Cancer vaccine using common cancer antigen cocktail, TCR / CAR-T cell therapeutic agent, companion diagnosis method, and cancer onset risk diagnosis method based on detection of circulating cancer cells in blood

Abstract

The present invention addresses the problem of providing a cancer vaccine using a common cancer antigen cocktail, a TCR / CAR-T cell therapeutic agent, a companion diagnosis method, and a cancer onset risk diagnosis method based on the detection of circulating cancer cells in blood. According to the present invention, provided is a cancer vaccine comprising: (1) a general-purpose cancer antigen comprising three or more species selected from the group consisting of GPC3, ROBO1, EPHB4, CLDN1, and LAT1; (2) a partial peptide of the three or more universal cancer antigens having CTL inducibility; (3) dendritic cells stimulated with the partial peptides; or (4) mRNA encoding the universal cancer antigen or the partial peptide.

Description

Technical Field

[0001] The present invention relates to a cancer vaccine using a universal cancer antigen cocktail. The present invention also relates to a TCR / CAR-T cell therapy drug, a companion diagnostic method, and a cancer risk diagnosis method based on the detection of circulating cancer cells in the blood. Background Art

[0002] Even if they receive radical resection surgery, many patients diagnosed with cancer cannot survive for more than 10 years. One million people in Japan are diagnosed with cancer and 400,000 die each year. If cancer vaccines, TCR / CAR-T cell therapy drugs, companion diagnostic methods, and cancer risk diagnosis methods based on the detection of circulating cancer cells in the blood are developed for cancer, it is believed that most of the one million patients each year will be eligible. In addition, if it can be used in the future as a cancer risk diagnosis during blood sampling or a cancer prevention vaccine for those at risk, all citizens can be eligible.

[0003] However, the development of vaccine therapy for cancer has failed so far. TCR / CAR-T cell therapy is also a treatment for solid tumors, but a promising method has not yet emerged. In addition, the technology for capturing CTCs is not yet complete, and there is no technology that can reliably prove that cells considered to be CTCs are cancer cells and diagnose the risk of cancer recurrence / onset. Vaccines or TCR-T cell therapy using neoantigens that have attracted attention are also personalized treatments, and the development cost and regulatory difficulties are high. Summary of the invention

[0004] Problems to be solved by the invention

[0005] In order to develop cancer vaccines suitable for most people or introduce TCR genes into T cell therapy, antigens expressed in target cancer cells are very important. As antigens, it is difficult to use individual corresponding neoantigens, and so-called universal cancer antigens that are frequently expressed in cancer and not expressed in most normal organs are very important. In addition, for CAR-T cell therapy or antibody treatment, or to capture circulating cancer cells in the blood, universal cancer antigens expressed on the cell membrane are particularly needed. Most of the famous cancer antigens such as cancer testis antigens reported in the past are not expressed on the cell membrane, and there is a problem of low expression frequency or expression in normal tissues.

[0006] The present invention aims to provide a cancer vaccine, a TCR / CAR-T cell therapy drug, a companion diagnostic method, and a cancer risk diagnosis method based on the detection of circulating cancer cells in the blood, which utilize a universal cancer antigen cocktail.

[0007] Means for solving problems

[0008] In order to solve the above problems, the present inventors conducted intensive research and identified 10 universal cancer antigens that are frequently expressed in various solid tumors and are almost not expressed in normal tissues through immunohistochemical analysis. Five of them are membrane protein antigens. Among the solid tumors studied so far, such as head and neck cancer, lung cancer, liver cancer, biliary tract cancer, pancreatic cancer, and colorectal cancer, at least one of these five antigens is expressed in most cases of cancer, proving that these universal cancer antigen cocktails can cover all solid tumors. The present invention was completed based on the above findings.

[0009] That is, according to the present invention, the following inventions are provided.

[0010] <1> A cancer vaccine comprising:

[0011] (1) containing three or more universal cancer antigens selected from the group consisting of GPC3, ROBO1, EPHB4, CLDN1, and LAT1 (SLC7A5);

[0012] (2) partial peptides of the above three or more universal cancer antigens, which have CTL inducibility;

[0013] (3) dendritic cells stimulated with the above partial peptides; or

[0014] (4) mRNA encoding the above-mentioned universal cancer antigen or the above-mentioned partial peptide.

[0015] <2> <1> The cancer vaccine described above, wherein the universal cancer antigen further comprises one or more of AFP, TGFBI, SPARC, HSP105α (HSPH1) and FOXM1.

[0016] <3> <1> The cancer vaccine, wherein the universal cancer antigens include all of GPC3, ROBO1, EPHB4, CLDN1 and LAT1.

[0017] <4> <1> The cancer vaccine, wherein the universal cancer antigens include all of GPC3, ROBO1, EPHB4, CLDN1, LAT1, AFP, TGFBI, SPARC, HSP105α and FOXM1.

[0018] <5> <1> ~ <4> The cancer vaccine according to any one of the preceding claims, wherein the partial peptide is a peptide having an amino acid sequence as described in any one of SEQ ID NOs: 1 to 80.

[0019] <6> A cancer vaccine comprising:

[0020] (1) comprising three or more universal cancer antigens selected from the group consisting of GPC3, ROBO1, EPHB4, CLDN1, LAT1, AFP, TGFBI, SPARC, HSP105α, and FOXM1;

[0021] (2) partial peptides of the above three or more universal cancer antigens, which have CTL inducibility;

[0022] (3) dendritic cells stimulated with the above partial peptides; or

[0023] (4) mRNA encoding the above-mentioned universal cancer antigen or the above-mentioned partial peptide.

[0024] <7> A peptide having an amino acid sequence as described in any one of SEQ ID NOs: 5, 7 to 10, 14 to 22, 24 to 38, 40, 42, 48, 49, 52 to 80.

[0025] <8> A CAR-T cell therapy comprising a mixture of T cells having a chimeric antigen receptor (CAR) against a universal cancer antigen, wherein the universal cancer antigen comprises three or more selected from the group consisting of GPC3, ROBO1, EPHB4, CLDN1, and LAT1.

[0026] <9> <8> The CAR-T cell therapeutic agent, wherein the universal cancer antigen further comprises one or more of AFP, TGFBI, SPARC, HSP105α and FOXM1.

[0027] <10> <8> The CAR-T cell therapeutic agent, wherein the universal cancer antigen includes all of GPC3, ROBO1, EPHB4, CLDN1 and LAT1.

[0028] <11> <8> The CAR-T cell therapeutic agent, wherein the universal cancer antigens include all of GPC3, ROBO1, EPHB4, CLDN1, LAT1, AFP, TGFBI, SPARC, HSP105α and FOXM1.

[0029] <12> A CAR-T cell therapy comprising a mixture of T cells having a chimeric antigen receptor (CAR) against a universal cancer antigen, wherein the universal cancer antigen comprises three or more selected from GPC3, ROBO1, EPHB4, CLDN1, LAT1, AFP, TGFBI, SPARC, HSP105α and FOXM1.

[0030] <13> A TCR-T cell therapeutic agent comprises a mixture of T cells having a T cell receptor (TCR) capable of recognizing MHC class I binding antigen peptides derived from a universal cancer antigen, wherein the universal cancer antigen comprises three or more of GPC3, ROBO1, EPHB4, CLDN1 and LAT1.

[0031] <14> <13> The TCR-T cell therapeutic agent, wherein the universal cancer antigen further comprises one or more of AFP, TGFBI, SPARC, HSP105α and FOXM1.

[0032] <15> <13> The TCR-T cell therapeutic agent, wherein the universal cancer antigen includes all of GPC3, ROBO1, EPHB4, CLDN1 and LAT1.

[0033] <16> <13> The TCR-T cell therapeutic agent, wherein the universal cancer antigens include all of GPC3, ROBO1, EPHB4, CLDN1, LAT1, AFP, TGFBI, SPARC, HSP105α and FOXM1.

[0034] <17> A TCR-T cell therapeutic agent comprising a mixture of T cells having a T cell receptor (TCR) that can recognize MHC class I binding antigen peptides from a universal cancer antigen, wherein the universal cancer antigen comprises three or more of GPC3, ROBO1, EPHB4, CLDN1, LAT1, AFP, TGFBI, SPARC, HSP105α and FOXM1.

[0035] <18> <13> ~ <17> The TCR-T cell therapeutic agent according to any one of the preceding claims, wherein the T cell receptor (TCR) is any one of the following heterodimers:

[0036] A heterodimer composed of a combination of the protein of SEQ ID NO: 103 and the protein of SEQ ID NO: 104;

[0037] A heterodimer composed of a combination of the protein of SEQ ID NO: 105 and the protein of SEQ ID NO: 106;

[0038] A heterodimer composed of a combination of the protein of SEQ ID NO: 107 and the protein of SEQ ID NO: 108;

[0039] a heterodimer composed of a combination of the protein of SEQ ID NO: 109 and the protein of SEQ ID NO: 110;

[0040] A heterodimer composed of a combination of the protein of SEQ ID NO: 111 and the protein of SEQ ID NO: 112;

[0041] A heterodimer composed of a combination of the protein of SEQ ID NO: 123 and the protein of SEQ ID NO: 124;

[0042] A heterodimer composed of a combination of the protein of SEQ ID NO: 125 and the protein of SEQ ID NO: 126;

[0043] a heterodimer composed of a combination of the protein of SEQ ID NO: 127 and the protein of SEQ ID NO: 128;

[0044] A heterodimer composed of a combination of the protein of SEQ ID NO: 129 or 130 and the protein of SEQ ID NO: 131;

[0045] A heterodimer composed of a combination of the protein of SEQ ID NO: 132 and the protein of SEQ ID NO: 133;

[0046] a heterodimer composed of a combination of the protein of SEQ ID NO: 134 and the protein of SEQ ID NO: 135;

[0047] a heterodimer composed of a combination of the protein of SEQ ID NO: 136 and the protein of SEQ ID NO: 137;

[0048] A heterodimer composed of a combination of the protein of SEQ ID NO: 138 or 139 and the protein of SEQ ID NO: 140;

[0049] A heterodimer composed of a combination of the protein of SEQ ID NO: 141 and the protein of SEQ ID NO: 142;

[0050] A heterodimer composed of a combination of the protein of SEQ ID NO: 143 and the protein of SEQ ID NO: 144;

[0051] a heterodimer composed of a combination of the protein of SEQ ID NO: 145 and the protein of SEQ ID NO: 146;

[0052] a heterodimer composed of a combination of the protein of SEQ ID NO: 147 and the protein of SEQ ID NO: 148;

[0053] A heterodimer composed of a combination of the protein of SEQ ID NO: 177 and the protein of SEQ ID NO: 178;

[0054] a heterodimer composed of a combination of the protein of SEQ ID NO: 179 and the protein of SEQ ID NO: 180;

[0055] A heterodimer composed of a combination of the protein of SEQ ID NO: 181 and the protein of SEQ ID NO: 182;

[0056] a heterodimer composed of a combination of the protein of SEQ ID NO: 183 and the protein of SEQ ID NO: 184;

[0057] A heterodimer composed of a combination of the protein of SEQ ID NO: 193 and the protein of SEQ ID NO: 194;

[0058] A heterodimer composed of a combination of the protein of SEQ ID NO: 195 and the protein of SEQ ID NO: 196;

[0059] A heterodimer composed of a combination of the protein of SEQ ID NO: 197 and the protein of SEQ ID NO: 198;

[0060] A heterodimer composed of a combination of the protein of SEQ ID NO: 199 or 200 and the protein of SEQ ID NO: 201;

[0061] A heterodimer composed of a combination of the protein of SEQ ID NO: 202 and the protein of SEQ ID NO: 203;

[0062] A heterodimer composed of a combination of the protein of SEQ ID NO: 216 and the protein of SEQ ID NO: 217; or

[0063] A heterodimer composed of a combination of the protein of SEQ ID NO: 218 and the protein of SEQ ID NO: 219.

[0064] <19> A protein having an amino acid sequence as recorded in any one of SEQ ID NOs: 103-112, 123-148, 177-184, 193-203, 215-219.

[0065] <20> T cell receptor (TCR), which is a heterodimer of any of the following:

[0066] A heterodimer composed of a combination of the protein of SEQ ID NO: 103 and the protein of SEQ ID NO: 104;

[0067] A heterodimer composed of a combination of the protein of SEQ ID NO: 105 and the protein of SEQ ID NO: 106;

[0068] A heterodimer composed of a combination of the protein of SEQ ID NO: 107 and the protein of SEQ ID NO: 108;

[0069] a heterodimer composed of a combination of the protein of SEQ ID NO: 109 and the protein of SEQ ID NO: 110;

[0070] A heterodimer composed of a combination of the protein of SEQ ID NO: 111 and the protein of SEQ ID NO: 112;

[0071] A heterodimer composed of a combination of the protein of SEQ ID NO: 123 and the protein of SEQ ID NO: 124;

[0072] A heterodimer composed of a combination of the protein of SEQ ID NO: 125 and the protein of SEQ ID NO: 126;

[0073] a heterodimer composed of a combination of the protein of SEQ ID NO: 127 and the protein of SEQ D NO: 128;

[0074] A heterodimer composed of a combination of the protein of SEQ ID NO: 129 or 130 and the protein of SEQ ID NO: 131;

[0075] A heterodimer composed of a combination of the protein of SEQ ID NO: 132 and the protein of SEQ ID NO: 133;

[0076] a heterodimer composed of a combination of the protein of SEQ ID NO: 134 and the protein of SEQ ID NO: 135;

[0077] a heterodimer composed of a combination of the protein of SEQ ID NO: 136 and the protein of SEQ ID NO: 137;

[0078] A heterodimer composed of a combination of the protein of SEQ ID NO: 138 or 139 and the protein of SEQ ID NO: 140;

[0079] A heterodimer composed of a combination of the protein of SEQ ID NO: 141 and the protein of SEQ ID NO: 142;

[0080] A heterodimer composed of a combination of the protein of SEQ ID NO: 143 and the protein of SEQ ID NO: 144;

[0081] a heterodimer composed of a combination of the protein of SEQ ID NO: 145 and the protein of SEQ ID NO: 146;

[0082] a heterodimer composed of a combination of the protein of SEQ ID NO: 147 and the protein of SEQ ID NO: 148;

[0083] a heterodimer composed of a combination of the protein of SEQ ID NO: 177 and the protein of SEQ ID NO: 178;

[0084] a heterodimer composed of a combination of the protein of SEQ ID NO: 179 and the protein of SEQ ID NO: 180;

[0085] A heterodimer composed of a combination of the protein of SEQ ID NO: 181 and the protein of SEQ ID NO: 182;

[0086] a heterodimer composed of a combination of the protein of SEQ ID NO: 183 and the protein of SEQ ID NO: 184;

[0087] A heterodimer composed of a combination of the protein of SEQ ID NO: 193 and the protein of SEQ ID NO: 194;

[0088] A heterodimer composed of a combination of the protein of SEQ ID NO: 195 and the protein of SEQ ID NO: 196;

[0089] A heterodimer composed of a combination of the protein of SEQ ID NO: 197 and the protein of SEQ ID NO: 198;

[0090] A heterodimer composed of a combination of the protein of SEQ ID NO: 199 or 200 and the protein of SEQ ID NO: 201;

[0091] A heterodimer composed of a combination of the protein of SEQ ID NO: 202 and the protein of SEQ ID NO: 203;

[0092] A heterodimer composed of a combination of the protein of SEQ ID NO: 216 and the protein of SEQ ID NO: 217; or

[0093] A heterodimer composed of a combination of the protein of SEQ ID NO: 218 and the protein of SEQ ID NO: 219.

[0094] <21> A gene having a nucleotide sequence of any one of SEQ ID NOs: 113-122, 149-176, 185-192, 204-214, 220-224.

[0095] <22> A companion diagnostic method, comprising the following steps: step 1, simultaneously determining the presence or absence of expression of three or more of GPC3, ROBO1, EPHB4, CLDN1 and LAT1 and the cell membrane expression of HLA class I in a sample from a subject by multiple fluorescent immunostaining; and step 2, determining the applicability of cancer immunotherapy based on the presence or absence of the above-mentioned expression.

[0096] <23> <22> The companion diagnostic method, wherein in step 1, further determines whether the expression of one or more of AFP, TGFBI, SPARC, HSP105α and FOXM1 exists.

[0097] <24> <22> The companion diagnostic method, wherein in step 1, the presence or absence of all expressions of GPC3, ROBO1, EPHB4, CLDN1 and LAT1 is determined.

[0098] <25> <22> The companion diagnostic method, wherein in step 1, the presence or absence of all expressions of GPC3, ROBO1, EPHB4, CLDN1, LAT1, AFP, TGFBI, SPARC, HSP105α and FOXM1 is determined.

[0099] <26> A method for diagnosing cancer risk, comprising: analyzing the expression of three or more of GPC3, ROBO1, EPHB4, CLDN1, and LAT1 in cells in a blood sample from a subject.

[0100] <27> <26> The method for diagnosing cancer risk further comprises: analyzing the expression of one or more of AFP, TGFBI, SPARC, HSP105α and FOXM1.

[0101] <28> <26> The method for diagnosing cancer risk comprises analyzing the overall expression of GPC3, ROBO1, EPHB4, CLDN1 and LAT1.

[0102] <29> <26> The method for diagnosing cancer risk comprises analyzing the expressions of all of GPC3, ROBO1, EPHB4, CLDN1, LAT1, AFP, TGFBI, SPARC, HSP105α and FOXM1.

