KR102753583B1 - Antigen Composition For Inducing KRAS Specific Activated T Cell - Google Patents

Abstract

The antigen composition for inducing KRAS-specific activated T cells of the present invention has the advantage of having an improved KRAS-specific activated T cell induction effect than when using conventional KRAS mutant epitope peptides as antigens, by sequentially dividing the KRAS mutant (G12D, G12V, and G13D) recombinant overlapping peptides, which are active ingredients, into a total of 12 epitopes (epitopes, n=1 to 12, provided that the last epitope (n=12) is 23 amino acids) in units of 30 amino acids in the amino acid sequence of KRAS, and designing such that 15 amino acid sequences overlap between the epitopes.

Description

{Antigen Composition For Inducing KRAS Specific Activated T Cell}

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

Treatment for cancer patients basically involves surgery, chemotherapy, and radiation therapy alone or in combination, and concurrent treatment with cytokines, peptide vaccines, and antibody treatments is also on the rise.

It has been proven that immunotherapy with anticancer effects through non-specific passive immune responses has the effect of suppressing recurrence after surgery for liver cancer patients, and as an example, CAR-T cell therapy has received clinical approval, and research on concurrent treatment with anticancer immunotherapy and immune checkpoint inhibitors is actively being conducted.

Common peptide vaccines used as anticancer treatments select highly immunogenic amino acid sequences as epitopes and optimize them for use. Peptide vaccines used as anticancer treatments have the advantages of being highly selective, effective, and having excellent tolerability.

The above peptide vaccine induces cancer cell death by educating or activating mostly CD8 T cells by loading onto the major histocompatibility complex (MHC) molecule of antigen presenting cells (APC) that bind to the T cell receptor (TCR) that recognizes cancer antigens.

However, since peptide vaccines depend on epitopes consisting of 9 to 11 amino acids, if the epitopes are mutated in cancer cells, they can not only evade immunity, but also have limitations in the activity and memory functions of CD8 cells because they do not receive help from CD4 T cells. In addition, since the peptide vaccines are designed as peptides that are loaded onto MHC class I molecules, there was a problem in that there were limitations depending on the patient's human leukocyte antigen (HLA) type.

To improve the above problems, overlapping peptide (OLP) vaccines have been developed. Unlike peptide vaccines, OLP vaccines have the characteristic of containing the entire antigen. Since OLP vaccines contain the entire antigen but are designed so that the amino acid sequences of the epitopes overlap, they are not limited to HLA types and can receive help from CD4 T cells, so they have the advantage of showing a superior immune response than conventional peptide vaccines.

However, the above OLP vaccine not only requires a process of selecting a peptide containing about 20 amino acid sequences with an excellent immune response from an OLP library for a specific antigen as an epitope, but also has manufacturing problems that require a lot of cost and time because the selected epitopes are designed to be 10 or more consecutive and overlapping. Recombinant overlapping peptide (ROP) was developed to improve the problems of the above 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, and lung cancer. However, despite long-term research and efforts, new treatments targeting K-ras in KRAS mutation-dependent tumors are not satisfactory in terms of their effects. This is because it is difficult to create antibodies that individually bind to K-ras mutants expressed by KRAS mutations, and thus, they have been limited to indirect treatments that inhibit or inactivate the function of K-ras. Therefore, if an antigen that can induce individual recognition of K-ras mutants using recombinant overlapping peptide (ROP) is developed, it is expected to greatly contribute to the development of treatments for KRAS mutation-dependent tumors.

The patent documents and references mentioned in this specification are incorporated by reference into this specification to the same extent as if each document was individually and specifically designated by reference.

Korean Patent Publication No. 10-2018-0010229 Korean Patent Publication No. 10-2020-0141994

Vaccines. 2014 Jul 2;2(3):515-36. Adv Protein Chem Struct Biol. 2015;99:1-14. Nat Rev Cancer. 2008 May;8(5):351-60).

The present invention aims to provide an antigen for inducing KRAS-specific activated T cells, which comprises the entire amino acid sequence of KRAS and KRAS mutations, and thus can directly bind to various KRAS mutations of KRAS mutation-dependent tumors and induce an immune response, and which is economically efficient because it is manufactured using recombinant technology.

