Antigen composition for inducing KRAS-specific activated t cells

Abstract

An antigen composition for inducing KRAS-specific activated T-cells according to the present invention divides KRAS mutant (G12D, G12V, and G13D) recombinant overlapping peptides from the KRAS amino acid sequence sequentially into a total of 12 epitopes (epitope, n = 1 to 12, here, the last epitope (n = 12) is 23 amino acids) in units of 30 amino acids, and is designed such that 15 amino acid sequences of the epitopes overlap, and thus has the advantage of having an enhanced KRAS-specific activated T-cell induction effect compared with the conventional art in which KRAS mutant epitope peptides are used as antigens.

Description

Antigen composition for KRAS-specific activated T cell induction

[0001] The present invention relates to an antigen composition for inducing KRAS-specific activated T cells.

[0002] Treatment for cancer patients basically involves surgical procedures, chemotherapy, and radiation therapy, either alone or in combination, and there is a growing trend of combination therapies such as cytokines, peptide vaccines, and antibody treatments.

[0003] Immunotherapy drugs that have an anticancer effect through a non-specific passive immune response have been proven to be effective in suppressing recurrence after surgery in liver cancer patients. As an example, CAR-T cell therapy has received clinical approval, and research on the combination of anticancer immunotherapy and immune checkpoint inhibitors is actively underway.

[0004] Common peptide vaccines used as anticancer treatments select amino acid sequences with high immunogenicity as epitopes and optimize them for use. Peptide vaccines used as anticancer treatments have the advantages of good selectivity, effectiveness, and excellent tolerability.

[0005] The above-mentioned peptide vaccine is carried on the Major histocompatibility complex (MHC) molecules of antigen-presenting cells (APCs) that bind to T cell receptors (TCRs) that recognize cancer antigens, and induces cancer cell death by educating or activating most CD8 T cells.

[0006] However, since peptide vaccines rely on epitopes composed of 9 to 11 amino acids, if these epitopes mutate in cancer cells, they can not only evade immunity but also have limitations in the activation and memory functions of CD8 cells due to a lack of assistance from CD4 T cells. In addition, since these peptide vaccines are designed as peptides that are loaded onto MHC class I molecules, there was a problem of limitations depending on the patient's human leukocyte antigen (HLA) type.

[0007] To address the aforementioned problems, overlapping peptide (OLP) vaccine therapies have been developed. Unlike peptide vaccines, OLP vaccines are characterized by containing the entire antigen. Since OLP vaccines are designed to contain the entire antigen while overlapping the amioline sequences of the epitopes, they not only have no limitations regarding HLA types but also can receive assistance from CD4 T cells, which has the advantage of showing a superior immune response compared to conventional peptide vaccines.

[0008] However, the above-mentioned OLP vaccine had manufacturing problems that required significant cost and time, as it required not only a process of selecting peptides containing approximately 20 amino acid sequences with excellent immune response from an OLP library for a specific antigen as epitopes, but also designing the selected epitopes to be continuous and overlapping with more than 10 of them. Recombinant overlapping peptides (ROPs) were developed to address the problems of the above-mentioned OLP vaccine therapeutics and have the advantage of saving cost and time through recombinant protein production technology.

[0009] Among cancer antigens, KRAS mutations are relatively common oncogenic mutations found in approximately 20% of solid tumors, most frequently occurring in adenocarcinomas of the pancreas and colorectal cancer, as well as lung cancer. However, despite extensive research and efforts, new therapies targeting K-ras in KRAS mutation-dependent tumors have not been satisfactory in terms of efficacy. This is because it is difficult to produce antibodies that individually bind to K-ras mutants expressed by KRAS mutations, limiting therapeutic approaches to indirect methods that inhibit or inactivate the function of K-ras. Therefore, if an antigen capable of inducing the individual recognition of K-ras mutants is developed using a recombinant overlapping peptide (ROP), it is expected to make a significant contribution to the development of treatments for KRAS mutation-dependent tumors.

[0010] The patent documents and references mentioned in this specification are incorporated by reference into this specification to the same extent that each document is individually and clearly identified by reference.

[0011] The present invention aims to provide an antigen for inducing KRAS-specific activated T cells that includes the entire KRAS amino acid sequence and KRAS mutations, and thus can induce KRAS-specific activated T cells capable of directly binding to various KRAS mutations in KRAS mutation-dependent tumors to trigger an immune response, and is also highly economically efficient as it is manufactured using recombinant technology.

[0012] Other objects and technical features of the present invention are more specifically presented by the following detailed description of the invention, claims, and drawings.

[0013] The present invention provides an antigen composition for inducing KRAS-specific activated T cells comprising a KRAS mutant recombinant nested peptide having the amino acid sequence of SEQ ID NO. 1 as an active ingredient.