[0103] Effects of the Invention

[0104] According to the present invention, a cancer vaccine, a TCR / CAR-T cell therapy drug, a companion diagnostic method, and a cancer risk diagnosis method based on the detection of circulating cancer cells in the blood are provided, which utilize a universal cancer antigen cocktail. BRIEF DESCRIPTION OF THE DRAWINGS

[0105] [ Figure 1 ] Figure 1 The results of immunohistochemical staining using paraffin-embedded tissue sections in order to confirm the expression of 10 universal cancer antigens in hepatocellular carcinoma and the like are shown.

[0106] [ Figure 2 ] Figure 2 The expression of cell membrane-expressed universal cancer antigens GPC3, EPHB4, CLDN1, LAT1, and ROBO1 in each cancer type is shown.

[0107] [ Figure 3 ] Figure 3 The expression of universal cancer antigens and HLA class I in childhood cancers such as neuroblastoma, Wilms tumor, and choriocarcinoma are shown.

[0108] [ Figure 4 ] Figure 4 The results of confirming the expression of each universal cancer antigen in the non-cancerous part of human clinical specimens of hepatocellular carcinoma are shown.

[0109] [ Figure 5 ] Figure 5 The table shows the expression of cell membrane-expressed universal cancer antigens GPC3, EPHB4, CLDN1, LAT1, and ROBO1 in 31 normal tissues using human normal tissue microarray.

[0110] [ Figure 6 ] Figure 6 Demonstrates the concept of developing an applicable diagnostic system, using multiplexed fluorescent immunostaining to determine which universal cancer antigens are effective when used as targets in immunotherapy such as TCR-T, CAR-T, and cancer vaccines.

[0111] [ Figure 7 ] Figure 7 In the data, PDXs were specimens from patients with hepatocellular carcinoma and lung cancer, and human clinical samples were specimens from patients with hepatocellular carcinoma, intrahepatic cholangiocarcinoma, and oropharyngeal cancer (HPV-). The results of multiplex fluorescent immunostaining using cell membrane-expressed universal cancer antigens such as GPC3, EPHB4, CLDN1, LAT1, and ROBO1, and HLA-I were shown.

[0112] [ Figure 8 ] Figure 8 The gene expression of each cancer antigen in hepatocellular carcinoma and primary colorectal cancer is shown.

[0113] [ Fig. 9 ] Fig. 9 Gene expression of each cancer antigen in hepatocellular carcinoma and colorectal cancer liver metastasis for which antigen prediction was performed is shown.

[0114] [ Fig.10 ] Fig.10 The predicted pipeline of epitope peptides from cancer antigens is shown.

[0115] [ Fig.11 ] Fig.11 The distribution of SCOREadj of peptides derived from each cancer antigen in hepatocellular carcinoma is shown.

[0116] [ Fig.12 ] Fig.12 The total number of cancer antigen peptides predicted in 7 cases of hepatocellular carcinoma is shown.

[0117] [ Fig.13 ] Fig.13 The present invention shows a method for identifying epitope peptides from universal cancer antigens that can induce antigen-specific T cells. The present invention shows the results of predicting 72 HLA-A*24:02-restricted epitope peptides and 73 HLA-A*02:01-restricted epitope peptides from the amino acid sequences of 10 universal cancer antigens using an algorithm established in joint research with Brightpath Bio, and then vaccinating transgenic mice with the synthesized peptides and evaluating the production of antigen-specific IFN-γ by the ELISpot method.

[0118] [ Fig.14 ] Fig.14 The results show that peptides predicted to be presented on HLA-A*02:01 were synthesized from the full-length amino acid sequences of 10 common cancer antigen proteins, and the peptide vaccine was administered once a week for a total of 3 times to mice expressing human HLA-A*02:01 in systemic cells. Multiple peptides that can induce CD8-positive killer T cells (CTLs) that react with the peptides in spleen cells were identified through screening.

[0119] [ Fig.15 ] Fig.15 The results show that peptides predicted to be presented on HLA-A*24:02 were synthesized from the full-length amino acid sequences of 10 common cancer antigen proteins, and the peptide vaccine was given once a week for a total of 3 times to mice expressing human HLA-A*24:02 in systemic cells. Multiple peptides that can induce CD8-positive killer T cells (CTLs) that react with the peptides in spleen cells were identified through screening.

[0120] [ Fig.16 ] Fig.16A method for evaluating whether antigen-specific T cells are induced by vaccinating transgenic mice with dendritic cells (DCs) sensitized with short peptides derived from universal cancer antigens confirmed to induce CTLs using transgenic mice introduced with each HLA gene is shown.

[0121] [ Fig.17 ] Fig.17 The results show that HLA-A*24:02 transgenic mice were vaccinated with dendritic cells (DCs) sensitized with short peptides derived from universal cancer antigens that were confirmed to induce CTLs, and whether antigen-specific T cells were induced was evaluated using transgenic mice into which each HLA gene was introduced.

[0122] [ Fig.18 ] Fig.18 The results show that HLA-A*02:01 transgenic mice were vaccinated with dendritic cells (DCs) sensitized with short peptides derived from universal cancer antigens that were confirmed to induce CTLs, and whether antigen-specific T cells were induced was evaluated using transgenic mice into which each HLA gene was introduced.

[0123] [ Fig.19 ] Fig.19 The results of verification using HLA-A*02:01 Tgm or HLA-A*24:02 Tgm are shown in order to confirm whether the peptide is actually processed in cells and presented on HLA using a long peptide containing a short peptide sequence derived from a universal cancer antigen confirmed to induce CTL.

[0124] [ Fig. 20 ] Fig. 20 The dosing regimen is shown for 3 administrations of LNP-Covid-19 Spike mRNA.

[0125] [ Fig.21 ] Fig.21 Shown are the results of Spike antigen-specific IFN-γ production in spleen cells of HLA-A*02:01 Tg mice given LNP-Covid-19 Spike mRNA three times.

[0126] [ Fig. 22 ] Fig. 22 The results of Spike antigen-specific IFN-γ production in spleen cells of HLA-A*02:01 Tg mice to which LNP-eGFP mRNA was administered three times are shown.

[0127] [ Fig.23 ] Fig.23The results of Spike antigen-specific IFN-γ production in inguinal lymph node cells of HLA-A*02:01 Tg mice given LNP-Covid-19 Spike mRNA or LNP-eGFP mRNA three times are shown.

[0128] [ Fig.24 ] Fig.24 Shown are the results of Spike antigen-specific IFN-γ production in spleen cells of HLA-A*24:02 Tg mice given LNP-Covid-19 Spike mRNA three times.

[0129] [ Fig.25 ] Fig.25 The results of Spike antigen-specific IFN-γ production in spleen cells of HLA-A*24:02 Tg mice to which LNP-eGFP mRNA was administered three times are shown.

[0130] [ Fig.26 ] Fig.26 The results of Spike antigen-specific IFN-γ production in inguinal lymph node cells of HLA-A*24:02 Tg mice given LNP-Covid-19 Spike mRNA or LNP-eGFP mRNA three times are shown.

[0131] [ Fig. 27 ] Fig. 27 The dosing regimen is shown with 2 administrations of LNP-Covid-19 Spike mRNA.

[0132] [ Fig.28 ] Fig.28 The results of Spike antigen-specific IFN-γ production in spleen cells of HLA-A*02:01 Tg mice given LNP-Covid-19 Spike mRNA twice are shown.

[0133] [ Fig.29 ] Fig.29 The results are shown for the production of Spike antigen-specific IFN-γ in spleen cells of HLA-A*02:01 Tg mice to which LNP-eGFP mRNA was administered twice.

[0134] [ Fig.30 ] Fig.30 The results show the production of Spike antigen-specific IFN-γ in inguinal lymph node cells of HLA-A*02:01 Tg mice given LNP-Covid-19 Spike mRNA or LNP-eGFP mRNA twice.

[0135] [ Fig.31] Fig.31 The results of Spike antigen-specific IFN-γ production in spleen cells of HLA-A*24:02 Tg mice given LNP-Covid-19 Spike mRNA twice are shown.

[0136] [ Fig.32 ] Fig.32 The results are shown for the production of spike antigen-specific FN-γ in spleen cells of HLA-A*24:02 Tg mice to which LNP-eGFP mRNA was administered twice.

[0137] [ Fig.33 ] Fig.33 The results show the production of Spike antigen-specific IFN-γ in inguinal lymph node cells of HLA-A*24:02 Tg mice given LNP-Covid-19 Spike mRNA or LNP-eGFP mRNA twice.

[0138] [ Fig.34 ] Fig.34 Shown are the results of an antibody assay against the Spike protein.

[0139] [ Fig.35 ] Fig.35 The administration method of LNP-hGPC3 mRNA is shown in which LNP-hGPC3 mRNA is administered three times.

[0140] [ Fig.36 ] Fig.36 The results show that LNP-hGPC3 mRNA was administered to HLA-A*02:01 Tg mice, and the production of antigen-specific IFN-γ in spleen cells was evaluated by the ELISPOT method.

[0141] [ Fig.37 ] Fig.37 The results show that LNP-luciferase mRNA was administered to HLA-A*02:01 Tg mice, and the production of antigen-specific IFN-γ in spleen cells was evaluated by the ELISPOT method.

[0142] [ Fig.38 ] Fig.38 The results show that LNP-hGPC3 mRNA was administered to HLA-A*24:02 Tg mice, and the production of antigen-specific IFN-γ in spleen cells was evaluated by the ELISPOT method.

[0143] [ Fig.39 ] Fig.39 The results show that LNP-luciferase mRNA was administered to HLA-A*24:02 Tg mice, and the production of antigen-specific IFN-γ in spleen cells was evaluated by the ELISPOT method.

[0144] [ Fig.40 ] Fig.40 The administration method of LNP-hROBO1 mRNA is shown in which LNP-hROBO1 mRNA is administered three times.

[0145] [ Fig.41 ] Fig.41 The present invention shows the results of administering LNP-hROBO1 mRNA or LNP-eGFP mRNA to HLA-A*02:01 Tg mice and evaluating the production of antigen-specific IFN-γ in spleen cells by the ELISPOT method.

[0146] [ Fig.42 ] Fig.42 The results show that LNP-hROBO1 mRNA or LNP-eGFP mRNA was administered to HLA-A*24:02 Tg mice, and the production of antigen-specific IFN-γ in spleen cells was evaluated by the ELISPOT method.

[0147] [ Fig.43 ] Fig.43 The administration method of LNP-hCLDN1 mRNA or LNP-hEPHB4 mRNA is shown as 3 administrations.

[0148] [ Fig.44 ] Fig.44 The present invention shows the results of administering LNP-hCLDN1 mRNA, LNP-hEPHB4 mRNA or LNP-eGFP mRNA to HLA-A*02:01 Tg mice and evaluating the production of antigen-specific IFN-γ in spleen cells by the ELISPOT method.

[0149] [ Fig.45 ] Fig.45 The present invention shows the results of administering LNP-hCLDN1 mRNA, LNP-hEPHB4 mRNA or LNP-eGFP mRNA to HLA-A*24:02 Tg mice and evaluating the production of antigen-specific IFN-γ in spleen cells by the ELISPOT method.

[0150] [ Fig.46 ] Fig.46 The administration method of LNP-hTGFBI mRNA or LNP-hSPARC mRNA is shown as 3 administrations.

[0151] [ Fig.47 ] Fig.47The present invention shows the results of administering LNP-hTGFBI mRNA, LNP-hSPARC mRNA or LNP-eGFP mRNA to HLA-A*02:01 Tg mice and evaluating the production of antigen-specific IFN-γ in spleen cells by the ELISPOT method.

[0152] [ Fig.48 ] Fig.48 The present invention shows the results of administering LNP-hTGFBI mRNA, LNP-hSPARC mRNA or LNP-eGFP mRNA to HLA-A*24:02 Tg mice and evaluating the production of antigen-specific IFN-γ in spleen cells by the ELISPOT method.

[0153] [ Fig.49 ] Fig.49 The administration method of LNP-hAFP mRNA or LNP-hHSP105α mRNA is shown as 3 administrations.

[0154] [ Fig.50 ] Fig.50 The present invention shows the results of administering LNP-hAFP mRNA, LNP-hHSP105α mRNA or LNP-eGFP mRNA to HLA-A*02:01 Tg mice and evaluating the production of antigen-specific IFN-γ in spleen cells by the ELISPOT method.

[0155] [ Fig.51 ] Fig.51 The present invention shows the results of administering LNP-hAFP mRNA, LNP-hHSP105α mRNA or LNP-eGFP mRNA to HLA-A*24:02 Tg mice and evaluating the production of antigen-specific IFN-γ in spleen cells by the ELISPOT method.

[0156] [ Fig.52 ] Fig.52 The administration method of LNP-(hCLDN1+hEPHB4) mRNA is shown as 3 administrations.

[0157] [ Fig.53 ] Fig.53 The figures show the results of administering LNP-(hCLDN1+hEPHB4) mRNA or LNP-luciferase mRNA to HLA-A*02:01 Tg mice and evaluating the production of antigen-specific IFN-γ in spleen cells by the ELISPOT method.

[0158] [ Fig.54 ] Fig.54The results show that LNP-(hCLDN1+hEPHB4) mRNA or LNP-luciferase mRNA was administered to HLA-A*02:01 Tg mice, and the production of antigen-specific IFN-γ in the inguinal lymph nodes was evaluated by the ELISPOT method.

[0159] [ Fig.55 ] Fig.55 The figures show the results of administering LNP-(hCLDN1+hEPHB4) mRNA or LNP-luciferase mRNA to HLA-A*24:02 Tg mice and evaluating the production of antigen-specific IFN-γ in spleen cells by the ELISPOT method.

[0160] [ Fig.56 ] Fig.56 The results show that LNP-(hCLDN1+hEPHB4) mRNA or LNP-luciferase mRNA was administered to HLA-A*24:02 Tg mice, and the production of antigen-specific IFN-γ in the inguinal lymph nodes was evaluated by the ELISPOT method.

[0161] [ Fig.57 ] Fig.57 The present invention shows a method for selecting high-affinity T cell clones by inhibiting the binding of CD8 molecules to MHC.

[0162] [ Fig.58 ] Fig.58 It was shown that peptide-specific CTL clones were established from the peripheral blood of patients who received the GPC3 peptide vaccine.

[0163] [ Fig.59 ] Fig.59 It was shown that peptide-specific CTL clones were established from the peripheral blood of patients who received FOXM1 peptide vaccine.

[0164] [ Fig.60 ] Fig.60 It was shown that peptide-specific CTL clones were established from the peripheral blood of patients who received the HSP105 peptide vaccine.

[0165] [ Fig.61 ] Fig.61 Identification of peptide-reactive TCR genes introduced into mice using human HLA genes is shown.

[0166] [ Fig.62 ] Fig.62 The results show that TCR-T cells that recognize a universal cancer antigen (FOXM1) were produced by electroporation.

[0167] [ Fig.63 ] Fig.63 Development of cocktail TCR-T cells that demonstrate the ability to overcome a variety of cancers.

[0168] [ Fig.64 ] Fig.64 Shows in vitro cytotoxicity evaluation using CAR-T cells.

[0169] [ Fig.65 ] Fig.65 In vivo cytotoxicity evaluation using CAR-T cells is shown.

[0170] [ Fig.66 ] Fig.66 Showing the development of cocktail CAR-T therapy.

[0171] [ Fig.67 ] Fig.67 The results of expression analysis of the membrane protein universal cancer antigen in various cancer cell lines are shown.

[0172] [ Fig.68 ] Fig.68 The results of expression analysis of the membrane protein universal cancer antigen in various cancer cell lines are shown.

[0173] [ Fig.69 ] Fig.69 The results of detecting CTC-like cells expressing any one of the membrane proteins universal cancer antigens GPC3, ROBO1, CLDN1, and EPHB4 on the cell membrane in blood samples collected from hepatocellular carcinoma cases are shown. DETAILED DESCRIPTION

[0174] Hereinafter, the present invention will be described in more detail.

[0175] The inventors have identified 10 universal cancer antigens that are frequently expressed in various solid tumors and hardly expressed in normal tissues through immunohistochemical analysis. Five of them are membrane protein antigens. Among the solid tumors studied so far, such as head and neck cancer, lung cancer, liver cancer, biliary tract cancer, pancreatic cancer, and colorectal cancer, at least one of these five antigens is expressed in most cases of cancer, confirming that these universal cancer antigen cocktails can cover all solid tumors. According to these results, cancer vaccines, TCR-T cell therapy, CAR-T cell therapy, their companion diagnosis, and cancer risk diagnosis based on the detection of circulating cancer cells in the blood using these universal cancer antigen cocktails can be achieved.

[0176] 85% of the Japanese population and 50% of the world's population have HLA-A24 or -A2. In the present invention, peptides predicted to be presented on HLA-A24 and peptides predicted to be presented on HLA-A2 were synthesized from the full-length amino acid sequences of 10 universal cancer antigen proteins, and mice expressing human HLA-A24 or -A2 in systemic cells were given peptide vaccines once a week for a total of 3 times, which proved that CD8 positive killer T cells (CTL) that react with peptides were induced in spleen cells. These peptides have excellent CTL induction ability and can be used for the treatment, recurrence prevention or prevention of cancer through peptide vaccines or dendritic cell vaccines with added peptides. In addition, the 10 universal cancer antigens contain multiple peptide sequences that can induce CTL and can also be used as mRNA vaccines.