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

The present invention provides an antigen composition for inducing KRAS-specific activated T cells, comprising a KRAS mutant recombinant overlapping peptide consisting of an amino acid sequence of sequence number 1 as an active ingredient.

The above KRAS mutant recombinant overlapping peptide comprises KRAS mutations including G12D, G12V, and G13D, and comprises a total of 12 types of epitopes (epitopes (n=1, 2, 3....10, 11, 12); here, n represents the order of the epitopes, and the epitopes (n=1 to 11) include 30 amino acid sequences and the last epitope (n=12) includes 23 amino acid sequences) sequentially listed as units from any one amino acid in the amino acid sequence of KRAS consisting of SEQ ID NO: 2, but is characterized in that the epitopes (n=2, 3,...12) excluding the epitope (n=1) are designed such that the N-terminal 15 amino acid sequence overlaps with the C-terminal 15 amino acid sequence of the epitope (n-1) of the immediately preceding order.

The above KRAS mutant recombinant overlapping peptide has the epitopes (n=1, 2, 3...10, 11, 12) sequentially positioned and the epitopes are connected by an LRMK-linker; the epitope (n=1) includes KRAS mutation G12V; at the N-terminal of the epitope (n=1), an epitope (n=1) including KRAS mutation G12D is further connected by an LRMK-linker; at the C-terminal of the epitope (n=12), an epitope (n=1) including KRAS mutation G13D is further connected by an LRMK-linker; The C-terminal of the epitope (n=1) containing the above KRAS mutation G13D is further connected to an epitope (n=1) not containing the KRAS mutation by a LRMK-linker.

The antigen composition for inducing KRAS-specific activated T cells of the present invention has the advantage of having an improved KRAS-specific activated T cell induction effect than when using conventional KRAS mutant epitope peptides as antigens, by sequentially dividing the KRAS mutant (G12D, G12V, and G13D) recombinant overlapping peptides, which are active ingredients, into a total of 12 epitopes (epitopes, n=1 to 12, provided that the last epitope (n=12) is 23 amino acids) in units of 30 amino acids in the amino acid sequence of KRAS, and designing such that 15 amino acid sequences overlap between the epitopes.

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 KRAS(M)-ROP concentration of the present invention.
Figure 4 shows the results of analyzing the antigen-specific CD3+ T cell ratio of LP-1 PBMCs for KRAS(M)-ROP, KRAS _1-24 Wild-type, and KRAS _1-24 m mutant 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.
Figure 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+) according to the conditions 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 an amino acid sequence of sequence number 1 as an active ingredient.

The above sequence number 1 is the amino acid sequence of the KRAS protein and consists of 189 amino acids. The above KRAS mutation means that the 12th amino acid is substituted from glycine (G) to aspartic acid (D), or the 12th amino acid is substituted from glycine (G) to valine (V), or the 13th amino acid is substituted from glycine (G) to aspartic acid (D).

The above recombination means inserting the genetic information of the designed antigen into a recombinant plasmid DNA, and when the recombinant plasmid DNA is transformed into a microorganism to express the protein and purify it, the antigen for inducing KRAS-specific activated T cells of the present invention is obtained.

The KRAS mutant recombinant overlapping peptide of the present invention comprises a total of 12 types of epitopes (epitopes (n=1, 2, 3....10, 11, 12); here, n represents the order of the epitopes, and the epitopes (n=1 to 11) include 30 amino acid sequences and the last epitope (n=12) includes 23 amino acid sequences) whose units are sequentially listed amino acid sequences from any one amino acid in the amino acid sequence of KRAS consisting of SEQ ID NO: 2, but the epitopes (n=2, 3,...12) excluding the epitope (n=1) are designed such that the N-terminal 15 amino acid sequence overlaps with the C-terminal 15 amino acid sequence of the epitope (n-1) of the immediately preceding order.

The above KRAS mutant recombinant overlapping peptide is characterized in that the epitopes (n=1, 2, 3...10, 11, 12) are sequentially located and the epitopes are connected by an LRMK-linker. The LRMK-linker is a linker composed of Leucine (L), Arginine (R), Methionine (M), and Lysine (K), and has an advantage in the antigen presentation process (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 KRAS mutation G12V; an epitope (n=1) including KRAS mutation G12D is further linked to the N-terminal of the epitope (n=1) via an LRMK-linker; an epitope (n=1) including KRAS mutation G13D is further linked to the C-terminal of the epitope (n=12) via an LRMK-linker; and an epitope (n=1) not including KRAS mutation is further linked to the C-terminal of the epitope (n=1) including KRAS mutation G13D via an LRMK-linker.