[0014] The above KRAS mutant recombinant overlapping peptide includes G12D, G12V, and G13D as KRAS mutants and is characterized by including a total of 12 types of epitopes (epitope(n=1, 2, 3....10, 11, 12); where n represents the number of the epitope, epitopes (n=1 to 11) contain 30 amino acid sequences, and the last epitope (n=12) contains 23 amino acid sequences) based on amino acid sequences sequentially arranged from any one amino acid in the amino acid sequence of KRAS consisting of SEQ ID NO. 2, wherein the epitopes (n=2, 3,...12), excluding epitope (n=1), are designed such that 15 amino acid sequences in the N-terminal direction overlap with 15 amino acid sequences in the C-terminal direction of the epitope (n-1) of the previous sequence.

[0015] The above KRAS mutant recombinant nested peptide is characterized in that the epitopes (n=1, 2, 3...10, 11, 12) are positioned in order and the epitopes are connected by an LRMK-linker; the epitope (n=1) contains the KRAS mutant G12V; the N-terminal of the epitope (n=1) is further connected by an LRMK-linker to an epitope (n=1) containing the KRAS mutant G12D; the C-terminal of the epitope (n=12) is further connected by an LRMK-linker to an epitope (n=1) containing the KRAS mutant G13D; and the C-terminal of the epitope (n=1) containing the KRAS mutant G13D is further connected by an LRMK-linker to an epitope (n=1) that does not contain the KRAS mutant.

[0016] The antigen composition for inducing KRAS-specific activated T cells according to the present invention has the advantage of having an improved KRAS-specific activated T cell induction effect compared to using conventional KRAS mutant epitope peptides as antigens, by designing the active ingredient, a KRAS mutant (G12D, G12V, and G13D) recombinant overlapping peptide, to be divided into a total of 12 epitopes (epitope, n=1 to 12, provided that the last epitope (n=12) has 23 amino acids) sequentially in units of 30 amino acids in the amino acid sequence of KRAS, with 15 amino acid sequences overlapping between the epitopes.

[0017] Figure 1 shows the amino acid sequence structure of the KRAS(M)-ROP of the present invention.

[0018] Figure 2 shows the results of analyzing the reactivity of PBMC to KRAS(M)-ROP of the present invention.

[0019] Figure 3 shows the results of analyzing the specific CD3+ T cell ratio of LP-1 PBMC according to the KRAS(M)-ROP concentration of the present invention.

[0020] FIG. 4 is the KRAS(M)-ROP of the present invention, KRAS 1-24 Wild-type, and KRAS 1-24 This shows the results of analyzing the antigen-specific CD3+ T cell ratio of LP-1 PBMCs against the m mutation.

[0021] Figure 5 shows the results of comparing the Fast-IVS process and the No-Cytokine process of the present invention.

[0022] Figure 6 shows the results of KRAS mutation epitope screening on ROP-T cells of the present invention.

[0023] Figure 7 shows the results of the HLA-DQ blocking essay of the present invention.

[0024] Figure 8 shows the ratio of CD3+ T cells secreting IFN-γ (IFN-γ+) under the conditions of the present invention.

[0025] The present invention provides an antigen composition for inducing KRAS-specific activated T cells comprising a KRAS mutant recombinant nested peptide having the amino acid sequence of SEQ ID NO. 1 as an active ingredient.

[0026] The above sequence number 1 is an amino acid sequence of the KRAS protein consisting of 189 amino acids. The above KRAS mutation means that 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), or the 13th amino acid is substituted from glycine (G) to aspartic acid (D).

[0027] The above recombinant means inserting the genetic information of the designed antigen into recombinant plasmid DNA containing the genetic information of the antigen, and when the recombinant plasmid DNA is transformed into a microorganism to express a protein and purified, the KRAS-specific activated T cell induction antigen of the present invention is obtained.

[0028] The KRAS mutant recombinant overlapping peptide of the present invention comprises a total of 12 types of epitopes (epitope(n=1, 2, 3....10, 11, 12); where n represents the number of the epitope, the epitopes (n=1 to 11) contain 30 amino acid sequences, and the last epitope (n=12) contains 23 amino acid sequences) based on amino acid sequences sequentially arranged from one amino acid in the amino acid sequence of KRAS sequence No. 2, wherein the epitopes (n=2, 3,...12), excluding the epitope (n=1), are designed such that 15 amino acid sequences in the N-terminal direction overlap with 15 amino acid sequences in the C-terminal direction of the epitope (n-1) of the previous sequence.

[0029] The above KRAS mutant recombinant nested peptide is characterized by the epitopes (n=1, 2, 3...10, 11, 12) being positioned in sequence and the epitopes being connected by LRMK-linkers, and the LRMK-linkers are composed of Leucine (L), Arginine (R), Methionine (M), and Lysine (K), which have the advantage of being favorable for the antigen presentation process (MHCI class I pathway) by dendritic cells.