[0177] Moreover, by cloning the T cell receptor (TCR), a T cell therapy (TCR-T therapy) in which the TCR is genetically introduced can be developed, and the TCR is located on the cell surface of CTLs induced by administering peptide vaccines from 10 universal cancer antigens and specifically reacting with peptides, and recognizes the complex of HLA and peptides on the surface of cancer cells. By increasing the TCR library (TCR repertory) that can react with each universal cancer antigen peptide, a cocktail TCR-T therapy that is most suitable for each patient or suitable for various cancers can be achieved. Any of the 10 membrane protein universal cancer antigens is expressed in most cancer tissues. This means that cocktail antibody therapy or cocktail CAR-T therapy for them can be used as a treatment method for most solid tumors.

[0178] <Universal Cancer Antigen>

[0179] GPC3, ROBO1, EPHB4, CLDN1, LAT1, AFP, TGFBI, SPARC, HSP105α and FOXM1 are known proteins. In the examples described below, proteins represented by the following amino acid sequences, isoforms thereof, or homologs thereof can be used:

[0180] hGPC3_isoform_2_NP_004475

[0181] hROBO1_isoform_X5_XP_006713340 (cytoplasmic form of hROBO1)

[0182] hROBO1_isoform_a_NP_002932 (membrane-localized form of hROBO1)

[0183] hEPHB4_NP_004435

[0184] hCLDN1_NP_066924

[0185] hSLC7A5_NP_003477

[0186] hAFP_isoform_1_NP_001125

[0187] hTGFBI_NP_000349

[0188] hSPARC_isoform_1_NP_003109

[0189] hFOXM1_isoform_2_NP_068772

[0190] hHSPH1_isoform_1_NP_006635.

[0191] Isoforms include, for example, splice variants, variants such as SNPs based on individual differences, etc. Specifically, the proteins include: a protein composed of an amino acid sequence having a sequence identity of preferably 95% or more, more preferably 98% or more, in the amino acid sequence described in the above examples; (2) a protein composed of an amino acid sequence in which one or more, preferably one to several, more preferably one to ten, one to five, one to three, one or two amino acids are substituted, deleted, added or inserted in the amino acid sequence described in the above examples.

[0192] <Peptides and proteins>

[0193] The peptides and proteins of the present invention can be produced by common genetic engineering techniques including DNA cloning based on the nucleotide sequence information of the genes encoding them, plasmid construction, host transfection, culture of transformants and recovery of proteins from the culture, using expression vectors or cloning vectors.

[0194] Recombinant vectors can be manufactured by inserting polynucleotides into appropriate vectors. The vector can be appropriately selected according to the type and purpose of use of the host. As vectors, for example, vectors from chromosomes, episomes or viruses can be listed. Specifically, for example, vectors from bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion elements, yeast chromosome elements, viruses (e.g., baculovirus, papovavirus, SV40, vaccinia virus, adenovirus and retrovirus, etc.) and vectors formed by their combination, vectors from genetic elements of plasmids and phages (e.g., cosmids and phagemids, etc.) can be listed.

[0195] By inserting the polynucleotide into a vector using a known method, a recombinant vector containing the polynucleotide can be obtained.

[0196] The recombinant vector inserted with the polynucleotide is introduced into a known host, such as bacteria such as Escherichia coli and Bacillus bacteria, yeast, insect cells or animal cells (for example, COS-7 cells, Vero cells, CHO cells, etc.) by a known method to obtain a transformant introduced with the recombinant vector.

[0197] Gene introduction can be performed by methods known to those skilled in the art, specifically, calcium phosphate transfection, DEAE-dextran-mediated transfection, microinjection, cationic lipid-mediated transfection, electroporation, transfection, infection, and the like.

[0198] <Cancer Vaccine>

[0199] The cancer vaccine of the present invention comprises:

[0200] (1) comprising three or more universal cancer antigens selected from the group consisting of GPC3, ROBO1, EPHB4, CLDN1 and LAT1;

[0201] (2) partial peptides of the above three or more universal cancer antigens, which have CTL inducibility;

[0202] (3) dendritic cells stimulated with the above partial peptides; or

[0203] (4) mRNA encoding the above-mentioned universal cancer antigen or the above-mentioned partial peptide.

[0204] The universal cancer antigen may include three or more selected from GPC3, ROBO1, EPHB4, CLDN1, and LAT1, or may include three, four, or five selected from GPC3, ROBO1, EPHB4, CLDN1, and LAT1. Preferably, the universal cancer antigen may include all of GPC3, ROBO1, EPHB4, CLDN1, and LAT1.

[0205] In addition to the substance selected from GPC3, ROBO1, EPHB4, CLDN1 and LAT1, the universal cancer antigen may further include one or more of AFP, TGFBI, SPARC, HSP105α and FOXM1. That is, the universal cancer antigen may further include one, two, three, four or five of AFP, TGFBI, SPARC, HSP105α and FOXM1.

[0206] Preferably, the universal cancer antigens may include all of GPC3, ROBO1, EPHB4, CLDN1, LAT1, AFP, TGFBI, SPARC, HSP105α, and FOXM1.

[0207] In the present invention, partial peptides of the three or more universal cancer antigens described above and having CTL inducibility can be used as cancer vaccines. That is, at least three or more partial peptides are used as partial peptides.

[0208] Specifically, as partial peptides, three or more selected from the group consisting of partial peptides of GPC3, partial peptides of ROBO1, partial peptides of EPHB4, partial peptides of CLDN1, and partial peptides of LAT1 may be included, and three, four, or five selected from the group consisting of partial peptides of GPC3, partial peptides of ROBO1, partial peptides of EPHB4, partial peptides of CLDN1, and partial peptides of LAT1 may be included. Preferably, as partial peptides, all of partial peptides of GPC3, partial peptides of ROBO1, partial peptides of EPHB4, partial peptides of CLDN1, and partial peptides of LAT1 may be included.

[0209] In addition to the partial peptide selected from the group consisting of the partial peptide of GPC3, the partial peptide of ROBO1, the partial peptide of EPHB4, the partial peptide of CLDN1, and the partial peptide of LAT1, the partial peptide may include one or more of the partial peptide of AFP, the partial peptide of TGFBI, the partial peptide of SPARC, the partial peptide of HSP105α, and the partial peptide of FOXM1. That is, the universal cancer antigen may further include one, two, three, four, or five of the partial peptide of AFP, the partial peptide of TGFBI, the partial peptide of SPARC, the partial peptide of HSP105α, and the partial peptide of FOXM1.

[0210] Preferably, the partial peptide may include all of the partial peptide of GPC3, the partial peptide of ROBO1, the partial peptide of EPHB4, the partial peptide of CLDN1, the partial peptide of LAT1, the partial peptide of AFP, the partial peptide of TGFBI, the partial peptide of SPARC, the partial peptide of HSP105α and the partial peptide of FOXM1.

[0211] The above-mentioned partial peptides are epitope peptides, which are peptides that bind to MHC (HLA in humans) to present antigens to the cell surface and have antigenicity (can be recognized by T cells). As epitope peptides, there are epitope peptides that bind to MHC class I to present antigens and are recognized by CD8 positive T cells, namely CTL epitope peptides, and epitope peptides that bind to MHC class II to present antigens and are recognized by CD4 positive T cells, namely auxiliary epitope peptides. The partial peptides in the present invention are preferably epitope peptides that bind to MHC class I to present antigens and are recognized by CD8 positive T cells, namely CTL epitope peptides.

[0212] Some of the peptides in the present invention are peptides from proteins specifically expressed in tumor cells and are tumor antigen peptides. Antigen presentation refers to the phenomenon that a peptide present in a cell binds to MHC and the MHC / antigen peptide complex is localized on the cell surface. After the antigen presented to the cell surface is recognized by T cells, etc., cellular immunity or humoral immunity is activated. Antigens presented to MHC class I activate cellular immunity and are recognized by the T cell receptors of naive T cells at the same time, inducing naive T cells into CTLs with cytotoxic activity. Therefore, as tumor antigen peptides used for immunotherapy, peptides that bind to MHC class I and present antigens are preferred.

[0213] As described above, the partial peptide of the present invention is a partial peptide of GPC3, ROBO1, EPHB4, CLDN1, LAT1, AFP, TGFBI, SPARC, HSP105α or FOXM1, and is a peptide that binds to MHC, particularly HLA, preferably a peptide that presents an antigen through MHC, particularly HLA, and more preferably a peptide that can induce CTL by presenting an antigen through MHC, particularly HLA. The partial peptide is preferably able to bind to HLA class I, and more preferably can bind to HLA-A02 and / or HLA-A24.

[0214] The amino acid length of the partial peptide is not particularly limited as long as it contains the amino acid sequence of the epitope, but is generally preferably about 8 to 14 amino acids in length, more preferably about 8 to 11 amino acids in length, and particularly preferably about 9 to 11 amino acids in length.

[0215] It is known that epitope peptides that bind to human MHC class I, i.e., HLA class I, are about 8 to 14 amino acids in length, preferably about 9 to 11 amino acids in length, and have a binding motif specific to the HLA to which they bind in their sequence. For example, a peptide that binds to HLA-A02 may have a binding motif in which the second amino acid from the N-terminus may be leucine, isoleucine, or methionine and / or the amino acid at the C-terminus may be valine, leucine, or isoleucine, and a peptide that binds to HLA-A24 may have a binding motif in which the second amino acid from the N-terminus may be tyrosine, phenylalanine, methionine, or tryptophan and / or the amino acid at the C-terminus may be leucine, isoleucine, or phenylalanine, but are not limited to the above.

[0216] Therefore, the partial peptide preferably includes an epitope peptide, which is a partial peptide of GPC3, ROBO1, EPHB4, CLDN1, LAT1, AFP, TGFBI, SPARC, HSP105α or FOXM1, which may be a peptide consisting of 8 to 14 consecutive amino acids in the amino acid sequence of the above protein, and the second amino acid from the N-terminus may be leucine, isoleucine or methionine and / or the amino acid at the C-terminus may be valine, leucine or isoleucine. Alternatively, the partial peptide is preferably a partial peptide of GPC3, ROBO1, EPHB4, CLDN1, LAT1, AFP, TGFBI, SPARC, HSP105α or FOXM1, which is a peptide consisting of 8 to 14 consecutive amino acids in the amino acid sequence of the above protein, and the second amino acid from the N-terminus may be tyrosine, phenylalanine, methionine or tryptophan and / or the amino acid at the C-terminus may be leucine, isoleucine or phenylalanine, but is not limited to the above.

[0217] The N-terminus and / or C-terminus of the partial peptide may be modified. Specific examples of modifications include N-alkanoylation (e.g., acetylation), N-alkylation (e.g., methylation), C-terminal alkyl ester (e.g., ethyl ester), and C-terminal amide (e.g., formamide).

[0218] The synthesis of a partial peptide can be carried out according to a known method generally used in peptide chemistry.

[0219] As the partial peptide, a peptide having an amino acid sequence described in any one of SEQ ID NOs: 1 to 80 can be used.

[0220] Among the above peptides, a peptide having an amino acid sequence described in any one of SEQ ID NOs: 5, 7 to 10, 14 to 22, 24 to 38, 40, 42, 48, 49, and 52 to 80 is a novel peptide.

[0221] The above-mentioned partial peptides have CTL inducing activity and can be used as CTL inducing agents as tumor antigen peptides. That is, peripheral blood lymphocytes are separated from people who are positive for HLA-A02 antigen or HLA-A24 antigen, and the above-mentioned partial peptides are added in vitro for stimulation, so that CTLs that specifically recognize HLA-A02 antigen-positive cells or HLA-A24 antigen-positive cells presenting the above-mentioned partial peptides can be induced. Whether CTL induction exists can be confirmed by, for example, measuring the amount of various cytokines (such as IFN-γ) produced by the reaction of CTL with antigen peptide presenting cells using, for example, ELISA method. In addition, it can also be confirmed by measuring the toxicity of CTL to antigen peptide presenting cells labeled with 51Cr.

[0222] In the present invention, dendritic cells stimulated by partial peptides can be used as cancer vaccines. That is, by contacting partial peptides with dendritic cells in vitro, antigen presenting cells presenting the complex of HLA-A02 antigen or HLA-A24 antigen and partial peptides of the present invention on the cell surface can be manufactured. Preferably, as dendritic cells, cells separated from cancer patients can be used. Regarding dendritic cells, for example, lymphocytes can be separated from the peripheral blood of cancer patients by the Ficoll method, non-adherent cells are removed afterwards, and adherent cells are cultured in the presence of GM-CSF and IL-4 to induce dendritic cells. By contacting the dendritic cells separated from cancer patients as above with partial peptides of the present invention in vitro, antigen presenting cells presenting the complex of HLA-A02 antigen or HLA-A24 antigen and partial peptides on the cell surface can be manufactured.

[0223] In the present invention, mRNA encoding the universal cancer antigen or the partial peptide can be used as a cancer vaccine. As mRNA encoding the universal cancer antigen or the partial peptide, mRNA encoding multiple partial peptides in series can be used. In this case, multiple partial peptides are encoded on one mRNA.

[0224] The cancer vaccine of the present invention is effective for cancer patients, especially for HLA-A02 or HLA-A24 positive cancer patients. Specifically, for example, it is effective for patients with hepatocellular carcinoma, colorectal cancer, oropharyngeal cancer, esophageal cancer, uterine cancer, Wilms tumor, lung cancer, breast cancer, tongue cancer, intrahepatic bile duct cancer, renal cancer, neuroblastoma, choriocarcinoma, etc.

[0225] The cancer vaccine of the present invention can be used for the prevention or treatment of cancer. Cancer prevention includes not only preventing patients from developing cancer, but also preventing recurrence in patients whose tumors have been surgically removed from the primary lesion; preventing the metastasis of tumors that cannot be completely removed by cancer treatments such as surgery, radiotherapy, or drug therapy. Cancer treatment includes not only curing cancer / improving symptoms by shrinking the cancer, but also inhibiting the proliferation of cancer cells, the enlargement of tumors, or preventing the deterioration of cancer cells from metastasizing from the primary lesion.

[0226] The cancer vaccine of the present invention can be provided in the form of a pharmaceutical composition.

[0227] The dosage form of the cancer vaccine of the present invention may be oral or parenteral, and parenterals are generally preferred. Examples of parenterals include subcutaneous injections, intramuscular injections, intravenous injections, and suppositories.

[0228] When the cancer vaccine of the present invention is an oral administration agent, the above-mentioned active ingredients and pharmaceutically acceptable excipients can be formulated into a cancer vaccine. Examples of excipients include starch, mannitol, lactose, magnesium stearate, cellulose, polymerized amino acids, and albumin.

[0229] When the cancer vaccine of the present invention is a parenteral agent, the above-mentioned active ingredients and a pharmaceutically acceptable carrier can be formulated into a cancer vaccine. Examples of the carrier include water, salt, glucose, ethanol, glycerol, and DMSO.

[0230] The cancer vaccine of the present invention may further contain albumin, a wetting agent and / or an emulsifier, etc. as necessary.

[0231] In addition, in order to activate cellular immunity, the above-mentioned active ingredients may be used together with an appropriate adjuvant. The cancer vaccine of the present invention may contain the adjuvant.

[0232] As adjuvants, adjuvants known in the art can be used, specifically, the following substances can be listed. As gel type, aluminum hydroxide, aluminum phosphate and calcium phosphate can be listed. As bacterial type, CpG, monophosphoryl lipid A, cholera toxin, Escherichia coli heat-labile toxin, pertussis toxin and muramyl dipeptide can be listed. As oil emulsion type (emulsion preparation), Freund's incomplete adjuvant can be listed. As polymer nanoparticle type, liposomes, biodegradable microspheres and QS-21 from saponin can be listed. As synthetic type, non-ionic block copolymers, muramyl peptide analogs, polyphosphazenes and synthetic polynucleotides can be listed. As cytokine type, IFN-γ, IL-2 and IL-12 can be listed.

[0233] The dosage form of the cancer vaccine (drug composition) of the present invention is not particularly limited, and examples thereof include: oil emulsions (emulsion preparations), polymer nanoparticles, liposome preparations, granular preparations combined with beads having a diameter of several μm, preparations combined with lipids, microsphere preparations, microcapsule preparations, etc.

[0234] The administration method of the cancer vaccine (pharmaceutical composition) of the present invention includes intradermal administration, subcutaneous administration, intramuscular administration, intravenous administration, etc. The dosage of the active ingredient in the cancer vaccine can be appropriately adjusted according to the target disease to be treated, the age and weight of the patient, etc., and is usually 0.0001 mg to 1000 mg, preferably 0.001 mg to 1000 mg, and more preferably 0.1 mg to 10 mg per adult (weight 50 kg), and is preferably administered once every few days to several months.

[0235] In addition, in the case of using dendritic cells stimulated with partial peptides as a cancer vaccine, in order to stably maintain the above-mentioned dendritic cells, it is preferable to contain physiological saline, phosphate-buffered saline (PBS), culture medium, etc. As the administration method, intravenous administration, subcutaneous administration, intradermal administration, etc. can be cited. By returning the cancer vaccine containing such dendritic cells stimulated with partial peptides as an active ingredient to the patient's body, CTLs specific to cancer cells expressing the universal cancer antigen of the present invention can be efficiently induced in the body of a cancer patient. As a result, cancer can be prevented or treated.

[0236] <CAR-T cell therapy agent>

[0237] The CAR-T cell therapy agent of the present invention comprises a mixture of T cells having a chimeric antigen receptor (CAR) directed against three or more universal cancer antigens selected from GPC3, ROBO1, EPHB4, CLDN1, and LAT1.

