WO2023128379A1 - Composition pharmaceutique comprenant des lymphocytes t activés spécifiques de k-ras pour la prévention et le traitement du cancer du sein et son procédé de préparation - Google Patents

Composition pharmaceutique comprenant des lymphocytes t activés spécifiques de k-ras pour la prévention et le traitement du cancer du sein et son procédé de préparation Download PDF

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WO2023128379A1
WO2023128379A1 PCT/KR2022/019964 KR2022019964W WO2023128379A1 WO 2023128379 A1 WO2023128379 A1 WO 2023128379A1 KR 2022019964 W KR2022019964 W KR 2022019964W WO 2023128379 A1 WO2023128379 A1 WO 2023128379A1
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ras
cells
epitope
breast cancer
rop
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Korean (ko)
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이왕준
문현종
임선기
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의료법인 명지의료재단
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere

Definitions

  • the present invention relates to a pharmaceutical composition for preventing and treating breast cancer containing K-ras-specific activated T cells and a method for preparing the same.
  • Immunotherapy is a method of inducing cancer-specific CTL (Cytotoxic T Lymphocyte) endogenously or injecting it to kill cancer cells.
  • CTL Cytotoxic T Lymphocyte
  • LAK Lymphokine activate killer
  • the LAK immune cell therapy showed encouraging clinical results for cancer patients who had failed surgery, chemotherapy, and radiation therapy.
  • 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
  • IL-2 interleukin-2
  • 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.
  • the peptide vaccine can not only cause immune evasion when the target site of cancer cells mutates, but also have low immune reactivity because CD4 T cells do not help in the immune response, and are designed with peptides that are 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).
  • OLP vaccines designed by overlapping peptides containing the entire antigen and having high immunogenicity are being developed.
  • 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.
  • the OLP vaccine has a problem in that manufacturing cost is high and immunomodulation is relatively difficult.
  • a method for producing antigen-specific T cells using a vaccine is also an important factor.
  • 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.
  • 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.
  • 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.
  • 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 breast cancer containing cells and a method for preparing the same.
  • the present invention provides a pharmaceutical composition for preventing and treating breast cancer, comprising an antigen composition for inducing K-ras-specific activated T cells and K-ras-specific activated T cells induced using cytokine It is characterized by providing, and the cancer cells of the breast cancer are characterized in that K-ras, K-ras mutant G12V, K-ras mutant G12D, or K-ras mutant G13D are detected.
  • 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.
  • PBMC peripheral blood mononuclear cells
  • 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- ⁇ , interleukin-1 ⁇ , and prostaglandin E2.
  • a pharmaceutical composition for the prevention and treatment of breast cancer containing K-ras-specific activated T cells of the present invention comprises K-ras mutations (G12D, G12V, and G13D) recombinant overlapping peptides as an antigen composition.
  • epitopes epitopes 1 to 12
  • it is more effective in recognizing and killing breast cancer in which K-ras, K-ras mutant G12V, K-ras mutant G12D, or K-ras mutant G13D is detected.
  • the pharmaceutical composition for preventing and treating breast cancer containing K-ras-specific activated T cells of the present invention not only K-ras but also breast cancer in which K-ras mutations are detected, especially breast adenocarcinoma (breast cancer) adenocarcinoma) can be effectively prevented and treated.
  • 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.
  • FIG 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 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 results.
  • Figure 5 shows the results of comparing the Fast-IVS process and the No-Cytokine process of the present invention.
  • FIG 6 shows the results of K-ras mutant epitope screening for ROP-T cells of the present invention.
  • Figure 8 shows the ratio of CD3+ T cells secreting IFN- ⁇ (IFN- ⁇ +) 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.
  • FIG. 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 provides a pharmaceutical composition for preventing and treating breast cancer, comprising an antigen composition for inducing K-ras-specific activated T cells and K-ras-specific activated T cells induced using cytokine to provide.
  • 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 breast cancer of the present invention
  • treatment of the present invention means breast cancer 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 cancer.
  • the breast cancer 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
  • KRAS 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
  • the activity of K-ras abnormally increases and causes cancer.
  • the detection of K-ras, K-ras mutant G12V, K-ras mutant G12D, or K-ras mutant G13D in the breast cancer cancer cells of the present invention means that KRAS is expressed as an oncogene and K-ras activity is increased. This means that it is abnormally increased.
  • the breast cancer 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, more preferably K-ras mutant G13D. and K-ras mutant 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.
  • GAPs GTPase-activating proteins