By 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 the T cells specific for the KRAS mutations (G12D, G12V, G13D) can be used to treat cancer cells having KRAS mutations (G12D, G12V, G13D).

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

The antigen composition for inducing KRAS-specific activated T cells used as an antigen in the above T cell induction process may further contain cytokines, hormones, and buffers 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).

KRAS is a type of RAS protein, a small GTPases protein that plays an important role in the signaling system related to cell differentiation, proliferation, and survival. The RAS protein is well known as an oncogene found as a mutation in various cancers, and 85% of RAS-derived cancers are known to be caused by KRAS mutations. Therefore, if T cells that specifically recognize KRAS mutations are amplified, cancers with KRAS mutations can be eliminated and treated, or they can serve as a vaccine against cancer. The above cancers are not limited to cancers with KRAS mutations, and examples thereof include adrenocortical carcinoma (ACC), bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), cervical squamous cell carcinoma and cervical intraepithelial adenocarcinoma (CESC), colon adenocarcinoma (COAD), chronic lymphocytic leukemia (CLL), colorectal cancer (CRC), diffuse large B-cell lymphoma (DLBCL), glioblastoma multiforme (GBM), head and neck squamous cell carcinoma (HNSC), chromophobe kidney (KICH), renal clear cell carcinoma (KIRC), renal papillary cell carcinoma (KIRP), acute myeloid leukemia (LAML), hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), multiple myeloma (MM), ovarian serous adenocarcinoma (OV), pancreatic adenocarcinoma (PAAD), prostate adenocarcinoma (PRAD), rectal adenocarcinoma (READ), skin It may be melanoma (SKCM), gastric adenocarcinoma (STAD), testicular germ cell tumor (TGCT), thyroid adenocarcinoma (THCA), corpus endometrioid carcinoma (UCEC), or uterine carcinosarcoma (UCS).

The present invention is described in detail below through examples.

Example

1. Manufacturing 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 as shown in Table 1 below.

name Amino acid sequence (189aa) KRAS(WT) MTEYKLVVVG ¹⁰ AGGVGKSALT ²⁰ IQLIQNHFVD ³⁰ EYDPTIEDSY ⁴⁰

RKQVVIDGET ⁵⁰ CLLDILDTAG ⁶⁰ QEEYSAMRDQ ⁷⁰ YMRTGEGFLC ⁸⁰

VFAINNTKSF ⁹⁰ EDIHHYREQI ¹⁰⁰ KRVKDSEDVP ¹¹⁰ MVLVGNKCDL ¹²⁰

PSRTVDTKQA ¹³⁰ QDLARSYGIP ¹⁴⁰ FIETSAKTRQ ¹⁵⁰ RVEDAFYTLV ¹⁶⁰

REIRQYRLKK ¹⁷⁰ ISKEEKTPGC ¹⁸⁰ VKIKKCIIM ¹⁸⁹

The above KRAS-WT was treated with a restriction enzyme and placed into the pET30a vector to produce an expression vector, which was then transformed into E. coli to express the protein.

2. Manufacturing of KRAS(M)-ROP

The antigen of KRAS Mutant Recombinant Overlapping Peptide (KRAS (M)-ROP) was designed and an expression vector was prepared in the same manner 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).

The above KRAS(M)-ROP is characterized by having a 500 amino acid sequence, and the amino acids are sequentially separated into groups of 30, but each epitope is designed to have 15 amino acid sequences overlapping each other. Table 2 shows the amino acid sequence of KRAS(M)-ROP (SEQ ID NO: 1) and the amino acid sequence of the KRAS(M)-ROP epitope.