[0238] The universal cancer antigen only needs to contain three or more selected from GPC3, ROBO1, EPHB4, CLDN1, and LAT1, and may also contain three, four, or five selected from GPC3, ROBO1, EPHB4, CLDN1, and LAT1. Preferably, the universal cancer antigen may contain all of GPC3, ROBO1, EPHB4, CLDN1, and LAT1.

[0239] In addition to substances selected from GPC3, ROBO1, EPHB4, CLDN1, and LAT1, the universal cancer antigen may further contain one or more of AFP, TGFBI, SPARC, HSP105α, and FOXM1. That is, the universal cancer antigen may further contain one, two, three, four, or five of AFP, TGFBI, SPARC, HSP105α, and FOXM1.

[0240] Preferably, the universal cancer antigen may contain all of GPC3, ROBO1, EPHB4, CLDN1, LAT1, AFP, TGFBI, SPARC, HSP105α, and FOXM1.

[0241] CAR-T cell therapy refers to an immunotherapy in which a chimeric antigen receptor (CAR) modified by genetic manipulation of a part of a monoclonal antibody specific to a tumor antigen is introduced into T cells from a patient, and the gene-modified T cells are amplified and cultured in vitro and then infused into the patient. Specifically, after activating T cells by culturing peripheral blood mononuclear cells collected from a patient in the presence of anti-CD3 antibody and IL-2, etc., a gene encoding CAR is introduced into T cells using a transformation vector such as a retroviral vector or a lentiviral vector, thereby producing gene-modified T cells.

[0242] A chimeric antigen receptor is a chimeric protein molecule in which a single-chain antibody (scFv) formed by tandem binding of the light and heavy chains of an antibody variable region designed to have an antibody that recognizes a molecule present on the cell surface of cancer cells on the N-terminal side, and a CD3ζ chain in a molecule constituting the T cell receptor (TCR) / CD3 complex on the C-terminal side. When this chimeric antigen receptor recognizes a specific antigen in the scFv region, activation of T cells occurs via the CD3ζ chain. To enhance T cell activation, one or more co-stimulatory molecules (such as CD28, 4-1BB, ICOS, etc.) can be inserted between the scFv and the ζ chain. As the scFv, a TCR-like antibody (including an antibody molecule or a fragment thereof that can be designed from a TCR-like antibody) can be used to produce a CAR. A CAR that recognizes a complex of a peptide from a tumor antigen and MHC can recognize cancer cells presenting a tumor antigen peptide that can be targeted by CTLs, dendritic cells that phagocytose cancer cells to present the tumor antigen peptide on MHC class I, etc. Therefore, genetically modified T cells introduced with the above CAR can be used as a prophylactic and / or therapeutic agent for cancers specific to the above tumor antigens, just like artificial CTLs.

[0243] T cells having a chimeric antigen receptor (CAR-T cells) can be formulated directly or together with various carriers into a CAR-T cell therapeutic agent.

[0244] The dosage form of the CAR-T cell therapeutic agent can be either an oral dosage form or a parenteral dosage form. Usually, a parenteral dosage form is preferably used. Examples of parenteral dosage forms include: subcutaneous injection agents, intramuscular injection agents, intravenous injection agents, suppositories, etc.

[0245] In the case where the CAR-T cell therapeutic agent is an oral dosage form, the obtained CAR-T cells can be formulated together with a pharmaceutically acceptable excipient into a CAR-T cell therapeutic agent. Examples of such excipients include: starch, mannitol, lactose, magnesium stearate, cellulose, polymeric amino acids, and albumin, etc.

[0246] In the case where the CAR-T cell therapeutic agent is a parenteral dosage form, the obtained CAR-T cells can be formulated together with a pharmaceutically acceptable carrier into a CAR-T cell therapeutic agent. Examples of such excipients include: water, sodium chloride, glucose, ethanol, glycerol, and DMSO, etc.

[0247] <TCR-T cell therapeutic agent>

[0248] The TCR-T cell therapeutic agent of the present invention comprises a mixture of T cells having a T cell receptor (TCR) that can recognize an MHC class I-binding antigen peptide from a common cancer antigen comprising three or more of GPC3, ROBO1, EPHB4, CLDN1, and LAT1.

[0249] The universal cancer antigen may include at least three selected from GPC3, ROBO1, EPHB4, CLDN1, and LAT1, and may include three, four, or five selected from GPC3, ROBO1, EPHB4, CLDN1, and LAT1. Preferably, the universal cancer antigen may include all of GPC3, ROBO1, EPHB4, CLDN1, and LAT1.

[0250] In addition to the substance selected from GPC3, ROBO1, EPHB4, CLDN1 and LAT1, the universal cancer antigen may further include one or more of AFP, TGFBI, SPARC, HSP105α and FOXM1. That is, the universal cancer antigen may further include one, two, three, four or five of AFP, TGFBI, SPARC, HSP105α and FOXM1.

[0251] Preferably, the universal cancer antigens may include all of GPC3, ROBO1, EPHB4, CLDN1, LAT1, AFP, TGFBI, SPARC, HSP105α, and FOXM1.

[0252] T cell receptors (TCRs) that can recognize MHC class I-binding antigenic peptides derived from universal cancer antigens can recognize a complex of the partial peptide (antigenic peptide) of the present invention and HIA.

[0253] T cell receptor (TCR) is usually a heterodimer of TRA and TRB.

[0254] As specific examples of T cell receptors (TCR), the T cell receptors described in Tables 2 to 11 in the Examples described below can be used. Specifically, specific T cell receptors (TCR) include:

[0255] A heterodimer composed of a combination of the protein of SEQ ID NO: 103 and the protein of SEQ ID NO: 104;

[0256] A heterodimer composed of a combination of the protein of SEQ ID NO: 105 and the protein of SEQ ID NO: 106;

[0257] A heterodimer composed of a combination of the protein of SEQ ID NO: 107 and the protein of SEQ ID NO: 108;

[0258] a heterodimer composed of a combination of the protein of SEQ ID NO: 109 and the protein of SEQ ID NO: 110;

[0259] A heterodimer composed of a combination of the protein of SEQ ID NO: 111 and the protein of SEQ ID NO: 112;

[0260] A heterodimer composed of a combination of the protein of SEQ ID NO: 123 and the protein of SEQ ID NO: 124;

[0261] A heterodimer composed of a combination of the protein of SEQ ID NO: 125 and the protein of SEQ ID NO: 126;

[0262] a heterodimer composed of a combination of the protein of SEQ ID NO: 127 and the protein of SEQ ID NO: 128;

[0263] A heterodimer composed of a combination of the protein of SEQ ID NO: 129 or 130 and the protein of SEQ ID NO: 131;

[0264] A heterodimer composed of a combination of the protein of SEQ ID NO: 132 and the protein of SEQ ID NO: 133;

[0265] a heterodimer composed of a combination of the protein of SEQ ID NO: 134 and the protein of SEQ ID NO: 135;

[0266] a heterodimer composed of a combination of the protein of SEQ ID NO: 136 and the protein of SEQ ID NO: 137;

[0267] A heterodimer composed of a combination of the protein of SEQ ID NO: 138 or 139 and the protein of SEQ ID NO: 140;

[0268] A heterodimer composed of a combination of the protein of SEQ ID NO: 141 and the protein of SEQ ID NO: 142;

[0269] A heterodimer composed of a combination of the protein of SEQ ID NO: 143 and the protein of SEQ ID NO: 144;

[0270] a heterodimer composed of a combination of the protein of SEQ ID NO: 145 and the protein of SEQ ID NO: 146;

[0271] a heterodimer composed of a combination of the protein of SEQ ID NO: 147 and the protein of SEQ ID NO: 148;

[0272] A heterodimer composed of a combination of the protein of SEQ ID NO: 177 and the protein of SEQ ID NO: 178;

[0273] a heterodimer composed of a combination of the protein of SEQ ID NO: 179 and the protein of SEQ ID NO: 180;

[0274] A heterodimer composed of a combination of the protein of SEQ ID NO: 181 and the protein of SEQ ID NO: 182;

[0275] a heterodimer composed of a combination of the protein of SEQ ID NO: 183 and the protein of SEQ ID NO: 184;

[0276] A heterodimer composed of a combination of the protein of SEQ ID NO: 193 and the protein of SEQ ID NO: 194;

[0277] A heterodimer composed of a combination of the protein of SEQ ID NO: 195 and the protein of SEQ ID NO: 196;

[0278] A heterodimer composed of a combination of the protein of SEQ ID NO: 197 and the protein of SEQ ID NO: 198;

[0279] A heterodimer composed of a combination of the protein of SEQ ID NO: 199 or 200 and the protein of SEQ ID NO: 201;

[0280] A heterodimer composed of a combination of the protein of SEQ ID NO: 202 and the protein of SEQ ID NO: 203;

[0281] A heterodimer composed of a combination of the protein of SEQ ID NO: 216 and the protein of SEQ ID NO: 217; or

[0282] A heterodimer composed of a combination of the protein of SEQ ID NO: 218 and the protein of SEQ ID NO: 219.

[0283] The amino acid sequence and nucleotide sequence of the above-mentioned T cell receptor (TCR) are both novel sequences.

[0284] According to the present invention, a protein having an amino acid sequence described in any one of SEQ ID NOs: 103 to 112, 123 to 148, 177 to 184, 193 to 203, and 215 to 219 is provided.

[0285] According to the present invention, a gene having a nucleotide sequence of any one of SEQ ID NOs: 113 to 122, 149 to 176, 185 to 192, 204 to 214, and 220 to 224 is provided.

[0286] By contacting the partial peptides of the universal cancer antigen of the present invention with peripheral blood lymphocytes in vitro, T cells with T cell receptors (TCR) can be induced. The obtained T cells with T cell receptors (TCR) can specifically damage cells presenting the universal cancer antigen of the present invention. Preferably, the partial peptides of the universal cancer antigen of the present invention can be used to stimulate the patient's peripheral blood lymphocytes in vitro to increase T cells with T cell receptors (TCR) (i.e., tumor-specific CTL), and then the T cells with T cell receptors (TCR) are returned to the patient.

[0287] As described above, T cells with T cell receptors (TCR) can become the active ingredients of therapeutic or preventive drugs for cancer, and can be used as TCR-T cell therapeutic agents. TCR-T cell therapeutic agents preferably include physiological saline, phosphate buffered saline (PBS), culture medium, etc. to stably maintain TCR-T cells. As methods of administration, examples include: intravenous administration, subcutaneous administration, intradermal administration, etc. By returning such a therapeutic or preventive drug for cancer formed by containing TCR-T cells as an active ingredient to the patient's body, the toxic effect of cancer cells is promoted by TCR-T cells in the body of a patient suffering from a cancer positive for the universal cancer antigen of the present invention, and cancer cells are destroyed, thereby treating cancer.

[0288] The TCR-T cells of the present invention can exert cytotoxic activity by targeting the complex of the partial peptide and HLA presented by the antigen to tumor cells. That is, the T cell receptor (TCR) of the TCR-T cells of the present invention recognizes the complex of the partial peptide and HLA of the present invention.

[0289] T cells (TCR-T cells) having T cell receptors (TCR) can be formulated directly or together with various carriers into TCR-T cell drugs.

[0290] The dosage form of the TCR-T cell therapy agent can be any of an oral administration agent or a parenteral administration agent. Parenteral administration agents are generally preferred. Examples of parenteral administration agents include subcutaneous injections, intramuscular injections, intravenous injections, suppositories, and the like.

[0291] When the TCR-T cell therapeutic agent is an oral agent, the obtained TCR-T cell therapeutic agent can be formulated into a TCR-T cell therapeutic agent together with a pharmaceutically acceptable excipient. Examples of the excipient include starch, mannitol, lactose, magnesium stearate, cellulose, polymerized amino acids, and albumin.

[0292] When the TCR-T cell therapeutic agent is a parenteral agent, the obtained TCR-T cell therapeutic agent can be formulated with a pharmaceutically acceptable carrier to prepare a TCR-T cell therapeutic agent. Examples of the excipient include water, salt, glucose, ethanol, glycerol, and DMSO.

[0293] <Companion diagnostic method>

[0294] The companion diagnostic method of the present invention comprises the following steps: step 1, simultaneously determining the presence or absence of expression of three or more (i.e., three, four or five) of GPC3, ROBO1, EPHB4, CLDN1 and LAT1 and the cell membrane expression of HLA class I in a sample from a subject by multiple fluorescent immunostaining; and step 2, determining the applicability of cancer immunotherapy based on the presence or absence of the above-mentioned expression.

[0295] Preferably, in step 1, the presence or absence of expression of one or more (ie, one, two, three, four or five) of AFP, TGFBI, SPARC, HSP105α and FOXM1 can be further determined.

[0296] Preferably, in step 1, the presence or absence of expression of all of GPC3, ROBO1, EPHB4, CLDN1 and LAT1 can be determined.

[0297] Preferably, in step 1, the presence or absence of expression of all of GPC3, ROBO1, EPHB4, CLDN1, LAT1, AFP, TGFBI, SPARC, HSP105α and FOXM1 can be determined.

[0298] The sample derived from the subject is not particularly limited, and examples thereof include liver, large intestine, oropharynx, esophagus, uterus, kidney, lung, breast, tongue, intrahepatic bile duct, nerve, blood, and the like.

[0299] The presence or absence of expression of the universal cancer antigen of the present invention and the cell membrane expression of HLA class I are simultaneously determined by multiple fluorescent immunostaining. Multiple fluorescent immunostaining can be any of a direct method or an indirect method. In the direct method, multiple primary antibodies directly labeled with fluorescence are used, and no labeled secondary antibody is used. In the direct method, cross-reactions caused by secondary antibodies can be avoided, and the system is simple and the operation time is short. In the indirect method, multiple primary antibodies are used, and labeled secondary antibodies against these primary antibodies are used. By using an amplification method and the like in combination, there is an advantage of being able to detect with a higher sensitivity than the direct method.

[0300] The fluorescent dye used in multiplex fluorescent immunostaining can be selected in consideration of the fluorescence spectra of the excitation wavelength and the fluorescence wavelength.

[0301] In the companion diagnostic method of the present invention, in a patient suffering from cancer, when a therapeutic drug is administered for the treatment of cancer, it is possible to examine or diagnose whether there is improvement or the extent thereof. In addition, the companion diagnostic method of the present invention can also be used to select patients to whom the cancer vaccine, TCR-T cell therapeutic drug or CAR-T cell therapeutic drug of the present invention can be effectively applied, or to predict or determine the therapeutic effect of the cancer vaccine, TCR-T cell therapeutic drug or CAR-T cell therapeutic drug of the present invention.

[0302] <Methods for diagnosing cancer risk>

[0303] The method for diagnosing cancer risk of the present invention comprises analyzing the expression of three or more (ie, three, four or five) of GPC3, ROBO1, EPHB4, CLDN1 and LAT1 in cells in a blood sample from a subject.

[0304] Preferably, the expression of one or more (ie, one, two, three, four or five) of AFP, TGFBI, SPARC, HSP105α and FOXM1 can be analyzed.

[0305] Preferably, the expression of all of GPC3, ROBO1, EPHB4, CLDN1 and LAT1 may be analyzed.

[0306] Preferably, the expression of all of GPC3, ROBO1, EPHB4, CLDN1, LAT1, AFP, TGFBI, SPARC, HSP105α, and FOXM1 can be analyzed.

[0307] In the method for diagnosing cancer risk of the present invention, typically, blood is collected from a subject, and the expression of three or more (i.e., three, four, or five) of GPC3, ROBO1, EPHB4, CLDN1, and LAT1 in the cells contained therein is analyzed. By detecting or measuring the expression level of the universal cancer antigens, the presence or absence of cancer or the degree of cancer can be detected, examined, or diagnosed.

[0308] Example

[0309] Example 1: Expression analysis of universal cancer antigens based on immunohistochemistry analysis and development of companion diagnostic methods in a multiplex fluorescent immunostaining system

[0310] <Method>

[0311] 4um paraffin sections were made from formalin-fixed paraffin blocks of various human cancer tissues. Microarray slides of normal tissues were commercially available (BioChain). Deparaffinization was performed with xylene and ethanol, and endogenous peroxidase was inactivated using 0.3% H2O2 / methanol. Antigen activation was performed by heat treatment in Target Retrieval Solution, pH9 (Agilent) or AR6 (AKOYA BIOSCIENCES) inactivation buffer using microwaves or pressure cookers. After blocking with pig serum, primary antibodies against each antigen were reacted. GPC3 (BIOMOSICS, 300-fold dilution), AFP (Agikent, ready-to-use), ROBO1 (PROTEINTECH, 300-fold dilution), EPHB4 (Cell Signaling Technology, 300-fold dilution), CLDN1 (Cell Signaling Technology, 300-fold dilution), FOXM1 (abcam, 150-fold dilution), HSP105α (abcam, 200-fold dilution), SPARC (Santa Cruz Biotechnology, 250-fold dilution), TGFBI (abcam, 100-fold dilution), HLA-ABC (Hokudo, 200-fold dilution), LAT1 (abcam, 200-fold dilution). Polymer reagents (EnVision+System-HRP-labeled polymer anti-rabbit or EnVision+System-HRP-labeled polymer anti-mouse) were used as secondary antibodies. Liquid DAB+substrate color development system was used for color development. After contrast staining with hematoxylin, the cells were dehydrated and transparentized with ethanol and xylene. The specimens were made into virtual slides using NanoZoomer (Hamamatsu Photonics KK).

