WO2023085657A1 - Antigen composition for inducing kras-specific activated t cells - Google Patents

Abstract

In an antigen composition for inducing KRAS-specific activated T cells, of the present invention, a recombinant KRAS-mutant (G12D, G12V, and G13D) overlapping peptide, which is an active ingredient, is designed so that 30 consecutive amino acids in a KRAS amino acid sequence are divided into a total of 12 epitopes as units (n=1 to 12, and the last epitope (n=12) consists of 23 amino acids) and 15 amino acid sequences overlap between the epitopes, and thus the present invention induces KRAS-specific activated T cells that are superior to those when common KRAS-mutant epitope peptides are used as antigens.

Description

Antigen composition for inducing KRAS-specific activated T cells

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

Treatment of cancer patients is basically performed alone or in parallel with surgical operation, chemotherapy, radiotherapy, etc., and concurrent treatment such as cytokine, peptide vaccine, antibody treatment is also increasing.

It has been proven that immune cell therapy with anticancer treatment effect by non-specific passive immune response has the effect of suppressing recurrence after surgery for liver cancer patients. Studies on the combination treatment of inhibitors are being actively conducted.

A general peptide vaccine used as an anti-cancer treatment uses an amino wire sequence with high immunogenicity selected as an epitope and optimized for use. Peptide vaccines used as anticancer drugs have the advantages of good selectivity, effectiveness, and excellent tolerability.

The peptide vaccine is carried on the major histocompatibility complex (MHC) molecule of the antigen presenting cell (APC) that binds to the T cell receptor (TCR) recognizing cancer antigens, and most of the CD8 T Induces cancer cell death by educating or inducing activation of cells.

However, since peptide vaccines depend on an epitope composed of 9 to 11 amino acids, when the epitope is mutated in cancer cells, immune evasion is possible and CD4 T cells do not help, limiting the activity and memory function of CD8 cells will have In addition, since the peptide vaccine is designed as a peptide loaded on an MHC class I molecule, there is a problem in that it is limited according to the type of human leukocyte antigen (HLA) of the patient.

In order to improve the above problems, overlapping peptide (OLP) vaccine therapeutics have been developed. Unlike peptide vaccines, OLP vaccines are characterized by including all antigens. OLP vaccines are designed to include all antigens but have overlapping epitope amioline sequences, so there is no restriction on HLA types, and they can receive help from CD4 T cells, so they have the advantage of showing better immune responses than conventional peptide vaccines. .

However, the OLP vaccine requires a process of selecting as an epitope a peptide containing about 20 amino acid sequences with excellent immune response among the OLP library for a specific antigen, and 10 or more selected epitopes are contiguous and overlapping. Because of the design, there were manufacturing issues that cost a lot of money and time. Recombinant overlapping peptide (ROP) was developed to improve the problems of the OLP vaccine treatment, and has the advantage of saving cost and time through recombinant protein production technology.

Among cancer antigens, KRAS mutation is a relatively common oncogenic mutation found in about 20% of solid cancers, and is most commonly found in adenocarcinoma of the pancreas and colon, lung cancer, and the like. However, despite long-term research and efforts, new therapies targeting K-ras in KRAS mutation-dependent tumors are not satisfactory in terms of their effectiveness. The reason is that it was difficult to make antibodies that individually bind to K-ras mutants expressed by KRAS mutations, so it was limited to indirect treatments that inhibit or inactivate the function of K-ras. Therefore, if an antigen capable of inducing individual recognition of K-ras mutants is developed using a recombinant overlapping peptide (ROP), it is expected to make a great contribution to the development of KRAS mutation-dependent tumor therapeutics.

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.

Since the present invention includes the entire KRAS amino acid sequence and KRAS mutations, it is possible to induce KRAS-specific activated T cells capable of inducing an immune response by directly binding to various KRAS mutations in KRAS mutation-dependent tumors, as well as to manufacture using recombinant technology Therefore, an object of the present invention is to provide an antigen for inducing KRAS-specific activated T cells with excellent economic efficiency.

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

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

The KRAS mutant recombinant overlapping peptide includes G12D, G12V, and G13D as KRAS mutations, and includes a total of 12 types of epitopes (epitope (n = 1, 2, 3....10, 11, 12); Here, n means the sequence of the epitope, and the epitope (n = 1 to 11) contains a sequence of 30 amino acids, and the last epitope (n = 12) includes a 23 amino acid sequence), but the epitope (n = 2, 3, ... 12) except for the epitope (n = 1) is the epitope of the immediately preceding sequence of 15 amino acids in the N-terminal direction It is characterized in that it is designed to overlap with the 15 amino acid sequence in the C-terminal direction of (n-1).

In the KRAS mutant recombinant overlapping peptide, the epitopes (n = 1, 2, 3...10, 11, 12) are located in sequence, and the epitopes are connected by an LRMK-linker; 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; 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 antigen composition for inducing KRAS-specific activated T cells of the present invention comprises a total of 12 epitopes (epitope , n = 1 to 12, but the last epitope (n = 12) is 23 amino acids), but designed so that 15 amino acid sequences overlap between epitopes, and conventional KRAS mutant epitope peptides are used as antigens It has the advantage of having an improved KRAS-specific activated T cell inducing effect.

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 provides an antigen composition for inducing KRAS-specific activated T cells comprising a KRAS mutant recombinant overlapping peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

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).

Induction of T cells specific for the KRAS mutations (G12D, G12V, G13D) can be performed through in vitro stimulation (IVS) or Fast-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, and the Fast-IVS is a maturation process and antigen (antigen composition for inducing KRAS-specific activated T cells) treatment for DC cells in PBMC. means to do it at the same time.

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).

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 evaluated. 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 antigen-specific CD3+ T-cell ratio 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.

When 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 agent can be developed.

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 (5)

1. An antigen composition for inducing KRAS-specific activated T cells comprising a KRAS mutant recombinant overlapping peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.
2. The antigen composition for inducing KRAS-specific activated T cells according to claim 1, wherein the KRAS mutations are G12D, G12V, and G13D.
3. According to claim 1, 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 the epitope (n = 2, 3, ... 12) is the 15 amino acid sequence in the N-terminal direction of the 15 amino acid sequence in the C-terminal direction of the immediately preceding epitope (n-1) Antigen composition for inducing KRAS-specific activated T cells, characterized in that designed to overlap with each other.
4. The method of claim 3, 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 An antigen composition for inducing KRAS-specific activated T cells.
5. 4. The method of claim 3, 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 antigen composition.

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