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

name Amino acid sequence (500aa) KRAS(M)-ROP MTEYKLVVVG ¹⁰ A D GVGKSALT ²⁰ IQLIQNHFVD ³⁰ LRMK ³⁴
MTEYKLVVVG ⁴⁴ A V GVGKSALT ⁵⁴ IQLIQNHFVD ⁶⁴ LRMK ⁶⁸
KSALTIQLIQ ⁷⁸ NHFVDEYDPT ⁸⁸ IEDSYRKQVV ⁹⁸ LRMK ¹⁰²
EYDPTIEDSY ¹¹² RKQVVIDGET ¹²² CLLDILDTAG ¹³² LRMK ¹³⁶
IDGETCLLDI ¹⁴⁶ LDTAGQEEYS ¹⁵⁶ AMRDQYMRTG ¹⁶⁶ LRMK ¹⁷⁰
QEEYSAMRDQ ¹⁸⁰ YMRTGEGGFLC ¹⁹⁰ VFAINNTKSF ²⁰⁰ LRMK ²⁰⁴
EGFLCVFAIN ²¹⁴ NTKSFEDIHH ²²⁴ YREQIKRVKD ²³⁴ LRMK ²³⁸
EDIHHYREQI ²⁴⁸ KRVKDSEDVP ²⁵⁸ MVLVGNKCDL ²⁶⁸ LRMK ²⁷²
SEDVPMVLVG ²⁸² NKCDLPSRTV ²⁹² DTKQAQDLAR ³⁰² LRMK ³⁰⁶
PSRTVDTKQA ³¹⁶ QDLARSYGIP ³²⁶ FIETSAKTRQ ³³⁶ LRMK ³⁴⁰
SYGIPFIETS ³⁵⁰ AKTRQRVEDA ³⁶⁰ FYTLVREIRQ ³⁷⁰ LRMK ³⁷⁴
RVEDAFYTLV ³⁸⁴ REIRQYRLKK ³⁹⁴ ISKEEKTPGC ⁴⁰⁴ LRMK ⁴⁰⁸
YRLKKISKEE ⁴¹⁸ KTPGCVKIKK ⁴²⁸ CIIM ⁴³² LRMK ⁴³⁶
MTEYKLVVVG ⁴⁴⁶ AG D VGKSALT ⁴⁵⁶ IQLIQNHFVD ⁴⁶⁶ LRMK ⁴⁷⁰
MTEYKLVVVG ⁴⁸⁰ AGGVGKSALT ⁴⁹⁰ IQLIQNHFVD ⁵⁰⁰ Epitope name Amino acid sequence
(Sequence numbers are based on KRAS WT) Epitope 1 (E1, n=1) MTEYKLVVVG ¹⁰ AGGVGKSALT ²⁰ IQLIQNHFVD ³⁰ Epitope 2 (E2, n=2) KSALT ²⁰ IQLIQNHFVD ³⁰ EYDPTIEDSY ⁴⁰ RKQVV ⁴⁵ Epitope 3 (E3, n=3) EYDPTIEDSY ⁴⁰ RKQVVIDGET ⁵⁰ CLLDILDTAG ⁶⁰ Epitope 4 (E4, n=4) IDGET ⁵⁰ CLLDILDTAG ⁶⁰ QEEYSAMRDQ ⁷⁰ YMRTG ⁷⁵ Epitope 5 (E5, n=5) QEEYSAMRDQ ⁷⁰ YMRTGEGGFLC ⁸⁰ VFAINNTKSF ⁹⁰ Epitope 6 (E6, n=6) EGFLC ⁸⁰ VFAINNTKSF ⁹⁰ EDIHHYREQI ¹⁰⁰ KRVKD ¹⁰⁵ Epitope 7 (E7, n=7) EDIHHYREQI ¹⁰⁰ KRVKDSEDVP ¹¹⁰ MVLVGNKCDL ¹²⁰ Epitope 8 (E8, n=8) SEDVP ¹¹⁰ MVLVGNKCDL ¹²⁰ PSRTVDTKQA ¹³⁰ QDLAR ¹³⁵ Epitope 9 (E9, n=8) PSRTVDTKQA ¹³⁰ QDLARSYGIP ¹⁴⁰ FIETSAKTRQ ¹⁵⁰ Epitope 10 (E10, n=10) SYGIP ¹⁴⁰ FIETSAKTRQ ¹⁵⁰ RVEDAFYTLV ¹⁶⁰ REIRQ ¹⁶⁵ Epitope 11 (E11, n=11) RVEDAFYTLV ¹⁶⁰ REIRQYRLKK ¹⁷⁰ ISKEEKTPGC ¹⁸⁰ Epitope 12 (E12, n=12) YRLKK ¹⁷⁰ ISKEEKTPGC ¹⁸⁰ VKIKKCIIM ¹⁸⁹ Epitope 1-G12D (E1-G12D) MTEYKLVVVG ¹⁰ A D GVGKSALT ²⁰ IQLIQNHFVD ³⁰ Epitope 1-G12V (E1-G12V) MTEYKLVVVG ¹⁰ A V GVGKSALT ²⁰ IQLIQNHFVD ³⁰ Epitope 1-G13D (E1-G13D) MTEYKLVVVG ¹⁰ AG D VGKSALT ²⁰ IQLIQNHFVD ³⁰