[0030] In detail, the antigen composition for inducing KRAS-specific activated T cells according to the present invention is designed as follows. The epitope (n=1) comprises a KRAS mutation G12V; the N-terminal of the epitope (n=1) is further connected to an epitope (n=1) comprising a KRAS mutation G12D via an LRMK-linker; the C-terminal of the epitope (n=12) is further connected to an epitope (n=1) comprising a KRAS mutation G13D via an LRMK-linker; and the C-terminal of the epitope (n=1) comprising the KRAS mutation G13D is further connected to an epitope (n=1) not comprising a KRAS mutation via an LRMK-linker.

[0031] Using the antigen composition for inducing KRAS-specific activated T cells of the present invention, T cells specific to KRAS mutations (G12D, G12V, G13D) can be induced, and said T cells specific to KRAS mutations (G12D, G12V, G13D) can be used to treat cancer cells having KRAS mutations (G12D, G12V, G13D).

[0032] The induction of T cells specific to the above KRAS mutations (G12D, G12V, G13D) can be performed through in vitro stimulation (IVS) or Fast-IVS. The above IVS refers to a method of obtaining monocyte-derived dendritic cells (moDCs) from monocytes isolated from blood through differentiation and maturation processes, and then co-cultureting them with T cells in an environment treated with the antigen composition for inducing KRAS-specific activated T cells according to the present invention. The above Fast-IVS refers to simultaneously performing a maturation process and antigen treatment (antigen composition for inducing KRAS-specific activated T cells) on DC cells within PBMCs.

[0033] The KRAS-specific antigen composition for inducing activated T cells used as an antigen in the above T cell induction process may further include cytokines, hormones, and buffer solutions necessary for the maturation and growth of DC cells. Preferably, the cytokines may be interleukin-4, interleukin-1β, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-α (TNF-α), and the hormone may be prostaglandin E2 (PGE2).

[0034] KRAS is a type of RAS protein and a small GTPases protein that plays an important role in signaling pathways related to cell differentiation, proliferation, and survival. The aforementioned RAS protein is well known as an oncogene found as a mutation in various types of cancer, and it is known that 85% of RAS-derived cancers are caused by KRAS mutations. Therefore, amplifying T cells that specifically recognize KRAS mutations can eliminate cancers with KRAS mutations, thereby treating them or serving as a vaccine against cancer. The above cancers are not limited to cancers with KRAS mutations, examples of which include adrenocortical carcinoma (ACC), bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), cervical squamous cell carcinoma and cervical adenocarcinoma (CESC), colorectal 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), chromophrenic kidney (KICH), clear cell carcinoma of the kidney (KIRC), papillary cell carcinoma of the kidney (KIRP), acute myeloid leukemia (LAML), hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), multiple myeloma (MM), ovarian serous cyst adenocarcinoma (OV), pancreatic adenocarcinoma (PAAD), prostate adenocarcinoma (PRAD), rectal adenocarcinoma (READ), and skin It may be melanoma (SKCM), gastric adenocarcinoma (STAD), testicular germ cell tumor (TGCT), thyroid adenocarcinoma (THCA), endometrioid carcinoma of the uterine body (UCEC), or uterine carcinosarcoma (UCS).

[0035] The present invention will be described in detail below through examples.

[0036]

[0037] Examples

[0038]

[0039] 1. Manufacture of KRAS(WT)

[0040] First, the KRAS amino acid sequence (Sequence No. 2) was inserted into the expression vector. KRAS(WT) consists of 189 amino acids, and the amino acid sequence is as shown in Table 1 below.

[0041] NameAmino acid sequence (189aa)KRAS(WT)MTEYKLVVVG AGGVGKSALT IQLIQNHFVD EYDPTIEDSYRKQVVIDGET CLLDILDTAG QEEYSAMRDQ YMRTGEGFLCVFAINNTKSF EDIHHYREQI KRVKDSEDVP MVLVGNKCDLPSRTVDTKQA QDLARSYGIP FIETSAKTRQ RVEDAFYTLVREIRQYRLKK ISKEEKTPGC VKIKKCIIM

[0042] After treating the above KRAS-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 a protein.

[0043]

[0044] 2. Manufacture of KRAS(M)-ROP

[0045] An antigen of KRAS Mutant Recombinant Overlapping Peptide (KRAS(M)-ROP) was designed, and an expression vector was prepared using the same method as KRAS(WT). The KRAS(M)-ROP includes G12D, in which G (Glycine) at the 12th amino acid position of KRAS is mutated to D (Asapartic acid); G12V, in which G (Glycine) at the 12th amino acid position of KRAS is mutated to V (Valine); or G13D, in which G (Glycine) at the 13th amino acid position of KRAS is mutated to D (Asapartic acid).

[0046] The above KRAS(M)-ROP is characterized by having a sequence of 500 amino acids, with the amino acids sequentially separated into groups of 30, and designed so that 15 amino acid sequences of each epitope overlap. Table 2 shows the amino acid sequence of KRAS(M)-ROP (Sequence No. 1) and the amino acid sequence of the KRAS(M)-ROP epitope.