[0312] In the implementation of multiple fluorescent immunohistochemical staining, deparaffinization was performed with xylene and ethanol, and endogenous peroxidase was inactivated with 0.3% H2O2 / methanol. Antigen activation was performed by heat treatment in Target Retrieval Solution, pH9 (Agilent) or AR6 (AKOYA BIOSCIENCES) inactivation buffer using microwaves. After blocking with pig serum, the primary antibodies against each antigen were reacted. GPC3 (BIOMOSICS, 600-fold dilution), ROBO1 (PROTEINTECH, 600-fold dilution), EPHB4 (Cell Signaling Technology, 300-fold dilution), CLDN1 (Cell Signaling Technology, 1200-fold dilution), HLA-ABC (Hokudo, 600-fold dilution), LAT1 (abcam, 800-fold dilution). Polymer reagents (EnVision+System-HRP-labeled polymer anti-rabbit or EnVision+System-HRP-labeled polymer anti-mouse) were used as secondary antibodies. Opal 520, 540, 570, 620, 650, and 690 (AKOYA BIOSCIENCES) were used as fluorescent dyes by diluting them 100-fold. After nuclear staining with DAPI, the specimens were sealed with a water-soluble sealant and observed using Vectra 3 (PerkinElmer).

[0313] <Results>

[0314] Figure 1 The results of immunohistochemical staining using paraffin-embedded tissue sections to confirm the expression of 10 universal cancer antigens in hepatocellular carcinoma, etc. are shown. GPC3, AFP, ROBO1, TGFBI, EPHB4, and CLDN1 were observed to be moderately to highly expressed in the cancerous part of hepatocellular carcinoma, and negative to weakly expressed in the non-cancerous part. In addition, the expression of HLA-I class on the cell membrane was observed in most cases, which shows that universal cancer antigens expressed in hepatocellular carcinoma are excellent targets for TCR-T, CAR-T and cancer vaccine therapy. SPARC, HSP105α, FOXM1, TGFBI, EPHB4, CLDN1, and LAT1 were also confirmed to be moderately to highly expressed in colorectal cancer liver metastasis, oropharyngeal cancer (HPV-), colorectal cancer, esophageal cancer, and uterine cancer.

[0315] Figure 2The expression of cell membrane-expressed universal cancer antigens GPC3, EPHB4, CLDN1, LAT1, and ROBO1 in various cancer types was shown. GPC3 was observed to be co-expressed in hepatocellular carcinoma and pediatric cancer Wilms tumor, and weak to moderate expression was observed in squamous cell carcinomas such as lung cancer and esophageal cancer. EPHB4, CLDN1, and ROBO1 were observed to be moderately to highly expressed in lung cancer, colorectal cancer, breast cancer, tongue cancer, intrahepatic bile duct cancer, kidney cancer, and esophageal cancer, and LAT1 tended to be strongly expressed in squamous cell carcinoma.

[0316] Figure 3 The expression of universal cancer antigens and HLA-I class in childhood cancers such as neuroblastoma, Wilms tumor, and choriocarcinoma was shown. Although moderate to high expression of each universal cancer antigen was observed, represented by the nuclear expression type FOXM1, the expression of HLA-I class in the cell membrane could not be confirmed. On the other hand, the cell membrane-expressed universal cancer antigens of GPC3, EPHB4, CLDN1, LAT1, and ROBO1 were moderately to highly expressed in the cell membrane of cancer cells of neuroblastoma, Wilms tumor, and choriocarcinoma, indicating that the non-restrictive nature of HLA-Class I may be applied to CAR-T therapy.

[0317] Figure 4 The results of confirming the expression of each universal cancer antigen in the non-cancerous part of the clinical specimen of hepatocellular carcinoma are shown. GPC3, AFP, ROBO1, FOXM1, and CLDN1 are negative, and EPHB4 and TGFBI are observed to be weakly expressed in the blood sinusoids of the liver parenchyma. It can be seen that HLA-I class is weakly expressed in the cell membrane of hepatocytes.

[0318] Figure 5 The expression of cell membrane-expressed universal cancer antigens GPC3, EPHB4, CLDN1, LAT1, and ROBO1 in 31 normal tissues is shown in the table using human normal tissue microarray. Although weak signals of EPHB4 and ROBO1 were detected in various tissues, clear cell membrane expression could not be confirmed. Clear expression of GPC3, CLDN1, and LAT1 was observed only in some tissues.

[0319] Figure 6 Demonstrates the concept of developing an applicable diagnostic system, using multiplexed fluorescent immunostaining to determine which universal cancer antigens are effective when used as targets in immunotherapy such as TCR-T, CAR-T, and cancer vaccines.

[0320] Figure 7In the PDX, specimens from patients with hepatocellular carcinoma and lung cancer were used, and specimens from patients with hepatocellular carcinoma, intrahepatic cholangiocarcinoma, and oropharyngeal cancer (HPV-) were used as human clinical specimens. Multiple fluorescent immunostaining was performed on the cell membrane-expressed universal cancer antigens of GPC3, EPHB4, CLDN1, LAT1, and ROBO1, and HLA-I class. In hepatocellular carcinoma PDX, GPC3 was strongly expressed, followed by the expression of CLDN1 and EPHB4. In lung cancer PDX, GPC3, EPHB4, CLDN1, and LAT1 were moderately to highly expressed in any combination. In human clinical specimens, not only GPC3, CLDN1, and LAT1 were observed in patients with hepatocellular carcinoma and oropharyngeal cancer (HPV-), but also the expression of HLA-I class was observed, showing the possibility of being applicable not only to CAR-T, but also to TCR-T and cancer vaccines. In patients with intrahepatic cholangiocarcinoma, LAT1, followed by EPHB4 and CLDN1 were confirmed as applicable antigens.

[0321] Example 2: Gene expression of universal cancer antigens and prediction of HLA binding peptides

[0322] <Method>

[0323] Figure 8 The gene expression of each cancer antigen in hepatocellular carcinoma and primary colorectal cancer is shown. The gene expression of each cancer antigen in 371 cases of hepatocellular carcinoma (left figure) and 478 cases of primary colorectal cancer (right figure) registered in TCGA (https: / / www.cancer.gov / ccg / research / genome-sequencing / tcga) is shown in a heat map. The expression level of each gene is standardized and evaluated by log2(FPKM+1). Regarding the name of each cancer antigen, antigens with high frequency or high expression are displayed in red, antigens with medium frequency or moderate expression are displayed in green, antigens with low frequency or low expression are displayed in blue, and antigens with no expression observed in most cases are displayed in red.

[0324] Fig. 9 The gene expression of each cancer antigen in hepatocellular carcinoma and primary colorectal cancer for which antigen prediction was performed is displayed. The gene expression of each cancer antigen in 43 cases of hepatocellular carcinoma and 24 cases of metastatic liver cancer from colorectal cancer that underwent surgical resection at the National Cancer Center East Hospital from 2016 to 2023 is represented by a heat map. The expression level of each gene is standardized and evaluated by log2(TPM+1). Hepatocellular carcinoma is divided into HBV positive (5 cases), HCV positive (10 cases), and non-viral (28 cases) for display.

[0325] Fig.10The prediction process of epitope peptides from cancer antigens is shown. Tumor and normal liver tissues are collected from surgical resection specimens of registered cases, DNA and RNA are extracted, and neoantigens and universal cancer antigens that are highly expressed in cancer are selected through whole exome sequencing and RNA sequencing. The HLA haplotype of each case is determined based on whole exome sequencing analysis, and peptides with high binding affinity to MHC class I (HLA-A, B, C) are predicted. A predictor that incorporates the binding affinity to MHC and the expression level of each antigen as factors is constructed, and the antigenicity of peptides from cancer antigens is calculated in the form of SCOREadj.

[0326] Fig.11 The SCOREadj distribution of peptides from each cancer antigen in hepatocellular carcinoma is displayed. The peptides from each cancer antigen predicted in one case of hepatocellular carcinoma are classified according to SCOREadj (1-2: light blue, 2-3: orange, 3-4: gray, 4-5: yellow, 5-6: blue, and 6 or more: green), and their numbers are displayed (upper section, bar graph). The expression of each cancer antigen gene is displayed in red (Log2_TPM=10) to blue (Log2_TPM=1). The bar graph in the lower section is a peptide from each cancer antigen, and only peptides with SCOREadj of 6 or more are extracted and displayed.

[0327] Fig.12 The total number of cancer antigen peptides predicted in 7 cases of hepatocellular carcinoma is shown. For 7 cases of hepatocellular carcinoma, the antigenicity of peptides from each cancer antigen was predicted using the same predictor. The total number of predicted peptides (the total number of peptides predicted to be from cancer antigens and bound to HLA-A, B, and C) was classified according to SCOREadj and displayed as a bar graph for each case.

[0328] <Results>

[0329] The gene expression of 12 common cancer antigens in hepatocellular carcinoma and colorectal cancer was confirmed using metadata registered in TCGA (hepatocellular carcinoma: TCGA-LIHC, colorectal cancer: TCGA-COAD). As a result, expression of CLDN1, SPARC, and TGFβI was confirmed in most cases. LAT1, FOXM1, EPHB4, and HSP105 were observed to be expressed in most cases of colorectal cancer. Although they were expressed at high frequencies in hepatocellular carcinoma, they were expressed at low frequencies in colorectal cancer. On the other hand, cases where GPC3 or AFP were highly expressed were observed in hepatocellular carcinoma, in contrast, the expression of these cancer antigens in colorectal cancer was low or could not be confirmed. Low expression of ROBO1 was often observed in hepatocellular carcinoma, but it was mostly low expression in colorectal cancer. NY-ESO-1 and WT1 were hardly expressed in hepatocellular carcinoma and colorectal cancer. Next, for each case of hepatocellular carcinoma and metastatic colorectal cancer we collected, we used DNA or RNA extracted from tumors and normal tissues to evaluate the expression of neoantigens derived from gene mutations and these universal cancer antigens through whole exome analysis and RNA sequencing analysis to study whether they can become candidates for cancer vaccines. The results showed that, similar to the analysis using TCGA metadata, CLDN1, GPC3, SPARC, and TGFβI were expressed at high frequencies in hepatocellular carcinoma. Regarding AFP, few cases with high expression were observed. In metastatic colorectal cancer, CLDN1, LAT1, HSP105, SPARC, and TGFβI were expressed at high frequencies, while the expression of hepatocellular carcinoma-specific cancer antigens such as ROBO1, GPC3, and AFP was hardly observed, showing the same tendency as primary colorectal cancer. On the other hand, EPHB4 and FOXM1 were observed to be expressed in only a portion of cases in metastatic colorectal cancer, indicating the possibility that cancer antigen expression is different from that in primary colorectal cancer. Based on the above results, these 10 cancer antigens other than NY-ESO-1 and WT1 are considered to be promising targets for innovative immunotherapy to overcome heterogeneity in solid tumors in cases of hepatocellular carcinoma or colorectal cancer including metastases.

[0330] Next, we verified the extent to which epitopes from cancer antigens that are targets of CD8+T cells that damage and eliminate cancer are present in each case when these cancer antigens are actually targeted. So far, we have developed a predictor through joint research with Brightpath Bio Co., Ltd., which combines existing HLA binding predictions such as netMHCPan (https: / / services.healthtech.dtu.dk / services / NetMHCpan-4.1 / ) and MHC Flurry (https: / / github.com / openvax / mhcflurry) with the protein proteasome cleavage prediction algorithm NetChop (https: / / services.healthtech.dtu.dk / services / NetChop-3.1 / ), and also incorporates cancer antigen expression information. This predictor optimizes these multiple factors using a linear prediction function and evaluates them as SCOREadj. So far, we have used 278 peptides of neoantigens predicted from 46 cases of hepatocellular carcinoma or metastatic colorectal cancer to verify the effectiveness of our predictor and reported it in an international journal (Cancer Sci. 2022Apr; 113(4): 1113-1124.) In this series of predictions, gene expression information in each case can be obtained, so not only neoantigens but also the expression of universal cancer antigens can be evaluated at the same time. Therefore, for cancer antigens that are actually highly expressed, it is verified to what extent peptides predicted to be highly antigenic are observed in each case. When predicting a case of hepatocellular carcinoma, a group of peptides predicted to bind to HLA for any antigen can be extracted. When extracting peptides with high SCOREadj, as expected, about 30 to 40 peptides that can be expected to become epitopes can be predicted for cancer antigens such as GPC3, ROBO1, FOXM1, and EPHB4. In the verification targeting neoantigens, it was found that if the peptides with strong immunogenicity were considered to be about 50% of the predicted number, a sufficient number of peptides from universal cancer antigens could be selected as candidates for cancer vaccines. In addition, in the case of high expression but a small number of predicted peptides like SPARC, or vice versa, when the expression level was the same but the number of peptides was large in ROBO1 and small in CLDN1, it was suggested that the number of predicted high SCOREadj peptides depends not only on the expression level of the antigen, but also on the binding affinity with HLA. Similarly, the results of the verification of 6 hepatocellular carcinoma cases predicted that in any case, about 100 peptides from universal cancer antigens could be used as candidates for cancer peptide vaccines.

[0331] Since the expression amount and expression pattern of cancer antigens vary, and HLA haplotypes also vary in each case, it is desirable to develop a cocktail vaccine targeting multiple cancer antigens as a personalized cancer vaccine that can prepare the best peptide combination predicted for each patient. On the other hand, the cost and time required for preparation or testing often become problems in personalized medicine. In addition to predicting peptides that can be expected to have effects from multiple universal cancer antigens, a peptide library with confirmed immunogenicity from these 10 universal cancer antigens can be installed and supplied as a stock, thereby providing a more effective cocktail peptide vaccine at low cost and in a short period of time.

[0332] Example 3: Identification of CTL-inducing peptides from universal cancer antigens and development of peptide vaccines / peptide-pulsed dendritic cell vaccines

[0333] <Method>

[0334] exist Fig.13 In the study, an algorithm established in joint research with Brightpath Bio was used to predict and synthesize 72 HIA-A*24:02 restricted epitope peptides and 73 HLA-A*02:01 restricted epitope peptides from the amino acid sequences of 10 universal cancer antigens. Whole exome analysis and RNA sequencing analysis were performed using a next-generation sequencer, and peptides that could bind to these HLAs were predicted in silico based on the obtained gene mutations and their expression data. Transgenic mice with introduced HLA genes were used to evaluate whether the predicted antigenic peptides could induce CTLs. Each peptide was dissolved in dimethyl sulfoxide (DMSO, Wako) to 10 mg / ml and used with an adjuvant to immunize HLA transgenic mice three times a week. The spleen was removed again one week later, and the production of IFN-γ was evaluated by IFN-γ ELISpot assay.

[0335] Fig.16 In this study, whether antigen-specific T cells can be induced by vaccinating transgenic mice with dendritic cells (DCs) sensitized with short peptides from universal cancer antigens that have been confirmed to induce CTLs was evaluated using transgenic mice into which each HLA gene was introduced. Each peptide was dissolved in dimethyl sulfoxide (DMSO, Wako) to 10 mg / ml, and 1×10 5 DCs sensitized with each peptide were prepared with PBS and immunized intradermally at the base of the tail of HLA-A*02:01 Tgm or HLA-A*24:02 Tgm using a 27G injection needle / 1ml syringe (Terumo) at a volume of 100μl. Immunization was performed three times a week in total, and the spleen was removed one week after the third vaccination, and the production of IFN-γ was evaluated by IFN-γ ELISpot assay.

[0336] DCs were collected from the femur of HLA-A*02:01 Tgm or HLA-A*24:02 Tgm. 2×10 6 Each DC was suspended in 10 ml of final culture medium (10% FBS / 1% L-glutamine / streptomycin-penicillin / NEAA / 10 mM HEPES / sodium pyruvate / 2-ME (Gibco)) + 2-ME (1000-fold dilution) + 20 ng / ml rmGM-CSF (PeproTech), seeded in a low-adhesion 10 cm Petri dish (AS ONE), and culture began. On the 3rd day from the start of culture, 10 ml of final culture medium + 20 ng / ml rmGM-CSF was added. On the 6th day, half of the culture medium was taken into a conical tube, centrifuged at 1,500 rpm for 5 minutes to remove the supernatant, and the pellet was suspended in a new 10 ml of final culture medium and returned to the same culture dish. On the 8th day, half of the culture medium was exchanged in the same manner as on the 6th day. On the 9th day, 10 mL of culture medium was collected into a conical tube. Non-adherent cells and loosely adherent cells were recovered by gentle pipetting and collected into the same conical tube. After centrifugation at 1500 rpm for 5 minutes, discard the supernatant. Resuspend with 10 mL of serum-free RPMI and centrifuge at 1500 rpm for 5 minutes. Repeat this operation twice (to remove FBS), discard the supernatant, resuspend with 10 mL of serum-free RPMI, add mature cytokines (20 ng / mL TNFα, 1 μg / mL PGE2, 20 ng / mL IL-4, 0.1 μM Zometa, 10 μg / mL antigen peptide or no peptide for negative control), and culture overnight. On the 10th day, the DCs sensitized by each peptide were washed twice with PBS and suspended into 1×10 5 / 100 μl, and administered to mice.

[0337] In the ELISpot assay, splenocytes from immunized mice were separated into CD8 positive T cells by MACS and the number of cells was 1×10 5 The spleen cells of unimmunized mice were irradiated (100 kV, 10 mA, 100 Gy) and suspended in the final medium supplemented with each peptide at a concentration of 20 ug / ml to form 2×10 6 The concentration of cells / well was then used as target cells.