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

3. KRAS(M)-ROP reactivity screening analysis

The reactivity of KRAS(M)-ROP was screened in peripheral blood mononuclear cells (PBMCs) from normal individuals. The screening was performed using an 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 leukapheresis of normal volunteers were seeded and cultured, and then treated with antigens. KRAS (M)-ROP 5 ㎍ / ㎖, 1.0 ㎍ / ㎖, and 0.1 ㎍ / ㎖ were used as the antigen, and anti-CD3 was used as a positive control. Cell culture was performed under the conditions of 37℃, CO ₂ 5%, 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 KRAS(M)-ROP-specific CD3+ T cell ratio according to the KRAS(M)-ROP concentration was analyzed for the above LP-1 PBMC. To this end, LP-1 PBMC was treated with antigen, and then IFN-γ capture staining was performed and analyzed.

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

First, LP-1 PBMC 1x10 ⁶ cells were seeded and cultured, and then treated with antigens at different concentrations (KRAS(M)-ROP 5 ㎍/㎖, KRAS(M)-ROP 1.0 ㎍/㎖, KRAS(M)-ROP 0.1 ㎍/㎖). In addition, LP-1 PBMC treated with 5 ㎍/㎖ and 1.0 ㎍/㎖ of tetanus toxoid vaccine (TTX) and anti-CD3 were used as positive controls. Cell culture was performed at 37℃, _CO2 5%, overnight (O/N).

IFN-γ capture staining was performed by treating LP-1 PBMCs treated with the ^1st capture antibody, incubating at 37℃ for 45 minutes, and then treating with ^2nd detection antibodies and CD3, CD4, CD8, and CD137. LP-1 PBMCs subjected to the IFN-γ capture staining were analyzed for their cell characteristics using a fluorescence activated cell sorter (FACS).

As a result of the experiment, it was confirmed that when LP-1 PBMCs were treated with antigens according to their concentration, the ratio of KRAS(M)-ROP-specific CD2+ T cells increased, which was well consistent with the above ELISpot (IFN-g) results. Therefore, it is judged that the reactivity of LP-1 PBMCs increases in a concentration-dependent manner to KRAS(M)-ROP. In addition, as a result of quantitatively evaluating the reactivity of LP-1 PBMCs to KRAS(M)-ROP, it was confirmed that the ratio of KRAS(M)-ROP-specific CD3+ T cells was about 2.4% when 5㎍/㎖ of KRAS(M)-ROP was treated.

5. Comparative analysis of antigen-specific CD3+ T cell ratios by antigen type

After treating LP-1 PBMCs with KRAS(M)-ROP(500aa), KRAS _1-24 Wild-type (Peptide Wt, 24aa), or KRAS _1-24 mutant (24aa), the proportion of antigen-specific CD3+ T cells was compared and analyzed.

Figure 4 shows the results of analyzing the antigen-specific CD3+ T-cell ratio of LP-1 PBMCs for 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 LP-1 PBMCs treated with KRAS(M)-ROP, KRAS _1-24 Wild-type, and KRAS _1-24 mutants. Panel B shows a graph of the percentage of IFN-γ secreting CD3+ T cells, and Panel C shows a table summarizing the graph of the percentage of antigen-specific CD3+ T cells. No Ag means that only the effector was used, and @CD3 means that anti-CD3 was used as a positive control.