[0047] Figure 1 shows the epitope structure of the KRAS(M)-ROP of the present invention.

[0048] NameAmino acid sequence (500aa)KRAS(M)-ROPMTEYKLVVVG ADGVGKSALT IQLIQNHFVD LRMKMTEYKLVVVG AVGVGKSALT IQLIQNHFVD LRMKKSALTIQLIQ NHFVDEYDPT IEDSYRKQVV LRMKEYDPTIEDSY RKQVVIDGET CLLDILDTAG LRMKIDGETCLLDI LDTAGQEEYS AMRDQYMRTG LRMKQEEYSAMRDQ YMRTGEGFLC VFAINNTKSF LRMKEGFLCVFAIN NTKSFEDIHH YREQIKRVKD LRMKEDIHHYREQI KRVKDSEDVP MVLVGNKCDL LRMKSEDVPMVLVG NKCDLPSRTV DTKQAQDLAR LRMKPSRTVDTKQA QDLARSYGIP FIETSAKTRQ LRMKSYGIPFIETS AKTRQRVEDA FYTLVREIRQ LRMKRVEDAFYTLV REIRQYRLKK ISKEEKTPGC LRMKYRLKKISKEE KTPGCVKIKK CIIM LRMKMTEYKLVVVG AGDVGKSALT IQLIQNHFVD LRMKMTEYKLVVVG AGGVGKSALT IQLIQNHFVD Epitope Name Amino Acid Sequence (Sequence numbers assigned based on KRAS WT) Epitope 1(E1, n=1)MTEYKLVVVG AGGVGKSALT IQLIQNHFVD Epitope 2(E2, n=2)KSALT IQLIQNHFVD EYDPTIEDSY RKQVV Epitope 3(E3, n=3)EYDPTIEDSY RKQVVIDGET CLLDILDTAG Epitope 4(E4, n=4)IDGET CLLDILDTAG QEEYSAMRDQ YMRTGEpitope 5 (E5, n=5)QEEYSAMRDQ YMRTGEGGFLC VFAINNTKSFEpitope 6 (E6, n=6)EGFLC VFAINNTKSF EDIHHYREQI KRVKDEpitope 7 (E7, n=7)EDIHHYREQI KRVKDSEDVP MVLVGNKCDL Epitope 8 (E8, n=8)SEDVP MVLVGNKCDL PSRTVDTKQA QDLAR Epitope 9 (E9,n=8)PSRTVDTKQA QDLARSYGIP FIETSAKTRQEpitop10(E10, n=10)SYGIP FIETSAKTRQ RVEDAFYTLV REIRQEpitop11(E11, n=11)RVEDAFYTLV REIRQYRLKK, ISKEEKTPGC epitope12(E12, n=12)YRLKK ISKEEKTPGC VKIKKCIIM Epitope1-G12D(E1-G12D)MTEYKLVVVG ADGVGKSALT IQLIQNHFVD Epitope1-G12V(E1-G12V)MTEYKLVVVG AVGVGKSALT IQLIQNHFVD Epitope1-G13D(E1-G13D)MTEYKLVVVG AGDVGKSALT IQLIQNHFVD

[0049] The above KRAS-ROP(M) was synthesized (Genescript Co. Ltd.), cloned into the pET30a vector, and expressed by transforming E. coli. The expressed protein was cleaved using APC (Activated protein C) to produce KRAS(M)-ROP.

[0050]

[0051] 3. KRAS(M)-ROP Reactivity Screening Analysis

[0052] The reactivity of KRAS(M)-ROP in peripheral blood mononuclear cells (PBMCs) of normal individuals was screened. The screening was performed using the enzyme-linked immune absorbent spot assay (ELISpot assay).

[0053] Figure 2 shows the results of analyzing the reactivity of PBMC to KRAS(M)-ROP of the present invention. The results of Figure 2 are graphs obtained by analyzing the SFC images of the ELISpot(IFN-γ) assay.

[0054] First, 1x10 PBMCs (LP-1 PBMC, LP-4 PBMC, and LP-6 PBMC) obtained from leukocyte splitting of healthy volunteers 5 Cells were seeded and cultured, after which antigens were applied. KRAS(M)-ROP at concentrations of 5 µg / ml, 1.0 µg / ml, and 0.1 µg / ml was used as the antigen, and anti-CD3 was used as a positive control. Cell culture was performed under conditions of 37°C, 25% CO₂, and overnight (O / N). The cultured cells were stained with IFN-γ, and Spot Forming Cells (SFCs) were identified and analyzed. The experimental results confirmed that among normal PBMCs, LP-1 showed the most excellent reactivity to KRAS(M)-ROP.

