WO2023085656A1 - Method for inducing antigen-specific t cells by using antigen composition for inducing kras-specific activated t cells - Google Patents

Abstract

A method for inducing KRAS-specific activated T cells by using an antigen composition for inducing KRAS-specific activated T cells, according to the present invention, uses a KRAS mutant (G12D, G12V, and G13D) recombinant overlapping peptide, which is an active ingredient of the antigen composition, designed such that, in the amino acid sequence of KRAS, a total of 12 epitopes (n=1 to 12, wherein the last epitope (n=12) is 23 amino acids) are grouped in a unit of 30 consecutive amino acids, and 15 amino acid sequences overlap between the epitopes. Thus, it is advantageous in that the method of the present invention has an improved effect of inducing KRAS-specific activated T cells, compared to conventional methods for inducing T cells by using wild-type KRAS or a mixture of KRAS mutant epitope peptides as an antigen composition. In addition, the Fast-IVS method of the present invention simultaneously carries out DC cell maturation and T cell induction processes, and thus has the advantage of more efficiently inducing KRAS-specific activated T cells, compared to conventional IVS methods.

Description

Antigen-specific T cell induction method using an antigen composition for inducing KRAS-specific activated T cells

The present invention relates to a method for inducing antigen-specific T cells using an antigen composition for inducing KRAS-specific activated T cells.

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 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).

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 several cancer antigens, KRAS mutations are oncogenic mutations found in about 20% of solid cancers, and are found mainly in adenocarcinomas of the pancreas and colon, lung cancers, and the like. Therapies targeting tumors dependent on KRAS mutant genes are indirect treatments that inhibit or inactivate the function of K-ras because it is difficult to make antibodies that individually bind to K-ras mutants expressed by KRAS mutant genes. was limited and its effectiveness was limited.

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 use an antigen for inducing KRAS-specific activated T cells, which is designed to include the entire KRAS amino acid sequence and mutations and is manufactured using recombinant technology and has excellent economic efficiency, but the DC cell maturation process and T cell culture and It is an object of the present invention to provide a method for inducing antigen-specific T cells capable of efficiently inducing KRAS-specific activated T cells by simultaneously performing the induction process.

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

The present invention cultivates peripheral blood mononuclear cells (PBMCs) in a medium containing an antigen composition for inducing KRAS-specific activated T cells and primary cytokines to mature dentritic cells. A first step of inducing KRAS-specific activated T cells; A second step of further inducing KRAS-specific activated T cells by adding and culturing a secondary cytokine to the cultured PBMC; and a third step of amplifying KRAS-specific activated T cells by culturing PBMCs cultured with the addition of the secondary cytokine; KRAS using an antigen composition for inducing KRAS-specific activated T cells comprising: A method for inducing specific activated T cells and T cells prepared using the same are provided.

The culturing in the first step is performed for 1 day, the culturing in the second step is performed for 2 days, and the culturing in the third step is performed for 10 days, and the primary cytokine is interleukin-4. (Interleukin-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF), and the secondary cytokines are tumor necrosis factor-α, interleukin-1β (Interleukin-1β) 1β), and prostaglandin E2.

The antigen composition for inducing KRAS-specific activated T cells is characterized in that it comprises a KRAS mutant recombinant overlapping peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient, and the KRAS mutations are G12D, G12V, and G13D.

The KRAS mutant recombinant overlapping peptide has a total of 12 types of epitopes (epitopes (n = 1, 2, 3... 10, 11, 12); where n means the order of the epitope, the epitope (n = 1 to 11) contains a 30 amino acid sequence, and the last epitope (n = 12) contains a 23 amino acid sequence.) Including but excluding the epitope (n = 1), the epitope (n = 2, 3, ... 12) has 15 amino acid sequences in the N-terminal direction and 15 amino acid sequences in the C-terminal direction of the immediately preceding epitope (n-1) It is characterized in that it is designed to overlap with the amino acid sequence.

The KRAS 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 epitope (n = 1) contains the KRAS mutation G12V; An epitope (n = 1) containing KRAS mutation G12D is further linked to the N-terminal of the epitope (n = 1) by an LRMK-linker; An epitope (n = 1) containing KRAS mutation 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 KRAS mutation G13D is characterized in that the epitope (n = 1) not containing the KRAS mutation is further linked with an LRMK-linker.