The above KRAS _1-24 mutations refer to the 12th amino acid G being substituted with D or V in KRAS _1-24 Wild-type, or the 12th amino acid G being replaced with D (Pep.G12D, Pep.G12V, Pep.G13D). The antigen-specific CD3+ T-cell ratio was 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.

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 significantly low at 0.31% (Pep.G12D), 0.11% (Pep.G12V), and 0.25% (Pep.G13D). Considering that the ratio of CD3+ T cells induced in response to KRAS(M)-ROP was 2.41%, the above results imply that the induction of antigen-specific CD3+ T cells is minimal with only an epitope consisting of a peptide composed of 24 amino acids.

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

Based on the above experimental results, CD3+ T cells (ROP-T cells) that react with KRAS(M)-ROP were produced. In the present invention, ROP-T cells were produced 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 characteristic analysis process of ROP-T cells. Panel B shows the results of comparing the IFNg+ CD3+ T cell ratios of T cells amplified under Fast-IVS process conditions and No-Cytokine process conditions.

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

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).

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 Day 5, 7, 9, 11, 12 Media Adding Media Adding Day 13 Harvest Harvest

The analysis results confirmed that the proportion of antigen-specific CD3+ T cells secreting IFN-γ was amplified approximately four times more in the Fast-IVS process than in the No-Cytokine process.

7. Optimization of Fast-IVS process for ROP-T cell production

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 optimization of the Fast-IVS process.

Example 1 Example 2 Example 3 Experimental conditions Scale PBMCs 10M@24well 10M@24well 10M@24well Cytokine manufacturing company JW Creagene JW Creagene JW Creagene KRAS(M)-ROP ㎍/㎖ 5.0 1.0 1.0 Fast-IVS (Badge AIM-V) period Days 5 5 7 Expansion period Days 10 10 10 Experimental Results Expansion Fold No Ag-T 45 34 55 ROP-T 55 49 65 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

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

8. Analysis of ROP-T cell characteristics

The characteristics of amplified ROP-T cells were analyzed by performing KRAS mutant epitope screening. To this end, autologous dendritic cells (ADCs) were induced from PBMCs, and antigen-sensitized ADCs were prepared to stimulate antigen-specific T cells, thereby confirming the reactivity of ROP-T cells. The reactivity was analyzed by calculating the re-stimulation IFN-γ secretion T cell 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 results of analyzing the cell ratio (%) of the conditionally restimulated IFN-γ secreting T cell ratio.

First, autologous DCs were cultured for 4 days and sensitized to antigen to prepare Ag pulsed DCs. The Ag pulsed DCs were dispensed into 96-well at ^5x103 cells/100㎕. KRAS(M)-ROP-specific CD3+ T cells were dispensed into the 96-well at ^1x105 cells/100㎕, with a cell ratio of 1:20 between the Ag pulsed DCs and KRAS(M)-ROP-specific CD3+ T cells. The medium containing Ag pulsed DCs and KRAS(M)-ROP-specific CD3+ T cells was cultured for 4 hours. The ratios (%) of CD3+, CD4+, CD137+, IFN-γ cap, and IFN-γ secreting T cells of the cultured cells were analyzed using FACS. The antigens used in the production of the above 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). In addition, DC using only the effector without the antigen (Ag) (NoAg_DC) was used for comparison.

The experimental results showed that when restimulated using ROP_DC, the ratios (%) of KRAS(M)-ROP-specific CD3+/CD4+ T cells and KRAS(M)-ROP-specific CD3+/CD8+ T cells in CD3+ ROP-T cells were 10% and 5%, respectively. When restimulated using G12D_DC, the ratios (%) 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% and 0.5%, respectively. 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 present in CD3+ ROP-T cells were confirmed to be 3.0% and 1.5%, respectively. In contrast, when restimulated with Wt_DC and G12V_DC, KRAS(M)-ROP-specific CD3+/CD4+ T cells and KRAS(M)-ROP-specific CD3+/CD8+ T cells were barely detected.

9. HLA restriction analysis of ROP-T cells

To confirm HLA restriction of KRAS G13D mutant-specific T cells among induced ROP-T, human leukocyte antigen DQ (HLA-DQ) assay was performed.