  • the pharmaceutical composition for preventing and treating breast cancer containing K-ras-specific activated T cells of the present invention is induced using an antigen composition for inducing K-ras-specific activated T cells and cytokine
  • an antigen composition for inducing K-ras-specific activated T cells and cytokine As it contains K-ras-specific activated T cells, it is suitable for cancer treatment 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.
  • K-ras, K-ras mutation G12V, K-ras mutation G12D, or K-ras mutation G13D is detected, and more preferably K-ras, K-ras mutation G12V, K-ras mutation G13D.
  • composition 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%.
  • 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.
  • 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 breast cancer 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.
  • the amount of active ingredient means that an appropriate dose within the indicated range can be obtained.
  • the pharmaceutical composition for preventing and treating breast cancer containing the K-ras-specific activated T cells of the present invention is formulated as a sterile composition for injection
  • a conventional preparation is performed using a vehicle such as distilled water for injection can be prescribed according to
  • 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.
  • 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.
  • the injection formulation examples 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 breast cancer 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.
  • 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 breast cancer 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 breast cancer of the present invention is a cancer type, patient's age, weight, sex, medical condition, disease severity, administration route, and various drugs such as separately administered It can be determined by considering factors.
  • the amount of the pharmaceutical composition for the prevention and treatment of breast cancer of the present invention administered to a patient may be determined by many factors such as an administration method, the patient's health condition, weight, and a doctor's prescription, which are in the art. It is within the knowledge of a person of ordinary skill.
  • the pharmaceutical composition for the prevention and treatment of breast cancer 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, 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 ml or more, about 9x10 7 cells/mL or more, about 1x10 8 cells/mL or more, about 2x10 8
  • 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.
  • 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 breast cancer of the present invention may be administered to a patient by bolus injection or continuous infusion.
  • the pharmaceutical composition for the prevention and treatment of breast cancer 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 day or more, 2 hours or more At least once every day, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 1 month, 3 months, 6 months , at least 2 times, at least 3 times, at least 4 times, or at least 5 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.
  • 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 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.
  • PBMC peripheral blood mononuclear cells
  • 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.
  • 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.
  • the cytokine is interleukin-4, interleukin-1 ⁇ , granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor- ⁇ (Tumor Necrosis Factor). - ⁇ , TNF- ⁇ ), and the hormone may be prostaglandin E2 (PGE2).
  • 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- ⁇ , interleukin-1 ⁇ , and prostaglandin E2 are secondary cytokines, immature DC ) was used to mature.
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • the culturing of the first step is performed for 1 day
  • the culturing of the second step is performed for 2 days
  • 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 consisting of 189 amino acids.
  • the 12th amino acid is substituted from glycine (G) to aspartic acid (D)
  • the 12th amino acid is substituted from glycine (G) to valine (V)
  • 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 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.
  • the antigen composition for inducing K-ras-specific activated T cells of the present invention is designed as follows.
  • 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
  • KRAS a gene of the ras protein
  • oncogene found as a mutation in various carcinomas
  • 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).
  • ACC adrenocortical carcinoma
  • BLCA bladder urothelial carcinoma
  • BRCA invasive breast carcinoma
  • CESC intracervical adenocarcinoma
  • colon adenocarcinoma COAD
  • chronic lymphocytic leukemia CLL
  • colorectal cancer CRC
  • DLBCL diffuse large B-cell lymphoma
  • GBM glioblastoma multiforme
  • HNSC head and neck squamous cell carcinoma
  • KICH chromophobe kidney
  • KIRC renal clear cell carcinoma
  • LAML renal papillary cell carcinoma
  • LIHC acute myelogenous leukemia
  • LAML acute myelogenous leukemia
  • LIHC acute myelogenous leukemia
  • LAML hepatocellular carcinoma
  • LAD lung adenocarcinoma
  • LUSC lung squamous cell carcinoma
  • MM multiple myeloma
  • OV pancreatic adenocarcinoma
  • PAAD prostate adenocarcinoma
  • PRAD rectal adenocarcinoma
  • READ skin melanoma
  • STAD gastric adeno
  • 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.
  • K-ras 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
  • 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.
  • 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.
  • 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.
  • APC activated protein C
  • PBMC peripheral blood mononuclear cells
  • 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- ⁇ ) assay and
  • panel B shows the SFC graph of the ELISpot (IFN- ⁇ ) assay.