[0055]

[0056] 4. Analysis of Specific CD3+ T Cell Ratios by KRAS(M)-ROP Concentration

[0057] The ratio of KRAS(M)-ROP-specific CD3+ T cells according to KRAS(M)-ROP concentration was analyzed for the above LP-1 PBMCs. To this end, LP-1 PBMCs were treated with antigens, IFN-γ capture staining was performed, and the results were analyzed.

[0058] Figure 3 shows the results of analyzing the ratio of KRAS(M)-ROP-specific CD3+ T cells in LP-1 PBMCs according to the KRAS(M)-ROP concentration of the present invention. Panel A shows a graph of the results of analyzing SFC images under different conditions of the ELISpot (IFN-γ) assay. Panel B shows the principle and method of IFN-γ capture staining. Panel C shows a graph of the ratio (%) of IFN-γ-secreting CD3+ T cells by analyzing the results of IFN-γ capture FACS analysis images.

[0059] First, LP-1 PBMC 1x10 6Cells were seeded and cultured, and then treated with antigens (KRAS(M)-ROP 5 µg / ml, KRAS(M)-ROP 1.0 µg / ml, KRAS(M)-ROP 0.1 µg / ml) at different concentrations. Additionally, LP-1 PBMCs 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, 25% CO₂, and overnight (O / N).

[0060] IFN-γ capture staining is performed on antigen-treated LP-1 PBMCs using a primary capture antibody (1 st After treatment with capture antibody), incubate at 37°C for 45 minutes, and secondary detection antibody (2 nd The procedure was performed using a detection and tibole) and CD3, CD4, CD8, and CD137 treatment method. The LP-1 PBMCs subjected to the above IFN-γ capture staining were analyzed for cell characteristics using a Fluorescence Activated Cell Sorter (FACS).

[0061] Experimental results confirmed that treating LP-1 PBMC with antigens at varying concentrations increased the proportion of KRAS(M)-ROP-specific CD2+ T cells, which was consistent with the results of the aforementioned ELISpot (IFN-g). Therefore, it is determined that the reactivity of LP-1 PBMC increases in a concentration-dependent manner with respect to KRAS(M)-ROP. Additionally, a quantitative evaluation of the reactivity of LP-1 PBMC to KRAS(M)-ROP revealed that when treated with 5 μg / ml of KRAS(M)-ROP, the proportion of KRAS(M)-ROP-specific CD3+ T cells was 2.4%.

[0062]

[0063] 5. Comparative analysis of antigen-specific CD3+ T cell ratios according to antigen type

[0064] KRAS(M)-ROP(500aa), KRAS on LP-1 PBMC 1-24 Wild-type (Peptide Wt, 24aa), or KRAS 1-24 After treatment with the mutation (24aa), the ratio of antigen-specific CD3+ T cells was compared and analyzed.

[0065] FIG. 4 is the KRAS(M)-ROP of the present invention, KRAS 1-24 Wild-type, and KRAS 1-24 This shows the results of the analysis of antigen-specific CD3+ T-cell ratios in LP-1 PBMCs for mutations. Panel A shows KRAS(M)-ROP, KRAS 1-24 Wild-type, and KRAS 1-24 The results of the IFN-γ capture FACS analysis of mutant-treated LP-1 PBMCs are displayed as a graph of the proportion of IFN-γ-secreting CD3+ T cells (%), and Panel B shows the results of the antigen-specific CD3+ T cell proportion (%) graph in a table.

[0066] The above KRAS 1-24 The mutation is KRAS 1-24 It refers to the case where the 12th amino acid G in the wild-type is substituted with D or V, or the 12th amino acid G is replaced with D (Pep.G12D, Pep.G12V, Pep.G13D). Antigen-specific CD3+ T-cell ratios were analyzed using the same method as above, and KRAS(M)-ROP(500aa), Peptide Wt, Pep.G12D, Pep.G12V, and Pep.G13D were used as antigens.

[0067] Experimental results showed that no CD+ T cells responded to Peptide Wt, and CD3+ T cells responded to Pep.G12D, Pep.G12V, and Pep.G13D were also found to be significantly low at 0.31% (Pep.G12D), 0.11% (Pep.G12V), and 0.25% (Pep.G13D). Considering that the proportion of CD3+ T cells induced by KRAS(M)-ROP is 2.41%, these results indicate that the induction of antigen-specific CD3+ T cells by epitopes alone—peptides composed of 24 amino acids—is minimal.

[0068]

[0069] 6. Preparation of ROP-T cells using KRAS(M)-ROP and Fast-IVS

[0070] Based on the above experimental results, CD3+ T cells (ROP-T cells) responsive to KRAS(M)-ROP were prepared. In the present invention, ROP-T cells were prepared by applying Fast-IVS (Fast-In vitro Stimulation).

[0071] Figure 5 shows the results of comparing the Fast-IVS process of the present invention with the No-Cytokine process. Panel A shows the manufacturing and evaluation process of ROP-T cells using Fast-IVS and the characterization process of ROP-T cells. Panel B shows the results of comparing the ratio of IFNg+ CD3+ T cells amplified under Fast-IVS process conditions and the No-Cytokine process conditions.