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

First, antigen-sensitized DC (Ag pulsed DC) was prepared. The antigens used in the preparation of the 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 on the prepared Ag pulsed DC by treating it with HLA-DQ antibody for 1 hour. After re-stimulation of ROP-T using the HLA-DQ blocked Ag pulsed DC, IFN-g+, CD3+, and CD4+ were analyzed through FACS.

The results of the analysis showed that the proportion of CD3+CD4+ T cells secreting IFN-γ was minimal for antigen-nonspecific T cells (NoAg-T), regardless of the type of DC used for restimulation and the presence or absence of HLA-DQ blocking on the DC. In contrast, when ROP-T cells were restimulated using ROP_DC, the proportion of CD3+CD4+ T cells secreting IFN-γ increased to more than 15%, regardless of the presence or absence of HLA-DQ blocking on the DC. In addition, when ROP-T cells were restimulated using HLA-DQ blocked G13D_DC, the proportion of CD3+CD4+ T cells secreting IFN-γ was confirmed to decrease by approximately 6% (7% → 1%) compared to when restimulated using HLA-DQ unblocked G13D_DC.

As a result, it is judged that the ROP-T cells of the present invention induce the amplification of CD4+ T cells that are specific for ROP antigens, restricted to HLA-DQ, and specific for the G13D mutation.

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

We verified that KRAS(M)-ROP induces a specific response to KRAS mutations.

Figure 8 shows the ratio of CD3+ T cells secreting IFN-γ (IFN-γ+) according to the conditions 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 an executor without an antigen. The induced T cells were restimulated using ROP_DC, and the ratio of IFN-γ+ CD3+ T cells was analyzed using FACS.

Table 5 below shows the specific response induction verification experimental method for KRAS mutations of KRAS(M)-ROP. In Table 5 below, KRAS epitope wild type means Native KRAS _1-24 (24aa); KRAS epitope G12D means KRAS _1-24 G12D(24aa); KRAS epitope G12V means KRAS _1-24 G12V(24aa); KRAS epitope G13D means 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=5㎍/㎖) Pep.-T KRAS epitope (24mer) 4 types mixture (wild type, G12D, G12V, G13D) (8.5 μM) WT-T Native KRAS (8.5 μM = 2 μg/ml)

The experimental results showed that the proportion of IFN-γ+ CD3+ T cells was approximately 8% in the case of T cells induced through Fast-IVS without using an antigen (No Ag-T). The proportion of IFN-γ+ CD3+ T cells was approximately 37.4% in the case of T cells induced through the Fast-IVS process using KRAS(M)-ROP as an antigen (ROP-T). The proportion of IFN-γ+ CD3+ T cells was approximately 18.4% in the case of T cells induced through the Fast-IVS process using Native KRAS as an antigen (WT-T). 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, the ratio of IFN-γ+ CD3+ T cells was confirmed to be 19.0%.

In summary, it was determined that the KRAS(M)-ROP antigen had a IFN-γ+ CD3+ T cell induction effect that was approximately twice as superior to that using epitopes containing native KRAS or KRAS mutations.

The specific embodiments 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 modifications and other uses of the present invention do not depart from the scope of the invention described in the claims of this specification.