The method for inducing KRAS-specific activated T cells using the antigen composition for inducing KRAS-specific activated T cells of the present invention is a KRAS mutant (G12D, G12V, and G13D) recombinant overlapping peptide, which is an active ingredient of the antigen composition, in the amino acid sequence of KRAS. Sequentially divided into a total of 12 epitopes (n=1 to 12, but the last epitope (n=12) is 23 amino acids) in units of 30 amino acids, but overlapping 15 amino acid sequences between epitopes Since it is designed and used as much as possible, it has an advantage of having an improved KRAS-specific activated T cell inducing effect compared to the conventional method of inducing T cells using a mixture of wild-type KRAS or KRAS mutant epitope peptides as an antigen composition.

In addition, the Fast-IVS method of the present invention has the advantage of being able to more efficiently induce KRAS-specific activated T cells because DC cell maturation and T cell induction processes are simultaneously performed compared to the conventional IVS method.

Therefore, when KRAS-specific activated T cells are prepared from PBMC by applying the method of inducing KRAS-specific activated T cells of the present invention, the conventional IVS method or wild-type KRAS protein or a mixture of KRAS epitope peptides containing as an active ingredient There is an advantage in that KRAS-specific activated T cells can be produced with improved efficiency compared to the induction method using an antigen composition.

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

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

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

Figure 4 shows the results of analyzing the ratio of antigen-specific CD3+ T cells in LP-1 PBMCs for KRAS(M)-ROP, KRAS _1-24 Wild-type, and KRAS _1-24 m 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.

Figure 6 shows the results of KRAS 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.

The present invention cultivates peripheral blood mononuclear cells (PBMCs) in a medium containing an antigen composition for inducing KRAS-specific activated T cells and primary cytokines to mature dentritic cells. A first step of inducing KRAS-specific activated T cells; A second step of further inducing KRAS-specific activated T cells by adding and culturing a secondary cytokine to the cultured PBMC; and a third step of amplifying KRAS-specific activated T cells by culturing PBMCs cultured with the addition of the secondary cytokine; KRAS using an antigen composition for inducing KRAS-specific activated T cells comprising: A method for inducing specific activated T cells is provided.

The method of inducing KRAS-specific activated T cells of the present invention is Fast-IVS (in vitro stimulation), which is different from conventional IVS. The IVS is a monocyte-derived dendritic cell (moDC) obtained from monocytes isolated from blood through a process of differentiation and maturation, and then the antigen for inducing KRAS-specific activated T cells of the present invention It refers to a method of co-culture with T cells in an environment treated with the composition. On the other hand, the Fast-IVS of the present invention has a difference in that the maturation process and antigen (antigen composition for inducing KRAS-specific activated T cells) treatment are simultaneously performed on DC cells in PBMC.

The antigen composition for inducing KRAS-specific activated T cells used as an antigen in the T cell induction process may further include cytokines, hormones, and buffers necessary 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 as a method capable of inducing KRAS-specific activated T cells most efficiently through the examples.

The present invention provides a method for inducing KRAS-specific activated T cells using an antigen composition for inducing KRAS-specific activated T cells, which comprises as an active ingredient a KRAS mutant recombinant overlapping peptide consisting of the amino acid sequence of SEQ ID NO: 1 .

SEQ ID NO: 1 is an amino acid sequence of the KRAS protein and consists of 189 amino acids. In the KRAS mutation, 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 valine (V). It means that an 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 KRAS-specific activated T of the present invention Antigens for cell derivation are obtained.

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

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

In detail, the antigen composition for inducing KRAS-specific activated T cells of the present invention is designed as follows. The epitope (n=1) includes the KRAS mutation G12V; An epitope (n = 1) containing KRAS mutation G12D is further linked to the N-terminal of the epitope (n = 1) by an LRMK-linker; An epitope (n = 1) containing KRAS mutation G13D is further linked to the C-terminal of the epitope (n = 12) by an LRMK-linker; An epitope (n = 1) without a KRAS mutation is further linked to the C-terminal of the epitope (n = 1) containing the KRAS mutation G13D by an LRMK-linker.