[0338] <Results>

[0339] exist Fig.14 , Fig.15In Table 1, peptides predicted to be presented on HLA-A*24:02 or HLA-A*02:01 were synthesized from the full-length amino acid sequences of 10 universal cancer antigen proteins. Peptide vaccines were administered once a week for a total of three times to mice expressing human HLA-A*24:02 or HLA-A*02:01 in systemic cells, and multiple peptides that can induce CD8-positive killer T cells (CTL) that react with the peptides were identified by screening in spleen cells. The CTL induction rate that reacts with the peptides is as follows: 30 / 72 in HLA-A*24:02-restricted universal cancer antigen short peptides, 38 / 73 in HLA-A*02:01-restricted universal cancer antigen short peptides, and 68 / 145 in total. The CTL induction ability of these peptides is superior to that of the peptides of the GPC3 peptide vaccine used in clinical trials, and can be used in the prevention and treatment of recurrence of various cancers in the future.

[0340] [Table 1]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346] Fig.17 and Fig.18 In this study, HLA-A*24:02 or HLA-A*02:01 transgenic mice were vaccinated with dendritic cells (DC) sensitized with short peptides from universal cancer antigens that have been confirmed to induce CTLs, and transgenic mice into which the respective HLA genes were introduced were used to evaluate whether antigen-specific T cells were induced. As a result, in most peptides, it was confirmed that universal cancer antigen peptide-sensitized dendritic cell vaccines induced antigen-specific CTLs by IFN-γ ELISpot assay. For each of the peptides used, three peptides that highly induced CTLs by peptide vaccines were selected from antigens that were highly expressed mainly in hepatocellular carcinoma. DCs were sensitized with the selected peptides, and transgenic mice were vaccinated, and the ability to induce antigen-specific CTLs was evaluated by IFN-γ ELISpot assay.

[0347] exist Fig.19In this study, a long peptide containing a short peptide sequence from a universal cancer antigen that has been confirmed to induce CTL was used. In order to confirm whether the peptide was actually processed in cells and presented on HLA, HLA-A*02:01 Tgm or HLA-A*24:02 Tgm was used for verification. A long peptide is a long peptide (15-21mer) designed to contain an amino acid sequence of a short peptide consisting of 9-10mer that can induce CTLs that react with the peptide. Whether the long peptide is actually processed in cells and bound to MHC molecules and presented on HLA to induce peptide-specific CTLs by vaccination was evaluated using IFN-γ ELISpot assay. In the HLA-A*24:02-restricted universal cancer antigen long peptide vaccine, 3 out of 21 synthesized long peptides were confirmed to induce CTLs that react with the short peptides contained in their sequences. In the HLA-A*02:01-restricted universal cancer antigen long peptide vaccine, 9 out of 21 synthesized long peptides were confirmed to induce CTLs that reacted with short peptides contained in their sequences.

[0348] Example 4: Development of universal cancer antigen mRNA vaccine

[0349] LNP-Covid-19 Spike mRNA was administered to evaluate the production of antigen-specific IFN-γ.

[0350] 2 μg or 10 μg of mRNA vaccine was administered intramuscularly (im) into the rectus femoris muscle of HLA-A*02:01 Tg mice and HLA-A*24:02 Tg mice, or intradermally (id) into the base of the tail. The mRNA vaccine was obtained by encapsulating the full-length mRNA encoding the Spike protein of Covid-19 (SARS-CoV-2) in LNP (lipid nanoparticle) (ARCTURUStherapeutics, non-patent literature (de Alwis R. et al., A single dose of self-transcribing and replicating RNA-based SARS-CoV-2 vaccine produces protective adaptive immunity in mice. Mol Ther. 2021; 29: 1970-1983.) conventional mRNA, and the following mRNA vaccines were also prepared in the same way). In the 3-dose regimen, the drug was administered 3 times on days 0, 7, and 14, and HLA-A*24:02 Tg mice were dissected on day 20, while HLA-A*02:01 Tg mice were dissected on day 21. In the 2-dose regimen, the drug was administered on days 0 and 28, and HLA-A*24:02 Tg mice were dissected on day 35, while HLA-A*02:01 Tg mice were dissected on day 36. Cells were modulated from spleen and inguinal lymph nodes (LN), and serum was collected from blood collected from the heart. Antigen-specific IFN-γ production from T cells was evaluated by ELISPOT according to the recommended protocol using the BD ELISPOT mouse IFN-γ panel and the BD ELISPOT AEC substrate panel (both BD). 2×10 6 splenocytes were seeded at 2×10 5LN cells were inoculated at 10 cells / well, and PMA (25 ng / mL) + ionomycin (1 μg / mL) were added, respectively; a cocktail of peptides that encapsulate Covid-19 Spike protein (PepMix S (jpt, PM-WCPV-S-1) in the 3-dose experiment and PepTivator SARC-CoV-2 Prot_S (Miltenyi Biotech, 130-127-953) in the 2-dose experiment, each peptide was 1 μg / mL); a cocktail of peptides that were predicted from the Covid-19 Spike protein sequence using the predictor described in the above paragraph, namely POOL1 (each peptide was 5 μg / mL) that was confirmed to produce peptide-specific IFN-γ in peptide immunization of HLA-A*02:01 Tg mice or HLA-A*24:02 Tg mice; and a cocktail of peptides that were predicted from the Covid-19 Spike protein sequence using the predictor described in the above paragraph and confirmed to produce peptide-specific IFN-γ in peptide immunization of HLA-A*02:01 Tg mice or HLA-A*24:02 Tg mice. The bone marrow of naive Tg mice was induced to differentiate in mouse GM-CSF (20 ng / mL) and matured by adding mouse TNF-α (20 ng / mL), mouse IL-4 (20 ng / mL), and PGE2 (1 μg / mL) 1 day before use. 5 cells / well), and at the same time of maturation, each 1×10 6 BMDCs (2×10 5 The cells were co-cultured for 20 hours to evaluate the production of IFN-γ. The spots were photographed with an ELISPOT counter AT-X (Minervatech) and the number of spots was counted. The number of spots was recorded at the lower left corner of each well.

[0351] Fig. 20 The dosing regimen is shown with 3 doses of LNP-Covid-19 Spike mRNA.

[0352] HLA-A*02:01 Tg mice and HLA-A*24:02 Tg mice were intramuscularly administered (im) into the rectus femoris muscle, or intradermally administered (id) into the base of the tail, for a total of three times on day 0, day 7, and day 14. The mRNA vaccine (ARCTURUS therapeutics) was obtained by encapsulating the full-length mRNA encoding the Covid-19 Spike protein in LNP. HLA-A*24:02 Tg mice were dissected on day 20, and HLA-A*02:01 Tg mice were dissected on day 21, and the production of antigen-specific IFN-γ was evaluated by the ELISPOT method.

[0353] Fig.21 and Fig. 22The results show the production of Spike antigen-specific IFN-γ in spleen cells of HLA-A*02:01 Tg mice given LNP-Covid-19 Spike mRNA three times.

[0354] In the spleen cells of HLA-A*02:01 Tg mice given LNP-Covid-19 Spike mRNA three times, T cells that produce IFN-γ were induced both by im and id against BMDCs added with PepMix S, POOL1, and LNP-Covid-19 Spike mRNA. In HLA-A*02:01 Tg mice given LNP-eGFP mRNA as a negative control three times in the same manner, almost no IFN-γ production was observed against BMDCs added with PepMix S, POOL1, and LNP-Covid-19 Spike mRNA, indicating that the above-mentioned IFN-γ production was specifically induced by the administration of LNP-Covid-19 Spike mRNA.

[0355] Fig.23 The results show the production of Spike antigen-specific IFN-γ in the inguinal lymph node cells of HLA-A*02:01 Tg mice that were given LNP-Covid-19 Spike mRNA three times.

[0356] In the inguinal lymph node cells of HLA-A*02:01 Tg mice given LNP-Covid-19 Spike mRNA three times, T cells that produced IFN-γ were induced both by im and id against BMDCs to which PepMix S and LNP-Covid-19 Spike mRNA were added. In HLA-A*02:01 Tg mice given LNP-eGFP mRNA as a negative control three times in the same manner, almost no IFN-γ production was observed against BMDCs to which PepMix S and LNP-Covid-19 Spike mRNA were added, indicating that the production of IFN-γ was specifically induced by the administration of LNP-Covid-19 Spike mRNA.

[0357] Fig.24 and Fig.25 The results show the production of Spike antigen-specific IFN-γ in spleen cells of HLA-A*24:02 Tg mice given LNP-Covid-19 Spike mRNA three times.

[0358] In the spleen cells of HLA-A*24:02 Tg mice that were given LNP-Covid-19 Spike mRNA three times, the 6 BMDCs to which 1 μg or 10 μg of PepMix S, POOL1, or LNP-Covid-19Spike mRNA was added per cell strongly induced IFN-γ-producing T cells both by im and id. In HLA-A*24:02 Tg mice to which LNP-eGFP mRNA as a negative control was similarly administered three times, the production of IFN-γ by BMDCs to which PepMix S, POOL1, or LNP-Covid-19Spike mRNA was added was small, indicating that the production of IFN-γ was specifically induced by administration of LNP-Covid-19 Spike mRNA.

[0359] Fig.26 The results show the production of Spike antigen-specific IFN-γ in the inguinal lymph node cells of HLA-A*24:02 Tg mice that were given LNP-Covid-19 Spike mRNA three times.

[0360] In the inguinal lymph node cells of HLA-A*24:02 Tg mice given LNP-Covid-19 Spike mRNA three times, T cells producing IFN-γ were induced both im and id against PepMixS and POOL1. In HLA-A*24:02 Tg mice given LNP-eGFP mRNA as a negative control three times, almost no IFN-γ production against PepMix S and POOL1 was observed, indicating that the production of IFN-γ was specifically induced by the administration of LNP-Covid-19 Spike mRNA.

[0361] Fig. 27 The dosing method of LNP-Covid-19 Spike mRNA is shown with 2 administrations.

[0362] 2 μg or 10 μg of mRNA vaccine encoding the full-length Covid-19 Spike protein encapsulated in LNP was administered intramuscularly (im) into the rectus femoris muscle of HLA-A*02:01 Tg mice and HLA-A*24:02 Tg mice or intradermally (id) into the base of the tail on day 0 and day 28. HLA-A*24:02 Tg mice were dissected on day 35, and HLA-A*02:01 Tg mice were dissected on day 36, and the production of antigen-specific IFN-γ was evaluated by ELISPOT method.

[0363] Fig.28 and Fig.29 The results show the production of Spike antigen-specific IFN-γ in spleen cells of HLA-A*02:01 Tg mice that were given LNP-Covid-19 Spike mRNA twice.

[0364] In the spleen cells of HLA-A*02:01 Tg mice that were given LNP-Covid-19 Spike mRNA twice, T cells that produced FN-γ were induced both by im and id against BMDCs that had been added with PepTi S, POOL1, and LNP-Covid-19 Spike mRNA. In HLA-A*02:01 Tg mice that were given LNP-eGFP mRNA twice as a negative control, only a small amount of IFN-γ production was observed against BMDCs that had been added with PepTi S, POOL1, and LNP-Covid-19 Spike mRNA, indicating that the production of IFN-γ was specifically induced by the administration of LNP-Covid-19 Spike mRNA.

[0365] Fig.30 The results show the production of Spike antigen-specific IFN-γ in the inguinal lymph node cells of HLA-A*02:01 Tg mice that were given LNP-Covid-19 Spike mRNA twice.

[0366] In the inguinal lymph node cells of HLA-A*02:01 Tg mice given LNP-Covid-19 Spike mRNA twice, T cells that produce IFN-γ were induced both by im and id for BMDCs to which PepTi S and LNP-Covid-19 Spike mRNA were added. In particular, high induction was observed in id. In HLA-A*02:01 Tg mice given LNP-eGFP mRNA as a negative control three times in the same manner, almost no IFN-γ production was observed for BMDCs to which PepTi S and LNP-Covid-19 Spike mRNA were added, indicating that the production of IFN-γ was specifically induced by the administration of LNP-Covid-19 Spike mRNA.

[0367] Fig.31 and Fig.32 The results show the production of Spike antigen-specific IFN-γ in spleen cells of HLA-A*24:02 Tg mice that were given LNP-Covid-19 Spike mRNA twice.

[0368] In the spleen cells of HLA-A*24:02 Tg mice given LNP-Covid-19 Spike mRNA twice, T cells that produced IFN-γ were strongly induced both by im and id against BMDCs added with PepTi S, POOL1, and LNP-Covid-19 Spike mRNA. In HLA-A*02:01 Tg mice given LNP-eGFP mRNA twice as a negative control, only a small amount of IFN-γ production was observed against BMDCs added with PepTi S, POOL1, and LNP-Covid-19 Spike mRNA, indicating that the above-mentioned IFN-γ production was specifically induced by the administration of LNP-Covid-19 Spike mRNA.

[0369] Fig.33 The results show the production of Spike antigen-specific IFN-γ in the inguinal lymph node cells of HLA-A*24:02 Tg mice that were given LNP-Covid-19 Spike mRNA twice.

[0370] In the inguinal lymph node cells of HLA-A*24:02 Tg mice given LNP-Covid-19Spike mRNA twice, T cells that produce IFN-γ were induced both by im and id for BMDCs to which PepTi S and LNP-Covid-19Spike mRNA were added. In particular, high induction was observed in id. In HLA-A*24:02 Tg mice given LNP-eGFP mRNA as a negative control three times, almost no IFN-γ production was observed for BMDCs to which PepTi S and LNP-Covid-19 Spike mRNA were added, indicating that the production of IFN-γ was specifically induced by the administration of LNP-Covid-19 Spike mRNA.

[0371] Fig.34 Shown are the results of an antibody assay against the Spike protein.

[0372] <Method>

[0373] On the MaxiSorp plate (ThrmoFisher), the Covid-19 Spike recombinant protein (Sino Biological, 40589-V08B1) was coated with 100ng / 50μL of PBS. After washing once with PBS containing 0.05% Tween20 (PBS-T), it was blocked with 3% skim milk at room temperature for 1 hour. Serum diluted with PBS containing 1% skim milk was added and incubated at 37°C for 1 hour. Washed 3 times with PBS-T, incubated with horseradish peroxidase (HRP) labeled goat anti-mouse IgG (SouthernNiotech, 6000-fold dilution) in PBS-T containing 1% skim milk at room temperature for 1 hour. After washing three times with PBS-T, 3,3',5,5'-tetramethylbenzidine substrate solution (Pierce) was added for color development, the reaction was stopped with 1.3NH2SO4, and the absorbance at 450nm was measured.

[0374] <Results>

[0375] No antibodies were induced when LNP-eGFP mRNA was administered as a negative control, but IgG antibodies against Covid-19 Spike protein were induced in either case of 2 μg or 10 μg of LNP-Covid-19Spike mRNA, in either case of im and id, in either case of 3 administrations or 2 administrations. Generally, IgG antibodies induced at 10 μg tended to be higher than at 2 μg, and at id than at im. In particular, high antibody induction was observed in 2 administrations of 10 μg i.d.

[0376] Example 5: Development of universal cancer antigen mRNA vaccine

[0377] LNP-hGPC3 mRNA, LNP-hROBO1 mRNA, LNP-hCLDN1 mRNA, LNP-hEphB4 mRNA, LNP-hTGFBI mRNA, LNP-hSPARC mRNA, LNP-hAFP mRNA, LNP-hHSP105α mRNA, and LNP-(hCLDN1+hEPHB4) mRNA were administered to evaluate the production of antigen-specific IFN-γ.

[0378] The genes encoding hGPC3 isoform 2 (RefSeq: NP_004475), hROBO1 isoform X5 (RefSeq: XP_006713340), hCLDN1 (RefSeq: NP_066924), hEphB4 (RefSeq: NP_004435), TGFBI (RefSeq: NP_000349), hSPARC isoform 1 (NP_003109), hAFP isoform 1 (NP_006713341), and hCLP1 (RefSeq: NP_0066924) were analyzed. mRNA vaccines (ARCTURUS therapeutics) encapsulated in LNP (lipid nanoparticles, ARCTURUS therapeutics) were administered intramuscularly (im) to the rectus femoris of HLA-A*02:01 Tg mice and HLA-A*24:02 Tg mice, or intradermally (id) to the base of the tail. Cells were modulated from the spleen and inguinal lymph nodes (LN). Antigen-specific IFN-γ production from T cells was evaluated by ELISPOT according to the recommended protocol using the BD ELISPOT Mouse IFN-γ Panel and the BD ELISPOT AEC Substrate Panel (both BD). 2 × 10 6 splenocytes were seeded at 2×10 5 LN cells were inoculated at 10 cells / well, and PMA (25 ng / mL) + ionomycin (1 μg / mL), a cocktail of short-chain peptides that can produce peptide-specific IFN-γ in peptide immunization of corresponding HLA Tg mice (10 μg / mL for each peptide), a cocktail of long-chain peptides that can produce peptide-specific IFN-γ in peptide immunization of corresponding HLA Tg mice (10 μg / mL for each peptide), and mouse cancer cells (MC38 or MCA205, 2×10 5 cells / well), mouse cancer cells (MC38 or MCA205, 2×10 5cells / well), bone marrow-derived dendritic cells (BMDCs) (2×10 cells / well) were induced to differentiate from the bone marrow of naive mice of the corresponding HLA Tg mice in mouse GM-CSF (20 ng / mL) and matured by adding mouse TNF-α (20 ng / mL), mouse IL-4 (20 ng / mL), and PGE2 (1 μg / mL) 1 day before use. 5 cells / well), and at the same time of maturation, each 5×10 6 BMDC (2×10 5 Cells / mL) were co-cultured for 20 hours to evaluate the production of IFN-γ. The spots were photographed using an ELISPOT counter AT-X (Minervatech). The following peptides were added as a cocktail of short-chain peptides.