<110> MYONGJI HOSPITAL, MYONGJI MEDICAL FOUNDATION <120> Antigen Composition For Inducing KRAS Specific Activated T Cell <130> MP21-0082 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 500 <212> PRT <213> Artificial Sequence <220> <223> KRAS antigen for KRAS Specific Activated Cell <400> 1 Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Asp Gly Val Gly Lys 1 5 10 15 Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe Val Asp Leu Arg 20 25 30 Met Lys Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Val Gly Val 35 40 45 Gly Lys Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe Val Asp 50 55 60 Leu Arg Met Lys Lys Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His 65 70 75 80 Phe Val Asp Glu Tyr Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln 85 90 95 Val Val Leu Arg Met Lys Glu Tyr Asp Pro Thr Ile Glu Asp Ser Tyr 100 105 110 Arg Lys Gln Val Val Ile Asp Gly Glu Thr Cys Leu Leu Asp Ile Leu 115 120 125 Asp Thr Ala Gly Leu Arg Met Lys Ile Asp Gly Glu Thr Cys Leu Leu 130 135 140 Asp Ile Leu Asp Thr Ala Gly Gln Glu Glu Tyr Ser Ala Met Arg Asp 145 150 155 160 Gln Tyr Met Arg Thr Gly Leu Arg Met Lys Gln Glu Glu Tyr Ser Ala 165 170 175 Met Arg Asp Gln Tyr Met Arg Thr Gly Glu Gly Phe Leu Cys Val Phe 180 185 190 Ala Ile Asn Asn Thr Lys Ser Phe Leu Arg Met Lys Glu Gly Phe Leu 195 200 205 Cys Val Phe Ala Ile Asn Asn Thr Lys Ser Phe Glu Asp Ile His His 210 215 220 Tyr Arg Glu Gln Ile Lys Arg Val Lys Asp Leu Arg Met Lys Glu Asp 225 230 235 240 Ile His His Tyr Arg Glu Gln Ile Lys Arg Val Lys Asp Ser Glu Asp 245 250 255 Val Pro Met Val Leu Val Gly Asn Lys Cys Asp Leu Leu Arg Met Lys 260 265 270 Ser Glu Asp Val Pro Met Val Leu Val Gly Asn Lys Cys Asp Leu Pro 275 280 285 Ser Arg Thr Val Asp Thr Lys Gln Ala Gln Asp Leu Ala Arg Leu Arg 290 295 300 Met Lys Pro Ser Arg Thr Val Asp Thr Lys Gln Ala Gln Asp Leu Ala 305 310 315 320 Arg Ser Tyr Gly Ile Pro Phe Ile Glu Thr Ser Ala Lys Thr Arg Gln 325 330 335 Leu Arg Met Lys Ser Tyr Gly Ile Pro Phe Ile Glu Thr Ser Ala Lys 340 345 350 Thr Arg Gln Arg Val Glu Asp Ala Phe Tyr Thr Leu Val Arg Glu Ile 355 360 365 Arg Gln Leu Arg Met Lys Arg Val Glu Asp Ala Phe Tyr Thr Leu Val 370 375 380 Arg Glu Ile Arg Gln Tyr Arg Leu Lys Lys Ile Ser Lys Glu Glu Lys 385 390 395 400 Thr Pro Gly Cys Leu Arg Met Lys Tyr Arg Leu Lys Lys Ile Ser Lys 405 410 415 Glu Glu Lys Thr Pro Gly Cys Val Lys Ile Lys Lys Cys Ile Ile Met 420 425 430 Leu Arg Met Lys Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Gly 435 440 445 Asp Val Gly Lys Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe 450 455 460 Val Asp Leu Arg Met Lys Met Thr Glu Tyr Lys Leu Val Val Val Gly 465 470 475 480 Ala Gly Gly Val Gly Lys Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn 485 490 495 His Phe Val Asp 500 <210> 2 <211> 189 <212> PRT <213> Homo sapiens <400> 2 Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Gly Gly Val Gly Lys 1 5 10 15 Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe Val Asp Glu Tyr 20 25 30 Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile Asp Gly 35 40 45 Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Gln Glu Glu Tyr 50 55 60 Ser Ala Met Arg Asp Gln Tyr Met Arg Thr Gly Glu Gly Phe Leu Cys 65 70 75 80 Val Phe Ala Ile Asn Asn Thr Lys Ser Phe Glu Asp Ile His His Tyr 85 90 95 Arg Glu Gln Ile Lys Arg Val Lys Asp Ser Glu Asp Val Pro Met Val 100 105 110 Leu Val Gly Asn Lys Cys Asp Leu Pro Ser Arg Thr Val Asp Thr Lys 115 120 125 Gln Ala Gln Asp Leu Ala Arg Ser Tyr Gly Ile Pro Phe Ile Glu Thr 130 135 140 Ser Ala Lys Thr Arg Gln Arg Val Glu Asp Ala Phe Tyr Thr Leu Val 145 150 155 160 Arg Glu Ile Arg Gln Tyr Arg Leu Lys Lys Ile Ser Lys Glu Glu Lys 165 170 175 Thr Pro Gly Cys Val Lys Ile Lys Lys Cys Ile Ile Met 180 185

Claims (5)

An antigen composition for inducing KRAS-specific activated T cells, comprising a KRAS mutant recombinant overlapping peptide consisting of an amino acid sequence of sequence number 1 as an active ingredient.
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