[0072] The above Fast-IVS process is characterized by simultaneously performing a process of inducing antigen-specific CD3+ T cells using an antigen and performing cell expansion by treating with cytokines. In contrast, the above No-Cytokine process is characterized by performing cell expansion on antigen-specific CD3+ T cells induced by the antigen without treating with cytokines.

[0073] The cytokines used for cell amplification in the above Fast-IVS process are Interleukin-4 (IL-4), Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), Tumor Necrosis Factor-α (TNF-α), Interleukin-1β (IL-1β), and Prostaglandin E2 (PGE2).

[0074] Table 3 below shows the Fast-IVS process and No-Cytokine process of the present invention.

[0075] Fast-IVS ProcessNo-Cytokine ProcessDay-0LP-1 PBMC Seeding with Ag, IL-4, and GM-CSFLP-1 PBMC Seeding Without CytokineDay-1Adding TNF-α, IL-1β and PGE2No Cytokine AddingDay 3ExpansionExpansionDay 5, 7, 9, 11, 12Media AddingMedia AddingDay 13HarvestHarvest

[0076] Analysis results confirmed that the proportion of antigen-specific CD3+ T cells secreting IFN-γ was amplified about four times more in the Fast-IVS process than in the No-Cytokine process.

[0077]

[0078] 7. Optimization of the Fast-IVS Process for ROP-T Cell Production

[0079] The Fast-IVS process for ROP-T cell production was optimized by changing the treatment concentration of KRAS(M)-ROP and the Fast-IVS process conditions. Table 4 below shows examples for the optimization of the Fast-IVS process.

[0080] Example 1 Example 2 Example 3 Experimental Conditions Scale PBMCs 10M @ 24well 10M @ 24well 10M @ 24well Cytokine Manufacturer JW Creagene JW Creagene JW Creagene KRAS(M)-ROP μg / ml 5.0 1.0 1.0 Fast-IVS (Medium AIM-V) Duration Days 5 5 7 Expansion Duration Days 10 10 10 Experimental Results Expansion Fold No Ag-T 45 3 4 5 5 ROP-T 55 4 9 6 5 Helper T cell No Ag-T 54.7 35.8 21.1 ROP-T 64.4 75.3 60.4 KRAS(M)-ROP specific T cell (IFN-g+, CD3+) (%) No Ag-T 4.6 1.1 1.2 ROP-T 19.7 18.6 52.9

[0081] Experimental results confirmed that ROP-T cells were amplified in all examples, and the optimal Fast-IVS process was confirmed to be a treatment concentration of KRAS(M)-ROP of 1.0 μg / ml and a Fast-IVS period of 7 days.

[0082]

[0083] 8. Analysis of ROP-T Cell Characteristics

[0084] The characteristics of amplified ROP-T cells were analyzed by performing KRAS mutant epitope screening. To this end, autologous dendritic cells were induced from PBMCs, and antigen-pulsed dendritic cells (Ag pulsed DCs) capable of stimulating antigen-specific T cells were prepared by sensitizing the autologous dendritic cells to antigens, and the responsiveness of ROP-T cells was confirmed. The responsiveness was analyzed by calculating the re-stimulation IFN-γ secretion T frequency (%).

[0085] Figure 6 shows the results of KRAS mutation epitope screening for ROP-T cells of the present invention. Figure 6 shows the results of analyzing FACS analysis images for IFG-γ+, CD3+, and CD4+ of the present invention as a graph of the cell ratio (%) of restimulated IFN-γ secreting T cells by condition.

[0086] First, autologous dendritic cells (Autologous DCs) were cultured for 4 days and sensitized to antigens to prepare Ag-pulsed DCs. The Ag-pulsed DCs were placed in 96-well plates containing 5 x 10⁶ 3 KRAS(M)-ROP-specific CD3+ T cells were dispensed at a density of 1 x 10⁶ cells / 100 µl. 5The cells were dispensed at a density of 100 µL / 100 µL, ensuring that the ratio of the Ag pulsed DCs to KRAS(M)-ROP-specific CD3+ T cells was 1:20. The medium mixed with Ag pulsed DCs and KRAS(M)-ROP-specific CD3+ T cells was cultured for 4 hours. The percentages of CD3+, CD4+, CD137+, IFN-γ cap, and IFN-γ-secreting T cells in the cultured cells were analyzed using FACS. The antigens used to prepare the Ag pulsed DCs were KRAS(M)-ROP(500aa) (ROP_DC) and KRAS 1-24 wild type peptide (WT_DC), KRAS 1-24 G12D mutant peptide (G12D_DC), KRAS 1-24 G12V mutant peptide (G12V_DC), and KRAS 1-24 It was a G13D mutant peptide (G13D_DC). In addition, for comparison, a DC using only an effector without an antigen (Ag) (NoAg_DC) was used.