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

KRAS, 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. 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 KRAS mutations. Therefore, when T cells specifically recognizing KRAS mutations are amplified, cancers with KRAS mutations can be eliminated and treated, or can serve as a vaccine against cancer. The cancer is not limited as long as the cancer has a KRAS mutation, and examples include adrenocortical carcinoma (ACC), bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), cervical squamous cell carcinoma and cervical adenocarcinoma (CESC), colon Adenocarcinoma (COAD), chronic lymphocytic leukemia (CLL), colorectal cancer (CRC), diffuse large B-cell lymphoma (DLBCL), glioblastoma multiforme (GBM), squamous cell carcinoma of the head and neck (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), endometrioid carcinoma of the uterus (UCEC) or uterine carcinosarcoma (UCS).

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

Example

1. Preparation of KRAS (WT)

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

name	Amino acid sequence (189aa)
KRAS(WT)	MTEYKLVVVG AGGVGKSALT IQLIQNHFVD EYDPTIEDSY RKQVVIDGET CLLDILDTAG QEEYSAMRDQ YMRTGEGFLC VFAINNTKSF EDIHHYREQI KRVKDSEDVP MVLVGNKCDL PSRTVDTKQA QDLARSYGIP FIETSAKTRQ RVEDAFYTLV REIRQYRLKK ISKEEKTPGC VKIKKCIIM

After treating the 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 the protein.

2. Preparation of KRAS(M)-ROP

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

The KRAS (M) -ROP is characterized by having a sequence of 500 amino acids, and the amino acids are sequentially divided into 30 units, but one epitope is designed such that 15 amino acid sequences overlap with each other. Table 2 shows the amino acid sequence of KRAS(M)-ROP (SEQ ID NO: 1) and the amino acid sequence of KRAS(M)-ROP epitope.

1 shows the epitope structure of KRAS(M)-ROP of the present invention.

name	Amino acid sequence (500aa)
KRAS(M)-ROP	MTEYKLVVVG ADGVGKSALT IQLIQNHFVD LRMK MTEYKLVVVG AVGVGKSALT IQLIQNHFVD LRMK KSALTIQLIQ NHFVDEYDPT IEDSYRKQVV LRMK EYDPTIEDSY RKQVVIDGET CLLDILDTAG LRMK IDGETCLLDI LDTAGQEEYS AMRDQYMRTG LRMK QEEYSAMRDQ YMRTGEGFLC VFAINNTKSF LRMK EGFLCVFAIN NTKSFEDIHH YREQIKRVKD LRMK EDIHHYREQI KRVKDSEDVP MVLVGNKCDL LRMK SEDVPMVLVG NKCDLPSRTV DTKQAQDLAR LRMK PSRTVDTKQA QDLARSYGIP FIETSAKTRQ LRMK SYGIPFIETS AKTRQRVEDA FYTLVREIRQ LRMK RVEDAFYTLV REIRQYRLKK ISKEEKTPGC LRMK YRLKKISKEE KTPGCVKIKK CIIM LRMK MTEYKLVVVG AGDVGKSALT IQLIQNHFVD LRMK MTEYKLVVVG AGGVGKSALT IQLIQNHFVD
epitope name	amino acid sequence (Sequence numbers were 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 YMRTG
Epitope 5 (E5, n=5)	QEEYSAMRDQ YMRTGEGFLC VFAINNTKSF
Epitope 6 (E6, n=6)	EGFLC VFAINNTKSF EDIHHYREQI KRVKD
Epitope 7 (E7, n=7)	EDIHHYREQI KRVKDSEDVP MVLVGNKCDL
Epitope 8 (E8, n=8)	SEDVP MVLVGNKCDL PSRTVDTKQA QDLAR
Epitope 9 (E9, n=8)	PSRTVDTKQA QDLARSYGIP FIETSAKTRQ
Epitope 10 (E10, n=10)	SYGIP FIETSAKTRQ RVEDAFYTLV REIRQ
Epitope 11 (E11, n=11)	RVEDAFYTLV REIRQYRLKK ISKEEKTPGC
Epitope 12 (E12, n=12)	YRLKK ISKEEKTPGC VKIKKCIIM
Epitope 1-G12D (E1-G12D)	MTEYKLVVVG ADGVGKSALT IQLIQNHFVD
Epitope 1-G12V (E1-G12V)	MTEYKLVVVG AVGVGKSALT IQLIQNHFVD
Epitope 1-G13D (E1-G13D)	MTEYKLVVVG AGDVGKSALT IQLIQNHFVD

The KRAS-ROP(M) was synthesized (Genescript Co. Ltd.), cloned into a pET30a vector, and then transformed into E.coli and expressed. KRAS(M)-ROP was prepared by cleaving the expressed protein using activated protein C (APC).