[0379] hGPC3 HLA-A*02: 01: CA02 11, CA02 13, CA02 15, A02-GPC3-10, A02-GPC3-12, A02-GPC3-14, A02-GPC3-18;

[0380] hGPC3 HLA-A*24:02:A24-GPC3-06, A24-GPC3-10, A24-GPC3-11, A24-GPC3-17;

[0381] hROBO1 HLA-A*02:01:CA02 91, CA02 92, CA02 95, CA02 97;

[0382] hROBO1 HLA-A*24:02:CA24 95, CA24 97, CA24 98;

[0383] hCLDN1 HLA-A*02:01: CLDN1-A02-2, CLDN1-A02-5, CLDN1-A02-6;

[0384] hCLDN1HLA-A*24:02: CLDN1-A24-2, CLDN1-A24-4, CLDN1-A24-5, CLDN1-A24-7;

[0385] hEPHB4 HLA-A*02:01:CA02 121, CA02 122, CA02 123, CA02 126, CA02 127;

[0386] hEPHB4 HLA-A*24:02:CA24 123, CA24 124, CA24 127;

[0387] hTGFBI HLA-A*02:01:CA02 81, CA02 82, CA02 83, CA02 86;

[0388] hTGFBI HLA-A*24:02:CA24 85;

[0389] hSPARC HLA-A*02:01:CA02 52, CA02 56;

[0390] hSPARC HLA-A*24:02:CA24 51, CA24 52, CA24 53;

[0391] hAFP HLA-A*02:01:CA02 101, CA02 103, CA02 104;

[0392] hAFP HLA-A*24:02:CA24 102, CA24 105;

[0393] hHSP105αHLA-A*02:01:CA02 21, CA02 24;

[0394] hHSP105αHLA-A*24:02: CA24 22, CA24 23, CA24 24, CA24 25;

[0395] hHSP105αHLA-A*24:02 short peptide;

[0396] CA24 22: PFSKVLTFL;

[0397] CA24 23:KLCGPYEKF;

[0398] CA24 24: PYPEAKIGRF;

[0399] CA24 25:NYGIYKQDL.

[0400] The following peptides were added as a cocktail of long-chain peptides.

[0401] hGPC3 HLA-A*02:01:CA02 11_L, CA02 15_L, A02-GPC3-14_L;

[0402] hGPC3 HLA-A*24:02:A24-GPC3-06_L, A24-GPC3-11_L, A24-GPC3-17_L;

[0403] hROBO1 HLA-A*02:01: CA02 95_L, CA02 97_L;

[0404] hROBO1 HLA-A*24:02: CA24 97_L, CA24 98_L;

[0405] hCLDN1 HLA-A*02:01: CLDN1-A02-2_L, CLIN1-A02-5_L;

[0406] hCLDN1 HLA-A*24:02: CLDN1-A24-2_L, CLDN1-A24-4_L, CLDN1-A24-5_L, CLDN1-A24-7_L;

[0407] hEPHB4 HLA-A*02:01: CA02 121_L, CA02 123_L;

[0408] hEPHB4 HLA-A*24:02: CA24 124_L;

[0409] hTGFBI HLA-A*02:01: CA02 83_L, CA02 86_L;

[0410] hSPARC HLA-A*02:01: CA02 56_L;

[0411] hSPARC HLA-A*24:02: CA24 51_L, CA24 53_L;

[0412] hAFP HLA-A*02:01: 02 103_L;

[0413] hAFP HLA-A*24:02: 24 105_L;

[0414] hHSP105α HLA-A*02:01: A2-7_L;

[0415] hHSP 105α HLA-A*24:02: A24-1_L, A24-7_L;

[0416] hHSP105α HLA-A*02:01 long peptide;

[0417] A2-7_L: VGLNCL[RLMNDMTAV]ALNYG;

[0418] hHSP105α HLA-A*24:02 long peptide;

[0419] A24-1_L:KNAVE[EYVYEFRDKL]CGPYE;

[0420] A24-7_L:AVAL[NYGIYKQDL]PSLDEK.

[0421] Fig.35 The administration method of LNP-hGPC3 mRNA is shown as 3 times.

[0422] HLA-A*02:01 Tg mice and HLA-A*24:02 Tg mice were injected with 2 μg or 10 μg of LNP-hGPC3 mRNA or 10 μg of LNP-luciferase mRNA three times on days 0, 7, and 14 by IM injection into the rectus femoris muscle or id injection into the base of the tail. HLA-A*24:02 Tg mice were dissected on day 20, while HLA-A*02:01 Tg mice were dissected on day 22, and the production of antigen-specific IFN-γ was evaluated by ELISPOT.

[0423] Figure 36 to Figure 39 Display the above results.

[0424] like Fig.36 and Fig.37 As shown, in spleen cells of HLA-A*02:01 Tg mice to which LNP-hGPC3 mRNA was administered three times, IFN-γ-producing T cells were induced against a cocktail of hGPC3 short-chain peptides that produced peptide-specific IFN-γ in peptide immunization of HLA-A*02:01 Tg mice, a cocktail of hGPC3 long-chain peptides that produced peptide-specific IFN-γ in peptide immunization of HLA-A*02:01 Tg mice, MC38 cancer cells expressing HLA-A*02:01HHD and hGPC3, and BMDCs transfected with hGPC3 mRNA. More IFN-γ was produced in the case of id administration than in the case of im administration, and strong IFN-γ induction was observed in particular when 2 μg was administered three times id. Compared with the IFN-γ production against the cancer cells MC38A02HHD mimics introduced with HLA-A*02:01HHD and empty vector, the IFN-γ production against the cancer cells MC38 A02HHD hGPC3 expressing HLA-A*02:01HHD and hGPC3 was significantly higher, indicating that T cells that specifically recognize and attack the cancer cells expressing hGPC3 were induced. In HLA-A*02:01 Tg mice to which LNP-luciferase mRNA as a negative control was similarly administered three times, only a small amount of IFN-γ production was observed against any of them, indicating that the above IFN-γ production was specifically induced by the administration of LNP-hGPC3 mRNA.

[0425] like Fig.38 and Fig.39 As shown, in spleen cells of HLA-A*24:02 Tg mice to which LNP-hGPC3 mRNA was administered three times, IFN-γ-producing T cells were induced against a cocktail of hGPC3 short-chain peptides that produced peptide-specific IFN-γ in peptide immunization of HLA-A*24:02 Tg mice, a cocktail of hGPC3 long-chain peptides that produced peptide-specific IFN-γ in peptide immunization of HLA-A*24:02 Tg mice, MC38 cancer cells expressing HLA-A*24:02 HHD and hGPC3, and BMDCs transfected with hGPC3 mRNA. There was a tendency for more IFN-γ to be produced in the case of id administration than in the case of im administration, and particularly, strong IFN-γ induction was observed in the case of 10 μg administered three times id. Compared with the IFN-γ production against the cancer cell MC38A24HHD mimic introduced with HLA-A*24:02HHD and an empty vector, the IFN-γ production against the cancer cell MC38 A24HHDhGPC3 expressing HLA-A*24:02HHD and hGPC3 was significantly higher, which shows that T cells that specifically recognize and attack the cancer cells expressing hGPC3 were induced. In HLA-A*24:02 Tg mice to which LNP-luciferase mRNA as a negative control was similarly administered three times, only a small amount of IFN-γ production was observed against any of them, which shows that the above IFN-γ production was specifically induced by the administration of LNP-hGPC3 mRNA.

[0426] Example 6: Development of universal cancer antigen mRNA vaccine

[0427] Fig.40 The administration method of LNP-hROBO1 mRNA is shown as 3 times of administration.

[0428] HLA-A*02:01 Tg mice and HLA-A*24:02 Tg mice were intramuscularly administered (im) into the rectus femoris muscle or intradermally administered (id) into the base of the tail. 10 μg of LNP-hROBO1 mRNA or LNP-eGFP mRNA was administered three times on day 0, day 7, and day 14. The mice were dissected on day 21, and the production of antigen-specific IFN-γ was evaluated by the ELISPOT method.

[0429] Fig.41 and Fig.42 Display the above results.

[0430] like Fig.41As shown, in the spleen cells of HLA-A*02:01 Tg mice administered LNP-hROBO1 mRNA three times, IFN-γ-producing T cells were induced against a cocktail of hROBO1 short-chain peptides that produced peptide-specific IFN-γ in peptide immunization of HLA-A*02:01 Tg mice, a cocktail of hROBO1 long-chain peptides that produced peptide-specific IFN-γ in peptide immunization of HLA-A*02:01 Tg mice, MC38 cancer cells expressing HLA-A*02:01HHD and hROBO1, and BMDCs transfected with hROBO1 mRNA. Compared with the IFN-γ production against the cancer cell MC38 A02HHD mimic introduced with HLA-A*02:01HHD and empty vector, the IFN-γ production against the cancer cell MC38 A02HHDhROBO1 expressing HLA-A*02:01HHD and hROBO1 in both im and id was significantly higher, which shows that T cells that specifically recognize and attack the cancer cells expressing hROBO1 were induced. In HLA-A*02:01 Tg mice to which LNP-eGFP mRNA as a negative control was similarly administered three times, only a small amount of IFN-γ production was observed against any of them, which shows that the above IFN-γ production was specifically induced by the administration of LNP-hROBO1 mRNA.

[0431] like Fig.42As shown, in the spleen cells of HLA-A*24:02 Tg mice administered LNP-hROBO1 mRNA three times, IFN-γ-producing T cells were induced against a cocktail of hROBO1 short-chain peptides that produced peptide-specific IFN-γ in peptide immunization of HLA-A*24:02 Tg mice, a cocktail of hROBO1 long-chain peptides that produced peptide-specific IFN-γ in peptide immunization of HLA-A*24:02 Tg mice, MC38 cancer cells expressing HLA-A*24:02 HHD and hROBO1, and BMDCs transfected with hROBO1 mRNA. Compared with the IFN-γ production against the cancer cell MC38 A24HHD mimic introduced with HLA-A2402HHD and empty vector, the IFN-γ production against the cancer cell MC38 A24HHDhROBO1 expressing HLA-A*24:02HHD and hROBO1 in both im and id was significantly higher, which shows that T cells that specifically recognize and attack the cancer cells expressing hROBO1 were induced. In HLA-A2402 Tg mice to which LNP-eGFP mRNA as a negative control was similarly administered three times, only a small amount of IFN-γ production was observed against any of them, which shows that the above IFN-γ production was specifically induced by the administration of LNP-hROBO1 mRNA.

[0432] Example 7: Development of universal cancer antigen mRNA vaccine

[0433] Fig.43 The administration method of LNP-hCLDN1 mRNA and LNP-hEPHB4 mRNA is shown as three times of administration.

[0434] HLA-A*02:01 Tg mice and HLA-A*24:02 Tg mice were intradermally administered (id) at the base of the tail with 10 μg of LNP-hCLDN1 mRNA, LNP-hEPHB4 mRNA or LNP-eGFP mRNA three times on day 0, day 7 and day 14. The mice were dissected on day 21 and the production of antigen-specific IFN-γ was evaluated by ELISPOT method.

[0435] Fig.44 and Fig.45 Display the above results.

[0436] like Fig.44As shown, in the spleen cells of HLA-A*02:01Tg mice administered LNP-hCLDN1 mRNA or LNP-hEPHB4 mRNA three times, IFN-γ-producing T cells were induced against a cocktail of short-chain peptides of hCLDN1 or hEPHB4 that produced peptide-specific FN-γ upon peptide immunization of HLA-A*02:01 Tg mice, a cocktail of long-chain peptides of hCLDN1 or hEPHB4 that produced peptide-specific IFN-γ upon peptide immunization of HLA-A*02:01 Tg mice, MC38 cancer cells expressing HLA-A*02:01HHD and hCLDN1, MCA205 cancer cells expressing HLA-A*02:01HHD and hEPHB4, and BMDCs transfected with hCLDN1 mRNA or hEPHB4 mRNA. Compared with IFN-γ production against MC38A02HHD mimics or MCA205A02HHD mimics introduced with HLA-A*02:01HHD and an empty vector, IFN-γ production against MC38 A02HHD hCLDN1 expressing HLA-A*02:01HHD and hCLDN1 or MCA205A02HHD hEPHB4 expressing HLA-A*02:01HHD and hEPHB4 was significantly higher, indicating that T cells that specifically recognize and attack cancer cells expressing hCLDN1 or hEPHB4 were induced. In HLA-A*02:01 Tg mice to which LNP-eGFP mRNA as a negative control was similarly administered three times, only a small amount of IFN-γ production was observed against either of them, indicating that the above IFN-γ production was specifically induced by administration of LNP-hCLDN1 mRNA or LNP-hEPHB4 mRNA.

[0437] like Fig.45As shown, in the spleen cells of HLA-A*24:02Tg mice administered LNP-hCLDN1 mRNA or LNP-hEPHB4 mRNA three times, IFN-γ-producing T cells were induced against a cocktail of short-chain peptides of hCLDN1 or hEPHB4 that produced peptide-specific IFN-γ upon peptide immunization of HLA-A*24:02Tg mice, a cocktail of long-chain peptides of hCLDN1 or hEPHB4 that produced peptide-specific IFN-γ upon peptide immunization of HLA-A*24:02 Tg mice, MC38 cancer cells expressing HLA-A*24:02HHD and hCLDN1, MCA205 cancer cells expressing HLA-A*24:02HHD and hEPHB4, and BMDCs transfected with hCLDN1 mRNA or hEPHB4 mRNA. Compared with the IFN-γ production against the cancer cells MC38 A24HHD hCLDN1 expressing HLA-A*24:02HHD and hCLDN1 or the cancer cells MCA205 A24HHD hEPHB4 expressing HLA-A*24:02HHD and hEPHB4, the IFN-γ production against the cancer cells MC38 A24HHD hCLDN1 expressing HLA-A*24:02HHD and hEPHB4 was higher than that against the cancer cells MC38 A24HHD mimic or MCA205 A24HHD mimic introduced with HLA-A*24:02HHD and an empty vector, indicating that T cells that specifically recognize and attack cancer cells expressing hCLDN1 or hEPHB4 were induced. The production of IFN-γ against the MC38 A24HHD mimic or MCA205 A24HHD mimic is considered to be due to the presentation of the peptides of mouse CLDN1 or EPHB4 on HLA-A*24:02HHD. In HLA-A*24:02 Tg mice to which LNP-eGFP mRNA as a negative control was similarly administered three times, only a small amount of IFN-γ production was observed for any of them, indicating that the production of IFN-γ was specifically induced by administration of LNP-hCLDN1 mRNA or LNP-hEPHB4 mRNA.

[0438] Example 8: Development of universal cancer antigen mRNA vaccine

[0439] Fig.46 The administration method of LNP-hTGFBI mRNA and LNP-hSPARC mRNA is shown as three administrations.

[0440] HLA-A*02:01 Tg mice and HLA-A*24:02 Tg mice were intradermally administered (id) at the base of the tail with 10 μg of LNP-hTGFBI mRNA, LNP-hSPARC mRNA or LNP-eGFP mRNA three times on day 0, day 7 and day 14. The mice were dissected on day 21 and the production of antigen-specific IFN-γ was evaluated by the ELISPOT method.

[0441] Fig.47 and Fig.48 Display the above results.

[0442] like Fig.47 As shown, in the spleen cells of HLA-A*02:01Tg mice administered LNP-hTGFBI mRNA or LNP-hSPARC mRNA three times, IFN-γ-producing T cells were induced against a cocktail of short-chain peptides of hTGFBI or hSPARC that produced peptide-specific IFN-γ upon peptide immunization of HLA-A*02:01Tg mice, a cocktail of long-chain peptides of hTGFBI or hSPARC that produced peptide-specific IFN-γ upon peptide immunization of HLA-A*02:01 Tg mice, MCA205 cancer cells expressing HLA-A*02:01HHD and hTGFBI, or MCA205 cancer cells expressing HLA-A*02:01HHD and hSPARC, and BMDCs transfected with hTGFBI mRNA or hSPARC mRNA. Compared with the IFN-γ production against the cancer cells MCA205 A02HHD hTGFBI expressing HLA-A*02:01HHD and the empty vector introduced, the IFN-γ production against the cancer cells MCA205 A02HHD hTGFBI expressing HLA-A*02:01HHD and the empty vector was significantly higher, indicating that T cells that specifically recognize and attack the cancer cells expressing hTGFBI or hSPARC were induced. In HLA-A*02:01 Tg mice to which LNP-eGFP mRNA as a negative control was similarly administered three times, the IFN-γ production against either was low, indicating that the IFN-γ production was specifically induced by the administration of LNP-hTGFBI mRNA or LNP-hSPARC mRNA.