[0087] Experimental results confirmed that when restimulated using ROP_DC, the ratio (%) of KRAS(M)-ROP-specific CD3+ / CD4+ T cells and KRAS(M)-ROP-specific CD3+ / CD8+ T cells present in CD3+ ROP-T cells was 10% and 5%, respectively. When restimulated using G12D_DC, the ratio (%) of KRAS(M)-ROP-specific CD3+ / CD4+ T cells and KRAS(M)-ROP-specific CD3+ / CD8+ T cells present in CD3+ ROP-T cells was 1.5% and 0.5%, respectively. When restimulated using G13D_DC, the ratio (%) of KRAS(M)-ROP-specific CD3+ / CD4+ T cells and KRAS(M)-ROP-specific CD3+ / CD8+ T cells present in CD3+ ROP-T cells was 3.0% and 1.5%, respectively. In contrast, when restimulated using Wt_DC and G12V_DC, KRAS(M)-ROP specific CD3+ / CD4+ T cells and KRAS(M)-ROP specific CD3+ / CD8+ T cells were hardly detected.

[0088]

[0089] 9. HLA restriction analysis of ROP-T cells

[0090] To confirm the HLA restriction of KRAS G13D mutant-specific T cells among induced ROP-Ts, a Human leukocyte antigen (HLA-DQ) assay was performed.

[0091] Figure 7 shows the results of the HLA-DQ blocking essay of the present invention.

[0092] First, antigen-sensitized DCs (Ag pulsed DCs) were prepared. The antigens used to prepare the Ag pulsed DCs were KRAS(M)-ROP(500aa)(ROP_DC) and KRAS 1-24 wild type peptide (WT_DC), KRAS 1-24G12D mutant peptide (G12D_DC), KRAS 1-24 G12V mutant peptide (G12V_DC), and KRAS 1-24 It was a G13D mutant peptide (G13D_DC). HLA-DQ blocking was performed on the Ag pulsed DC prepared above by treating it with an HLA-DQ antibody for 1 hour. After re-stimulating ROP-T using the HLA-DQ-blocked Ag pulsed DC, IFN-g+, CD3+, and CD4+ were analyzed via FACS analysis.

[0093] Analysis results confirmed that the proportion of CD3+CD4+ T cells secreting IFN-γ in antigen-nonspecific T cells (NoAg-T) was negligible, regardless of the type of DC used for restimulation or whether HLA-DQ blocking was performed on the DC. In contrast, it was confirmed that when ROP-T cells were restimulated using ROP_DC, the proportion of CD3+CD4+ T cells secreting IFN-γ increased to over 15%, regardless of whether HLA-DQ blocking was performed on the DC. Additionally, it was confirmed that when ROP-T cells were restimulated using HLA-DQ-blocked G13D_DC, the proportion of CD3+CD4+ T cells secreting IFN-γ decreased by approximately 6% (7% → 1%) compared to when they were restimulated using HLA-DQ-unblocked G13D_DC.

[0094] Consequently, it is determined that the ROP-T cells of the present invention induce the amplification of CD4+ T cells that are specific to the ROP antigen, restricted to HLA-DQ, and specific to the G13D mutation.

[0095]

[0096] 10. Comparison of control native KRAS-T cells and peptide mix-T cells

[0097] The induction of a specific response by KRAS(M)-ROP to KRAS mutations was verified.

[0098] Figure 8 shows the proportion of CD3+ T cells secreting IFN-γ (IFN-γ+) under the conditions of the present invention. First, T cells were induced using a Fast-IVS process using KRAS(M)-ROP or native KRAS(189aa) as antigens. In addition, KRAS 1-24 wild type peptide, KRAS 1-24 G12D peptide, KRAS 1-24 G12V peptide, and KRAS 1-24 T cells were induced using a Fast-IVS process with a G13D peptide mixture as the antigen. As a control, T cells were induced using a Fast-IVS process with only the implementer and no antigen. The induced T cells were restimulated using ROP_DC, and the proportion of IFN-γ+ CD3+ T cells was analyzed using FACS.

[0099] Table 5 below shows the experimental method for verifying the specific response induction of KRAS(M)-ROP for KRAS mutations. In Table 5 below, the KRAS epitope wild type is Native KRAS 1-24 It means (24aa); KRAS epitope G12D is KRAS 1-24 It refers to G12D(24aa); the KRAS epitope G12V is KRAS 1-24 It refers to G12V(24aa); the KRAS epitope G13D is KRAS 1-24 It means G13D(24aa).