3. KRAS(M)-ROP Reactivity Screening Assay

The reactivity of KRAS(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 KRAS (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 KRAS(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 KRAS(M)-ROP was the best in LP-1 among normal PBMCs.

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

The ratio of KRAS(M)-ROP-specific CD3+ T cells according to the KRAS(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.

Figure 3 shows the results of analyzing the ratio of KRAS (M) -ROP-specific CD3+ T cells in LP-1 PBMC according to the concentration of KRAS (M) -ROP of 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 (KRAS(M)-ROP 5μg/ml, KRAS(M)-ROP 1.0μg/ml, KRAS(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 KRAS(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-g) result. Therefore, it is believed that the reactivity of LP-1 PBMC increases in a concentration-dependent manner to KRAS(M)-ROP. In addition, as a result of quantitatively evaluating the reactivity to KRAS(M)-ROP in LP-1 PBMC, the ratio of KRAS(M)-ROP-specific CD3+ T cells was 2.4% when 5 μg/ml of KRAS(M)-ROP was treated. level was confirmed.

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

LP-1 PBMC were treated with KRAS(M)-ROP (500aa), KRAS _1-24 Wild-type (Peptide Wt, 24aa), or KRAS _1-24 mutant (24aa), and then the ratio of antigen-specific CD3+ T cells was measured. Comparative analysis was performed.

Figure 4 shows the results of analyzing the ratio of antigen-specific CD3+ T-cells of LP-1 PBMCs against KRAS(M)-ROP, KRAS _1-24 Wild-type, and KRAS _1-24 mutants of the present invention. Panel A shows the results of IFN-γ capture FACS analysis of KRAS(M)-ROP, KRAS _1-24 Wild-type, and KRAS _1-24 mutant-treated LP-1 PBMCs. 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.

The KRAS _1-24 mutation is a KRAS _1-24 Wild-type in which G, the 12th amino acid, is substituted with D or V, or G, the 12th amino acid, is replaced with D (Pep.G12D, Pep.G12V, Pep.G13D). means The ratio of antigen-specific CD3+ T-cells was analyzed in the same manner as above, and KRAS(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). 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, considering that the ratio of CD3+ T cells induced in response to KRAS(M)-ROP is 2.41%.

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

Based on the above experimental results, CD3+ T cells (ROP-T cells) responding to KRAS(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 IFNg+ CD3+ T cell ratio of 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 KRAS(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
	KRAS(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
	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

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 KRAS(M)-ROP treatment concentration of 1.0 μg/ml and Fast-IVS period of 7 days.

8. ROP-T cell characterization

KRAS 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-g secretion T frequency (%).

Figure 6 shows the results of KRAS mutant epitope screening for ROP-T cells of the present invention. Panel A shows the results of FACS analysis for IFG-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. KRAS(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 KRAS(M)-ROP-specific CD3+ T cells were dispensed at a ratio of 1:20. . Medium mixed with Ag pulsed DC and KRAS(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 Ag pulsed DC were KRAS (M)-ROP (500aa) (ROP_DC), 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 G13D mutant peptide (G13D_DC). Also, for comparison, DC (NoAg_DC) using only an effector without an antigen (Ag) was used.

As a result of the experiment, the ratio (%) of KRAS(M)-ROP-specific CD3+/CD4+ T cells and KRAS(M)-ROP-specific CD3+/CD8+ T cells in CD3+ ROP-T cells when restimulated with ROP_DC was 10% and 5% were confirmed. When restimulated with G12D_DC, the percentages (%) of KRAS(M)-ROP-specific CD3+/CD4+ T cells and KRAS(M)-ROP-specific CD3+/CD8+ T cells in CD3+ ROP-T cells were 1.5%, respectively. and 0.5%. When restimulated with G13D_DC, the percentages of KRAS(M)-ROP-specific CD3+/CD4+ T cells and KRAS(M)-ROP-specific CD3+/CD8+ T cells in CD3+ ROP-T cells were 3.0%, respectively. and 1.5%. In contrast, when restimulation 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.