  • PBMC peripheral blood mononuclear cell
  • LP-1 PBMC, LP-4 PBMC, and LP-6 PBMC peripheral blood mononuclear cells
  • antigen 5 ⁇ g/ml, 1.0 ⁇ g/ml, and 0.1 ⁇ g/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- ⁇ 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.
  • 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- ⁇ capture staining was performed after antigen treatment on LP-1 PBMCs, and this was analyzed.
  • Panel 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- ⁇ ) assay
  • Panel B shows the SFC graph for each condition of the ELISpot (IFN- ⁇ ) assay.
  • Panel C shows the principle light method of IFN- ⁇ capture staining
  • Panel D shows the results of IFN- ⁇ capture FACS analysis.
  • Panel E shows a graph of the percentage (%) of IFN- ⁇ secreting CD3+ T cells.
  • LP-1 PBMC 1x10 6 cells were seeded and cultured, and then antigens (K-ras(M)-ROP 5 ⁇ g/ml, K-ras(M)-ROP 1.0 ⁇ g/ml, K-ras(M)-ROP 0.1 ⁇ g/ml) was treated.
  • LP-1 PBMC treated with tetanus toxoid vaccine (TTX) at 5 ⁇ g/ml and 1.0 ⁇ g/ml and anti-CD3 were used as positive controls.
  • TTX tetanus toxoid vaccine
  • IFN- ⁇ 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.
  • secondary detection antibody 2nd detection andtibody
  • CD3, CD4 , CD8, and CD137 Cell characteristics of the LP-1 PBMCs subjected to the IFN- ⁇ capture staining were analyzed using a Fluorescence activated cell sorter (FACS).
  • FACS Fluorescence activated cell sorter
  • 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- ⁇ 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- ⁇ -secreting CD3+ T cell ratio (%)
  • 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.
  • 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).
  • D the 12th amino acid G is replaced with D
  • K-ras(M)-ROP 500aa
  • Peptide Wt Pep.G12D, Pep.G12V, and Pep.G13D were used as antigens.
  • CD+ T cells that responded to Peptide Wt were not identified, 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).
  • 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.
  • CD3+ T cells responding to K-ras(M)-ROP were prepared.
  • 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- ⁇ + 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.
  • 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- ⁇ (TNF- ⁇ ), Interleukin-1 ⁇ (IL-1 ⁇ ), and Prostaglandin E2 (PGE2).
  • IL-4 Interleukin-4
  • GM-CSF Granulocyte-Macrophage Colony-Stimulating Factor
  • TNF- ⁇ Tumor Necrosis Factor- ⁇
  • IL-1 ⁇ Interleukin-1 ⁇
  • PGE2 Prostaglandin E2
  • Table 3 below shows the Fast-IVS process and the No-Cytokine process of the present invention.
  • the Fast-IVS process for preparing ROP-T cells was optimized by changing the treatment concentration of K-ras(M)-ROP and 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 ⁇ g/mL 5.0 1.0 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- ⁇ +, CD3+) (%) No Ag-T 4.6 1.1 1.2 ROP-T 19.7 18.6 52.9
  • K-ras mutant epitope screening was performed to characterize the amplified ROP-T cells.
  • 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.
  • antigen pulsed dendritic cells Ag pulsed DC
  • the reactivity was analyzed by calculating the re-stimulation IFN- ⁇ secretion T frequency (%).
  • Panel 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- ⁇ +, CD3+, and CD4+.
  • Panel B shows the result of analyzing the cell ratio (%) of the restimulation IFN- ⁇ secreting T cell ratio for each condition.
  • 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 ⁇ l in 96 well.
  • K-ras(M)-ROP-specific CD3+ T cells were dispensed into the 96 well to be 1x10 5 cells/100 ⁇ l, but the Ag pulsed DC and K-ras(M)-ROP-specific CD3+ T cells had a cell count of 1:20. 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- ⁇ cap, and IFN- ⁇ 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).
  • DC (NoAg_DC) using only an effector without an antigen (Ag) was used.
  • 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.
  • HLA-DQ human leukocyte antigen DQ
  • 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- ⁇ +, CD3 +, and CD4 + were analyzed by FACS analysis.
  • antigen-specific T cells showed that the ratio of CD3+CD4+ T cells secreting IFN- ⁇ 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- ⁇ increased to 15% or more regardless of HLA-DQ blocking for DC.
  • 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.
  • FIG. 8 shows the ratio of CD3+ T cells secreting IFN- ⁇ (IFN- ⁇ +) for each condition of the present invention.
  • T cells were induced using the Fast-IVS process using K-ras(M)-ROP or native K-ras(189aa) as an antigen.
  • 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- ⁇ + CD3+ T cells was analyzed using FACS.
  • 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).
  • the K-ras(M)-ROP antigen has an IFN- ⁇ + CD3+ T cell induction effect that is about twice as good as that using native K-ras or an epitope containing K-ras mutation.
  • T cells induced through the Fast-IVS process were confirmed.
  • 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 kills each cancer cell. It was confirmed how soluble it was.
  • Table 6 below shows analysis information for each carcinoma cell line.
  • HLA-type HLA-A HLA-DR B1 HLA-DQ
  • 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,
  • 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.
  • 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.
  • Table 7 below shows test results for cancer cells (primary culture) obtained from colorectal cancer patients.
  • 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.
  • FIG. 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.
  • a pharmaceutical composition for preventing and treating breast cancer comprising the antigen composition for inducing K-ras-specific activated T cells of the present invention and K-ras-specific activated T cells induced using cytokine It can be used to prevent and treat breast cancer.

