	Expansion Fold	No Ag-T	45	34	55
		ROP-T	55	49	65
	Helper T cells	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

As a result of the experiment, it was confirmed that ROP-T cells were amplified in all examples, and it was confirmed that the optimal Fast-IVS process was a K-ras(M)-ROP treatment concentration of 1.0 μg/ml and a Fast-IVS period of 7 days.

8. ROP-T cell characterization

K-ras mutant epitope screening was performed to characterize the amplified ROP-T cells. To this end, autologous dendritic cells are induced from PBMC, and antigen pulsed dendritic cells (Ag pulsed DC) capable of stimulating antigen-specific T cells are prepared by sensitizing the autologous dendritic cells with an antigen. Thus, the reactivity of ROP-T cells was confirmed. The reactivity was analyzed by calculating the re-stimulation IFN-γ secretion T frequency (%).

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 result of analyzing the cell ratio (%) of the restimulation IFN-γ secreting T cell ratio for each condition.

First, autologous DCs were cultured for 4 days and Ag pulsed DCs were prepared by sensitizing them to an antigen. The Ag pulsed DC was dispensed at 5x10 ³ cells/100 μl in 96 well. K-ras(M)-ROP-specific CD3+ T cells were dispensed into the 96 well to be 1x10 ⁵ cells/100 μl, but the Ag pulsed DC and K-ras(M)-ROP-specific CD3+ T cells were divided into 1:20 cells. ratio was made. A medium containing Ag pulsed DC and K-ras(M)-ROP-specific CD3+ T cells was cultured for 4 hours. CD3+, CD4+, CD137+, IFN-γ cap, and IFN-γ secreting T cell ratios (%) of the cultured cells were analyzed using the FACS. 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). Also, for comparison, DC (NoAg_DC) using only an effector without an antigen (Ag) was used.

Experimental results Ratio 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 when restimulated with ROP_DC ( %) were found to be 10% and 5%, respectively. Ratio (%) 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 when restimulated with G12D_DC It was confirmed that these were 1.5% and 0.5%, respectively. Ratio (%) 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 when restimulated with G13D_DC It was confirmed that these were 3.0% and 1.5%, respectively. In contrast, when restimulation 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.

9. HLA restriction assay of ROP-T cells

In order 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.

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

First, antigen-primed DC (Ag pulsed DC) was prepared. 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 by treating the prepared Ag pulsed DC with an HLA-DQ antibody for 1 hour. After re-stimulation of ROP-T using HLA-DQ blocked Ag pulsed DC, IFN-γ +, CD3 +, and CD4 + were analyzed by FACS analysis.

As a result of the analysis, antigen-specific T cells (NoAg-T) showed that the ratio of CD3+CD4+ T cells secreting IFN-γ was insignificant, regardless of the type of DC used for restimulation and whether or not HLA-DQ blocking was performed on the DC. Confirmed. On the other hand, when ROP-T cells were re-stimulated using ROP_DC, it was confirmed that the ratio of CD3+CD4+ T cells secreting IFN-γ increased to 15% or more regardless of HLA-DQ blocking for DC. In addition, ROP-T cells, when restimulation was performed using HLA-DQ blocked G13D_DC, the ratio of CD3+CD4+ T cells secreting IFN-γ was 6 in preparation for restimulation using HLA-DQ unblocked G13D_DC. % reduction (7% → 1%) was confirmed.

As a result, it is determined that the ROP-T cells of the present invention induce the expansion of CD4+ T cells that are specific for the ROP antigen, restrictive for HLA-DQ, and show specificity for the G13D mutation.

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

Induction of a specific reaction of K-ras(M)-ROP to K-ras mutation was verified.

Figure 8 shows the ratio of CD3+ T cells secreting IFN-γ (IFN-γ+) for each condition 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, 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 was used Thus, T cells were induced. As a control, T cells were induced using the Fast-IVS process using only the effector without antigen. The induced T cells were re-stimulated using ROP_DC and the percentage of IFN-γ+ CD3+ T cells was analyzed using FACS.