9. HLA restriction assay of ROP-T cells

In order to confirm the HLA restriction of KRAS G13D mutant-specific T cells among the induced ROP-T, a 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 Ag pulsed DC were KRAS (M)-ROP (500aa) (ROP_DC), 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 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-g+, 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 KRAS-T cells and peptide mix-T cells

The specific response induction of KRAS(M)-ROP to KRAS 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 KRAS(M)-ROP or native KRAS(189aa) as an antigen. In addition, T cells were induced using the Fast-IVS process using a mixture of KRAS _1-24 wild type peptide, KRAS _1-24 G12D peptide, KRAS _1-24 G12V peptide, and KRAS _1-24 G13D peptide as antigens. 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 KRAS (M) -ROP to a KRAS mutation. In Table 5 below, KRAS epitope wild type means Native KRAS _1-24 (24aa); KRAS epitope G12D refers to KRAS _1-24 G12D (24aa); KRAS epitope G12V refers to KRAS _1-24 G12V (24aa); KRAS epitope G13D refers to KRAS _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	KRAS(M)-ROP (8.5μM=5μg/ml)
Pep. -T	KRAS epitope (24mer) 4 kinds mixture (wild type, G12D, G12V, G13D) (8.5 μM)
WT-T	Native KRAS (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 KRAS(M)-ROP as an antigen, it was confirmed that the ratio 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 KRAS as an antigen, the ratio of IFN-γ+ CD3+ T cells was confirmed to be 18.4%. In the case of T cells (Pep_T) induced through the Fast-IVS process using a mixture of KRAS _1-24 wild type peptide, KRAS _1-24 G12D peptide, KRAS _1-24 G12V peptide, and KRAS _1-24 G13D peptide as antigens It was confirmed that the ratio of IFN-γ+ CD3+ T cells was at the level of 19.0%.

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

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.

The present invention selectively amplifies endogenous immune cells capable of inducing a direct immune response by binding to K-ras mutations, including the most frequently occurring mutation sites of K-ras, thereby maximizing the cancer treatment effect and increasing the durability of the anti-cancer effect. It is expected to be used as a highly effective therapeutic agent.

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 (9)

1. By culturing peripheral blood mononuclear cells (PBMC) in a medium containing an antigen composition for inducing KRAS-specific activated T cells and primary cytokines, dentritic cells are matured and KRAS-specific A first step of inducing activated T cells;

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

   KRAS-specific activated T cells using an antigen composition for inducing KRAS-specific activated T cells, characterized in that it comprises; Method for inducing red activated T cells.
2. The method of claim 1, wherein the first step of culturing is performed for 1 day, the second step is cultured for 2 days, and the third step is cultured for 10 days. A method for inducing KRAS-specific activated T cells using an antigen composition for inducing activated T cells.
3. The method of claim 1, 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) KRAS-specific activated T cell induction method using an antigen composition for inducing KRAS-specific activated T cells .
4. The antigen for inducing KRAS-specific activated T cells according to claim 1, wherein the antigen composition for inducing KRAS-specific activated T cells comprises a KRAS mutant recombinant overlapping peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient. Method for inducing KRAS-specific activated T cells using the composition.
5. The antigen for inducing KRAS-specific activated T cells according to claim 1, wherein the antigen composition for inducing KRAS-specific activated T cells comprises a KRAS mutant recombinant overlapping peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient. Method for inducing KRAS-specific activated T cells using the composition.
6. The method of claim 4, wherein the KRAS 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 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) A method for inducing KRAS-specific activated T cells using an antigen composition for inducing KRAS-specific activated T cells, characterized in that it is designed to overlap with 15 amino acid sequences in the C-terminal direction.
7. The method of claim 6, wherein the KRAS 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 A method for inducing KRAS-specific activated T cells using an antigen composition for inducing KRAS-specific activated T cells.
8. 7. The method of claim 6, wherein the epitope (n=1) comprises the KRAS mutation G12V; An epitope (n = 1) containing KRAS mutation G12D is further linked to the N-terminal of the epitope (n = 1) by an LRMK-linker; An epitope (n = 1) containing KRAS mutation G13D is further linked to the C-terminal of the epitope (n = 12) by an LRMK-linker; For induction of KRAS-specific activated T cells, characterized in that an epitope (n = 1) without a KRAS mutation is further linked with an LRMK-linker to the C-terminal of the epitope (n = 1) containing the KRAS mutation G13D A method for inducing KRAS-specific activated T cells using an antigen composition.
9. KRAS-specific activated T cells prepared by the method of any one of claims 1 to 8.

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