[0100] conditionsFast-IVSExpansionAntigenCytokineDaysMediaDaysNo Ag-T-D0:IL-4, GM-CSFD+1:TNF-a, IL-1b, PGE27 DaysAlys+IL-2+SR3%10 DaysROP-TKRAS(M)-ROP(8.5μM=5μg / ml)Pep.-TKRAS epitope(24mer) 4 types of mixture (wild type, G12D, G12V, G13D) (8.5μM) WT-TNative KRAS (8.5μM=2μg / ml)

[0101] Experimental results confirmed that the proportion of IFN-γ+ CD3+ T cells was approximately 8% for T cells induced via Fast-IVS without the use of an antigen (No Ag-T). For T cells induced via the Fast-IVS process using KRAS(M)-ROP as the antigen (ROP-T), the proportion of IFN-γ+ CD3+ T cells reached 37.4%. For T cells induced via the Fast-IVS process using Native KRAS as the antigen (WT-T), the proportion of IFN-γ+ CD3+ T cells was found to be approximately 18.4%. KRAS 1-24 wild type peptide, KRAS 1-24 G12D peptide, KRAS 1-24 G12V peptide, and KRAS 1-24 In the case of T cells (Pep_T) induced by the Fast-IVS process using a G13D peptide mixture as an antigen, the proportion of IFN-γ+ CD3+ T cells was found to be 19.0%.

[0102] In summary, it is determined that the KRAS(M)-ROP antigen has an IFN-γ+ CD3+ T cell induction effect that is about twice as superior as using an epitope containing Native KRAS or a KRAS mutation.

[0103] The specific embodiments described herein represent preferred embodiments or examples of the invention and do not limit the scope of the invention. It is evident to those skilled in the art that variations and other uses of the invention do not deviate from the scope of the invention as described in the claims of this specification.

[0104] If an antigen capable of inducing individual recognition of K-ras mutants is developed using the recombinant overlapping peptide (ROP) of the present invention, a KRAS mutation-dependent tumor treatment can be developed.

[0105] 1. Amino acid sequence of SEQ ID NO. 1

[0106] MTEYKLVVVG ADGVGKSALT IQLIQNHFVD LRMKMTEYKL VVVGAVGVGK SALTIQLIQN 60

[0107] HFVDLRMKKS ALTIQLIQNH FVDEYDPTIE DSYRKQVVLR MKEYDPTIED SYRKQVVIDG 120

[0108] ETCLLDILDT AGLRMKIDGE TCLLDILDTA GQEEYSAMRD QYMRTGLRMK QEEYSAMRDQ 180

[0109] YMRTGEGFLC VFAINNTKSF LRMKEGFLCV FAINNTKSFE DIHHYREQIK RVKDLRMKED 240

[0110] IHHYREQIKR VKDSEDVPMV LVGNKCDLLR MKSEDVPMVL VGNKCDLPSR TVDTKQAQDL 300

[0111] ARLRMKPSRT VDTKQAQDLA RSYGIPFIET SAKTRQLRMK SYGIPFIETS AKTRQRVEDA 360

[0112] FYTLVREIRQ LRMKRVEDAF YTLVREIRQY RLKKISKEEK TPGCLRMKYR LKKISKEEKT 420

[0113] PGCVKIKKCI IMLRMKMTEY KLVVVGAGDV GKSALTIQLI QNHFVDLRMK MTEYKLVVVG 480

[0114] AGGVGKSALT IQLIQNHFVD

[0115]

[0116] 2. Amino acid sequence of Sequence No. 2

[0117] MTEYKLVVVG AGGVGKSALT IQLIQNHFVD EYDPTIEDSY RKQVVIDGET CLLDILDTAG 60

[0118] QEEYSAMRDQ YMRTGEGFLC VFAINNTKSF EDIHHYREQI KRVKDSEDVP MVLVGNKCDL 120

[0119] PSRTVDTKQA QDLARSYGIP FIETSAKTRQ RVEDAFYTLV REIRQYRLKK ISKEEKTPGC 180

[0120] VKIKKCIIM

Claims

1. An antigen composition for inducing KRAS-specific activated T cells comprising, as an active ingredient, a KRAS mutant recombinant overlapping peptide composed of the amino acid sequence of SEQ ID NO. 1, wherein the KRAS mutant recombinant overlapping peptide comprises a total of 12 types of epitopes (epitope(n=1, 2, 3....10, 11, 12); where n represents the sequence number of the epitope, epitopes (n=1 to 11) contain 30 amino acid sequences, and the last epitope (n=12) contains 23 amino acid sequences) based on amino acid sequences sequentially arranged from any one amino acid in the amino acid sequence of KRAS composed of SEQ ID NO. 2, wherein the epitopes (n=2, 3,...12) are designed such that 15 amino acid sequences in the N-terminal direction overlap with 15 amino acid sequences in the C-terminal direction of the epitope (n-1) of the immediately preceding sequence number. An antigen composition for inducing KRAS-specific activated T cells, characterized in that the KRAS mutant recombinant nested peptide has the epitopes (n=1, 2, 3...10, 11, 12) positioned in sequence and the epitopes are connected by an LRMK-linker.

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