Table 5 below shows an experimental method for verifying the induction of a specific reaction of K-ras(M)-ROP to the K-ras mutation. 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=5μg/ml)
Pep. -T	K-ras epitope (24mer) 4 kinds mixture (wild type, G12D, G12V, G13D) (8.5μM)
WT-T	Native K-ras (8.5μM=2μg/ml)

As a result of the experiment, in the case of T cells (No Ag-T) induced through Fast-IVS without using an antigen, it was confirmed that the ratio of IFN-γ+ CD3+ T cells was 8%. In the case of T cells (ROP-T) induced through the Fast-IVS process using K-ras(M)-ROP as an antigen, the proportion of IFN-γ+ CD3+ T cells reached 37.4%. In the case of T cells (WT-T) induced through the Fast-IVS process using native K-ras as an antigen, the proportion of IFN-γ+ CD3+ T cells was confirmed to be 18.4%. 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 In the case of one T cell (Pep_T), the ratio of IFN-γ+ CD3+ T cells was confirmed to be 19.0%.

In summary, it is judged that the K-ras(M)-ROP antigen has an IFN-γ+ CD3+ T cell induction effect that is about twice as good as that using native K-ras or an epitope containing a K-ras mutation.

11. Confirmation of cancer cytotoxic effect of ROP-T cells

Cancer cell lysis (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 and ROP are used as antigens. T cells induced using the Fast-IVS process (ROP-T) or T cells induced using the Fast-IVS process (LAK-T) that do not use antigens are co-cultured so that each T cell can kill each cancer cell. It was confirmed how soluble it was.

Table 6 below shows analysis information for each carcinoma cell line.

cell line name	carcinoma	K-ras mutation	HLA-type
			HLA-A	HLA-DR (B1)	HLA-DQ (B1)
MCF7	breast adenocarcinoma (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 adenocarcinoma (breast adenocarcinoma)	G13D	02:17, 02:01	13:05, 07:01	03:04, 03:04
SW480	colorectal adenocarcinoma (colorectal adenocarcinoma)	G12V	24:02, 02:01	13:27, 15:01	06:03, 05:01
NCI-H441	lung papillary adenocarcinoma (lung papillary adenocarcinoma)	G12V	03:01, 02:01	13:23, 14:10	06:07, 06:07
T3M-10	lung 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, MCF7, a cell line of breast carcinoma, and 526mel, a cell line of melanoma, confirmed that K-ras was detected, but no mutation was detected. ras G13D mutation; SW480, a cell line of colorectal adenocarcinoma, and NCI-H441, a cell line of lung papillary adenocarcinoma, had K-ras G12V mutation; and T3M-10, a lung large cell carcinoma cell line, were found to contain the K-ras G12D mutation.

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

As a result of the experiment, the ROP-T cells of the present invention showed a significant level of toxicity against breast adenocarcinoma cells, melanoma cells, colon adenocarcinoma cells, lung papillary adenocarcinoma cells, and lung colon adenocarcinoma cells compared to LAK-T cells not subjected to ROP antigen treatment. confirmed to be high.

After obtaining cancer tissues from colorectal cancer patients, tests for HLA-typing and K-ras mutation were performed.

Table 7 below shows test results for cancer cells (primary culture) obtained from colorectal 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

Colorectal cancer cells are obtained by performing primary culture from the cancer tissue of the colorectal cancer patient, and T cells (ROP-T) induced using the Fast-IVS process using ROP as an antigen or Fast-IVS process using no antigen T cells (LAK-T) induced using were cultured together to confirm how much each T cell inhibits the growth of colon cancer cells.

10 shows the results of analyzing the growth curve of the ROP-T cells of the present invention for cancer cells of colorectal 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 the LAK-T cells.

Specific examples 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 variations and other uses of the present invention do not depart from the scope of the invention described in the claims.

Prevention and treatment of lung papillary adenocarcinoma comprising K-ras-specific activated T cells induced using the antigen composition for inducing K-ras-specific activated T cells of the present invention and cytokine The pharmaceutical composition can be used to treat lung papillary adenocarcinoma.