Abstract

Composition pharmaceutique comprenant des lymphocytes T activés spécifiques de K-ras pour la prévention et le traitement du cancer du sein, selon la présente invention, étant une composition d'antigènes dans laquelle des peptides K-ras mutants (G12D, G12V et G13D) recombinants se chevauchant sont divisés séquentiellement en un total de 12 épitopes (n = 1 à 12, à condition que le dernier épitope (n = 12) soit de 23 acides aminés) de 30 acides aminés dans une séquence d'acides aminés de K-ras et étant conçue de telle sorte que des séquences de 15 acides aminés se chevauchent entre les épitopes. Ainsi, la composition pharmaceutique présente l'avantage d'être plus efficace dans la reconnaissance et la destruction du cancer du sein dans lequel le K-ras, le mutant K-ras G12V, le mutant K-ras G12D ou le mutant K-ras G13D est détecté. Par conséquent, lorsque la Composition pharmaceutique comprenant des lymphocytes T activés spécifiques de K-ras pour la prévention et le traitement du cancer du sein selon la présente invention est utilisée, il existe des avantages de prévention et de traitement efficace du cancer du sein, spécifiquement, d'un adénocarcinome du sein, dans lequel K-ras ainsi que des mutants K-ras sont détectés.
PCT/KR2022/019964 2021-12-29 2022-12-08 Composition pharmaceutique comprenant des lymphocytes t activés spécifiques de k-ras pour la prévention et le traitement du cancer du sein et son procédé de préparation WO2023128379A1 (fr)

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