1. Amino acid sequence of SEQ ID NO: 1

MTEYKLVVVG ADGVGKSALT IQLIQNHFVD LRMKMTEYKL VVVGAVGVGK SALTIQLIQN 60

HFVDLRMKKS ALTIQLIQNH FVDEYDPTIE DSYRKQVVLR MKEYDPTIED SYRKQVVIDG 120

ETCLLDILDT AGLRMKIDGE TCLLDILDTA GQEEYSAMRD QYMRTGLRMK QEEYSAMRDQ 180

YMRTGEGFLC VFAINNTKSF LRMKEGFLCV FAINNTKSFE DIHHYREQIK RVKDLRMKED 240

IHHYREQIKR VKDSEDVPMV LVGNKCDLLR MKSEDVPMVL VGNKCDLPSR TVDTKQAQDL 300

ARLRMKPSRT VDTKQAQDLA RSYGIPFIET SAKTRQLRMK SYGIPFIETS AKTRQRVEDA 360

FYTLVREIRQ LRMKRVEDAF YTLVREIRQY RLKKISKEEK TPGCLRMKYR LKKISKEEKT 420

PGCVKIKKCI IMLRMKMTEY KLVVVGAGDV GKSALTIQLI QNHFVDLRMK MTEYKLVVVG 480

AGGVGKSALT IQLIQNHFVD

2. Amino acid sequence of SEQ ID NO: 2

MTEYKLVVVG AGGVGKSALT IQLIQNHFVD EYDPTIEDSY RKQVVIDGET CLLDILDTAG 60

QEEYSAMRDQ YMRTGEGFLC VFAINNTKSF EDIHHYREQI KRVKDSEDVP MVLVGNKCDL 120

PSRTVDTKQA QDLARSYGIP FIETSAKTRQ RVEDAFYTLV REIRQYRLKK ISKEEKTPGC 180

VKIKKCIIM

Claims (8)

1. A pharmaceutical composition for preventing and treating lung papillary adenocarcinoma, comprising an antigen composition for inducing K-ras-specific activated T cells and K-ras-specific activated T cells induced using cytokine .
2. The lung papilla according to claim 1, wherein K-ras, K-ras mutant G12V, K-ras mutant G12D, or K-ras mutant G13D is detected in the lung papillary adenocarcinoma cancer cells. A pharmaceutical composition for preventing and treating lung papillary adenocarcinoma.
3. The pulmonary papillary adenocarcinoma according to claim 1, wherein 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 SEQ ID NO: 1 as an active ingredient ( A pharmaceutical composition for preventing and treating lung papillary adenocarcinoma).
4. The method of claim 3, wherein the K-ras mutant recombinant overlapping peptide is a total of 12 types of epitopes (epitope (n = 1, 2, 3...10, 11, 12); where n means the sequence of epitopes, the epitopes (n = 1 to 11) contain 30 amino acid sequences, and the last epitope (n = 12) contains a 23 amino acid sequence.), but the epitope (n = 2, 3, ... 12) except for the epitope (n = 1) is the epitope (n = 12) of the 15 amino acid sequence in the N-terminal direction A pharmaceutical composition for preventing and treating lung papillary adenocarcinoma, characterized in that it is designed to overlap with the 15 amino acid sequences in the C-terminal direction of -1).
5. The method of claim 4, wherein the K-ras mutant recombinant overlapping peptides are located in the order of the epitopes (n = 1, 2, 3...10, 11, 12), and the epitopes are linked by an LRMK-linker A pharmaceutical composition for the prevention and treatment of lung papillary adenocarcinoma.
6. 5. The method of claim 4, wherein the epitope (n=1) comprises the K-ras mutation G12V; An epitope (n = 1) containing K-ras mutant G12D is further linked to the N-terminal of the epitope (n = 1) by an LRMK-linker; An epitope (n = 1) containing K-ras mutant G13D is further linked to the C-terminal of the epitope (n = 12) by an LRMK-linker; Lung papillary adenocarcinoma, characterized in that an epitope (n = 1) not containing a K-ras mutation is further linked with an LRMK-linker to the C-terminal of the epitope (n = 1) containing the K-ras mutation G13D A pharmaceutical composition for the prevention and treatment of (lung papillary adenocarcinoma).
7. The method of claim 1, wherein the K-ras-specific activated T cells are obtained from peripheral blood mononuclear cells in a medium containing the antigen composition for inducing K-ras-specific activated T cells and a primary cytokine. A first step of culturing PBMC) to mature dentritic cells and inducing K-ras-specific activated T cells;

   a second step of further inducing K-ras-specific activated T cells by adding and culturing a secondary cytokine to the cultured PBMC; and

   Antigen composition for inducing K-ras-specific activated T cells, characterized in that it comprises a; A pharmaceutical composition for preventing and treating lung papillary adenocarcinoma, characterized in that it is prepared by a method for inducing K-ras-specific activated T cells using.
8. The method of claim 7, wherein the primary cytokines are interleukin-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF), and the secondary cytokines are tumor necrosis factor-α (Tumor Necrosis Factor-α), interleukin-1β (Interleukin-1β), and prostaglandin E2 (Prostaglandin E2) characterized in that the lung papillary adenocarcinoma (lung papillary adenocarcinoma) for preventing and treating a pharmaceutical composition.

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