[0443] like Fig.48As shown, in the spleen cells of HLA-A*24:02Tg mice administered LNP-hTGFBI mRNA or LNP-hSPARC mRNA three times, IFN-γ-producing T cells were induced against a cocktail of short-chain peptides of hTGFBI or hSPARC that produced peptide-specific IFN-γ upon peptide immunization of HLA-A*24:02Tg mice, a cocktail of long-chain peptides of hTGFBI or hSPARC that produced peptide-specific IFN-γ upon peptide immunization of HLA-A*24:02 Tg mice, MCA205 cancer cells expressing HLA-A*24:02HHD and hTGFBI, or MCA205 cancer cells expressing HLA-A*24:02HHD and hSPARC, and BMDCs transfected with hTGFBI mRNA or hSPARC mRNA. Compared with the case of the MCA205 A02HHD mimics introduced with HLA-A*02:01HHD and empty vector, the hTGFBI of the cancer cells MCA205 A02HHD expressing HLA-A*02:01HHD and hTGFBI was significantly higher, which shows that T cells that specifically recognize and attack the cancer cells expressing hTGFBI were induced. The IFN-γ production against the MCA205A24HHD mimics in the LNP-hSPARC mRNA-administered mice is believed to be due to the presentation of the mouse SPARC peptide on HLA-A*24:02HHD. In the HLA-A*02:01 Tg mice to which LNP-eGFP mRNA as a negative control was similarly administered three times, the production of IFN-γ against either was low, which shows that the production of the above IFN-γ was specifically induced by the administration of LNP-hTGFBI mRNA or LNP-hSPARC mRNA.

[0444] Example 9: Development of universal cancer antigen mRNA vaccine

[0445] Fig.49 The administration method of LNP-hAFP mRNA and LNP-hHSP105α mRNA is shown as three times of administration.

[0446] HLA-A*02:01 Tg mice and HLA-A*24:02 Tg mice were intradermally administered (id) at the base of the tail with 10 μg of LNP-hAFP mRNA, LNP-hHSP105α mRNA or LNP-eGFP mRNA administered three times on day 0, day 7 and day 14. The mice were dissected on day 21 and the production of antigen-specific IFN-γ was evaluated by the ELISPOT method.

[0447] Fig.50 and Fig.51 Display the above results.

[0448] like Fig.50 As shown, in the spleen cells of HLA-A*02:01 Tg mice administered LNP-hAFP mRNA or LNP-hHSP105α mRNA three times, IFN-γ-producing T cells were induced against cancer cells MC38 expressing HLA-A*02:01HHD and hAFP or cancer cells MC38 expressing HLA-A*02:01HHD and hHSP105α, and BMDCs transfected with hAFP mRNA or hHSP105α mRNA. Compared with the IFN-γ production against the cancer cell MC38 A02HHD mimic introduced with HLA-A*02:01HHD and an empty vector, the IFN-γ production against the cancer cells MC38 A02HHD hAFP expressing HLA-A*02:01HHD and hAFP or the cancer cells MC38 A02HHD hHSP105α expressing HLA-A*02:01HHD and hHSP105α was significantly higher, indicating that T cells that specifically recognize and attack cancer cells expressing hAFP or hHSP105α were induced. In HLA-A*02:01 Tg mice to which LNP-eGFP mRNA as a negative control was similarly administered three times, the production of IFN-γ against MC38 was high in one individual, while the production of IFN-γ against BMDCs transfected with the antigen mRNA was low. It is believed that the production of IFN-γ against BMDCs transfected with the antigen mRNA was specifically induced by the administration of LNP-hAFP mRNA or LNP-hHSP105α mRNA.

[0449] like Fig.51As shown, in the spleen cells of HLA-A*24:02 Tg mice to which LNP-hAFP mRNA or LNP-hHSP105α mRNA was administered three times, IFN-γ-producing T cells were induced against cancer cells MC38 expressing HLA-A*24:02HHD and hAFP, or cancer cells MC38 expressing HLA-A*24:02HHD and hHSP105α, and BMDCs transfected with hAFP mRNA or hHSP105α mRNA. The IFN-γ production against cancer cells MC38 A24HHD hAFP expressing HLA-A*24:02HHD and hAFP was significantly higher than that against cancer cells MC38 A24HHD hAFP introduced with HLA-A*24:02HHD and an empty vector, indicating that T cells that specifically recognize and attack cancer cells expressing hAFP were induced. The IFN-γ production against the MC38 A24HHD mimic was observed to be similar to the IFN-γ production against the cancer cells MC38 A24HHD hHSP105α expressing HLA-A*24:02HHD and hHSP105α, and it is considered that this is because the peptide of mouse HSP105α is presented on HLA-A*24:02HHD. In HLA-A*24:02 Tg mice to which LNP-eGFP mRNA as a negative control was similarly administered three times, the production of IFN-γ against either was low, indicating that the above IFN-γ production was specifically induced by the administration of LNP-hAFP mRNA or LNP-hHSP105α mRNA.

[0450] Example 10: Development of universal cancer antigen mRNA vaccine

[0451] Fig.52 The administration method of LNP-(hCLDN1+hEPHB4) mRNA is shown as three administrations.

[0452] HLA-A*02:01 Tg mice and HLA-A*24:02 Tg mice were intradermally administered (id) at the base of the tail with 10 μg or 20 μg of LNP-(hCLDN1+hEPHB4) mRNA or 20 μg of LNP-luciferase mRNA administered three times on day 0, day 7, and day 14. The mice were dissected on day 21 and the production of antigen-specific IFN-γ was evaluated by the ELISPOT method.

[0453] Figure 53 to Figure 56 Display the above results.

[0454] like Fig.53 and Fig.54As shown, in the spleen cells and inguinal lymph nodes of HLA-A*02:01 Tg mice to which LNP-(hCLDN1+hEPHB4) mRNA was administered three times, T cells producing IFN-γ were induced against MCA205 cancer cells expressing HLA-A*02:01HHD and hCLDN1 or MCA205 cancer cells expressing HLA-A*02:01HHD and hEPHB4, and BMDCs transfected with hCLDN1 mRNA or hEPHB4 mRNA. The IFN-γ production against MCA205A02HHD hEPHB4 cancer cells expressing HLA-A*02:01HHD and hEPHB4 was significantly higher than that against MCA205 A02HHD mimics into which HLA-A*02:01HHD and an empty vector were introduced, indicating that T cells that specifically recognize and attack cancer cells expressing hEPHB4 were induced. The IFN-γ production against the MCA205 A02HHD mimic was observed to be similar to the IFN-γ production against the cancer cell MCA205 A02HHD hCLDN1 expressing HLA-A*02:01HHD and hCLDN1, and it is considered that this is because the peptides of mouse CLDN1 or EPHB4 are presented on HLA-A*02:01HHD. In HLA-A*02:01 Tg mice to which LNP-luciferase mRNA as a negative control was similarly administered three times, the IFN-γ production against either was low, indicating that the above IFN-γ production was specifically induced by the administration of LNP-(hCLDN1+hEPHB4) mRNA.

[0455] like Fig.55 and Fig.56As shown, in the spleen cells and inguinal lymph nodes of HLA-A*24:02 Tg mice to which LNP-(hCLDN1+hEPHB4) mRNA was administered three times, T cells producing IFN-γ were induced against MCA205 cancer cells expressing HLA-A*24:02HHD and hCLDN1 or MCA205 cancer cells expressing HLA-A*24:02HHD and hEPHB4, and BMDCs transfected with hCLDN1 mRNA or hEPHB4 mRNA. The IFN-γ production against MCA205A24HHDhEPHB4 cancer cells expressing HLA-A*24:02HHD and hEPHB4 was significantly higher than that against MCA205 A24HHDmock cancer cells introduced with HLA-A*24:02HHD and an empty vector, indicating that T cells that specifically recognize and attack cancer cells expressing hEPHB4 were induced. The IFN-γ production against the MCA205 A02HHD mimic was observed to be similar to the IFN-γ production against the cancer cell MCA205 A24HHDhCLDN1 expressing HLA-A*24:02HHD and hCLDN1, and it is considered that this is because the peptides from mouse CLDN1 or EPHB4 are presented on HLA-A*24:02HHD. In HLA-A*02:01 Tg mice to which LNP-luciferase mRNA as a negative control was similarly administered three times, the IFN-γ production against either was low, indicating that the above IFN-γ production was specifically induced by the administration of LNP-(hCLDN1+hEPHB4) mRNA.

[0456]

[0457]

[0458]

[0459]

[0460]

[0461]

[0462]

[0463]

[0464]

[0465]

[0466]

[0467]

[0468]

[0469]

[0470]

[0471]

[0472]

[0473]

[0474]

[0475]

[0476]

[0477]

[0478]

[0479]

[0480]

[0481]

[0482]

[0483] Example 11: Development of TCR-T cell therapy and cocktail TCR-T cell therapy targeting peptides derived from universal cancer antigens

[0484] <Method>

[0485] Fig.57 A method for selecting high-affinity T cell clones by inhibiting the binding of CD8 molecules to MHC is shown.

[0486] Fig.57 A shows the interaction between Dextramer and TCR molecules in the presence of anti-CD8 antibodies. When the MHC molecule / cancer antigen peptide complex bound to Dextramer binds to the TCR molecule, the anti-CD8 antibody is added in advance to prevent the non-specific binding of the peptide to the CD8 molecule and the MHC molecule, so that only the T cell clones with the TCR molecule with stronger binding force are stained.

[0487] Fig.57 B shows a comparison of Dextramer staining based on whether CD8 binding is inhibited. T cell clones A and B show almost equal Dextramer staining when CD8 binding is not inhibited, but clone A shows stronger staining than clone B when CD8 binding is inhibited.

[0488] Fig.57 C is to select high affinity clones by inhibiting CD8 binding. Using the above principle, T cells that recognize cancer antigen peptides can be purified by Dextramer staining, thereby establishing T cell clones with TCR genes that have higher affinity for cancer antigen peptides.

[0489] <Results>

[0490] It is known that in the binding of MHC / peptide complexes to TCR, CD8 molecules on T cells play an auxiliary role and enhance their binding. In this method, anti-CD8 antibodies are used to block the binding of CD8 molecules to MHC molecules before Dextramer staining, thereby reducing nonspecific staining that depends on the binding of CD8 molecules, and successfully detecting clones with TCR genes that have strong binding ability when alone. In fact, when Dextramer staining was performed on two T cell clones that reacted with GPC3 peptide without blocking CD8, the staining intensity of clone A and clone B was the same, but a clear difference in staining was observed under the condition of blocking CD8. In this subject, based on the same principle, in order to select T cells expressing peptide-reactive TCR genes with anti-affinity from the patient's peripheral blood, a Dextramer-based staining method was used in combination with CD8 blocking.

[0491] Fig.58 The present invention shows the establishment of peptide-specific CTL clones from the peripheral blood of patients who received the GPC3 peptide vaccine. Dextramer-positive T cells were single-sorted from the peripheral blood of patients who received the cancer peptide vaccine using a GPC3-derived peptide under CD8 binding-blocking conditions and cultured on allogeneic feeder cells to establish T cell clones.

[0492] Fig.60 Figures A and B show the evaluation of TCR binding capacity by Dextramer staining of HLA-A2 restricted GPC3 specific T cell clones. A. In the absence of CD8 binding blocker. B. Comparison study in the presence of CD8 binding blocker.

[0493] Fig.60C and D show the evaluation of TCR binding capacity by Dextramer staining of HLA-A24 restricted GPC3 specific T cell clones. C. In the absence of CD8 binding blocker. D. Comparison and study in the presence of CD8 binding blocker.

[0494] Fig.60 E shows the evaluation of the antigen-specific IFN-γ production ability of HLA-A24-restricted GPC3-specific T cell clones. The established T cell clones were co-cultured with GPC3-positive HepG2 liver cancer cell lines, GPC3-negative SK-Hep cell lines (SK-vec) and GPC3 forced expression cell lines (SK-GPC3) for 20 hours, and the production of IFN-γ was evaluated by the ELISPOT method. Cells obtained by exogenously binding the peptide from GPC3 (GPC3 A24 prp.) used in the peptide vaccine to the TAP-deficient cell line, namely T2-A24 cells, were used as positive controls, and cells bound with peptides from HIV were used as negative controls to evaluate antigen specificity.

[0495] <Results>

[0496] In order to isolate GPC3 peptide-specific TCR genes, peripheral blood lymphocytes provided by HLA-A2 haplotype patients and HLA-A24 haplotype patients who received GPC3 peptide vaccines were cultured with peptide stimulation, and anti-affinity T cells were selected by a Dextramer staining method based on CD8 obstruction to establish T cell clones. When the Dextramer binding of each clone established was compared under the presence or absence of CD8 obstruction, although the staining intensity of the A2-restricted clone was reduced as a whole, the staining was maintained in any clone. In contrast, in the A24-restricted clone, as seen in the e1D8 clone, clones with significantly reduced staining were confirmed. Based on the above results, it is believed that by selecting T cell clones that are not affected by the presence or absence of CD8 obstruction and show higher Dextramer staining, TCR genes with high affinity can be efficiently identified. Next, the IFN-γ production ability of the established HLA-A24-restricted T cell clones was evaluated. All T cell clones showed strong IFN-γ production when co-cultured with T2-A24 cells to which peptides were exogenously bound. On the other hand, when a forced expression cell line (SK-GPC3) that endogenously expresses GPC3 was co-cultured with a HepG2 cell line, differences in reactivity were clearly seen in each clone. In addition, its IFN-γ production ability may not be consistent with Dextramer staining. Although the e1D8 described above has low Dextramer staining, it shows strong IFN-γ production for endogenous GPC3-expressing cells. Although the eC7 clone has high Dextramer staining intensity, the result is that the IFN-γ production ability produced by co-culture with endogenous GPC3-expressing cell lines is low. It is believed that this is because the IFN-γ production ability of T cells is determined not only by the binding force of TCR, but also by the differences in various functions within the cells of each clone. When selecting TCR genes that recognize endogenous antigens and can eliminate tumors, it is necessary to construct TCR-T cells for treatment and evaluate their functions again. The table shows the amino acid sequence and gene sequence of the TCR gene isolated from each clone.

[0497] [Table 2]

[0498] Table 2: Amino acid sequences of HLA-A2 restricted T cell receptors recognizing a peptide from GPC3 (FVGEFFTDV)

[0499]

[0500]

[0501] [Table 3]

[0502] Table 3: Gene sequences of HLA-A2 restricted T cell receptors recognizing peptides from GPC3 (FVGEFFTDV)

[0503]

[0504]

[0505] [Table 4]

[0506] Table 4: Amino acid sequences of HLA-A24 restricted T cell receptors recognizing a peptide (EYILSLEEL) from GPC3

[0507]

[0508]

[0509]

[0510] [Table 5]

[0511] Table 5: Gene sequences of HLA-A24 restricted T cell receptors recognizing peptides from GPC3 (EYILSLEEL)

[0512]

[0513]

[0514]

[0515]

[0516] Fig.59 The peptide-specific CTL clones were established from the peripheral blood of patients who received the FOXM1 peptide vaccine. Dextramer-positive T cells were single-sorted from the peripheral blood of patients who received the cancer peptide vaccine using a peptide derived from FOXM1 under CD8 binding-blocking conditions and cultured on allogeneic feeder cells to establish T cell clones.

[0517] Fig.59 Figures A and B show the evaluation of TCR binding capacity by Dextramer staining of HLA-A24 restricted FOXM1 specific T cell clones. A. In the absence of CD8 binding blocker. B. Comparison study in the presence of CD8 binding blocker.

[0518] <Results>

[0519] Peripheral blood of patients who received FOXM1 peptide vaccine was stimulated and cultured in the presence of peptide, and reactive clones were established by HLA-A24 Dextramer staining. The binding ability of TCR of FOXM1-specific reactive T cell clones established by Dextramer staining was evaluated. Among the four clones, three clones showed good staining even under CD8 blocking conditions, but the staining of 21E7 clone was significantly reduced due to CD8 blocking. The table shows the amino acid sequence and gene sequence of the TCR gene isolated from each clone.

[0520] [Table 6]

[0521] Table 6: Amino acid sequences of HLA-A24 restricted T cell receptors recognizing a peptide (IYTWIEDHF) from FOXM1

[0522]

[0523] [Table 7]

[0524] Table 7: Gene sequences of HLA-A24 restricted T cell receptors recognizing peptides from FOXM1 (IYTWIEDHF)

[0525]

[0526]

[0527] Fig.60 The results show that peptide-specific CTL clones were established from the peripheral blood of patients who received the HSP105 peptide vaccine. The HSP105-HLA-A2 Dextramer staining was insufficient and could not be used to detect T cells that showed binding. Therefore, the peripheral blood of patients who received a cancer peptide vaccine using a peptide derived from HSP105 was stimulated in the presence of antigen, and CD8 T cells that showed degranulation reactions were detected by CD107a measurement. CD107a-positive CD8 T cells were single-sorted and cultured on allogeneic feeder cells to establish T cell clones.

[0528] Fig.60 A shows the responsiveness of HLA-A2 restricted HSP105 specific T cell clones to produce IFN-γ by ELISPOT method. Each established T cell clone was co-cultured with target cells (T2 cells) exogenously bound to peptides from HSP105 for 20 hours to evaluate the production of IFN-γ dependent on antigen stimulation. Cells bound to peptides from HIV were used as negative controls to evaluate antigen specificity.

[0529] Fig.60B and C show the cytotoxic activity of HLA-A2 restricted HSP105 specific T cell clones. Target cells were labeled with a fluorescent dye (calcein) and co-cultured with T cell clones for 4 hours. The cytotoxic activity on target cells was evaluated using the decay of fluorescence as an indicator. B. T2 cells exogenously bound to peptides were used as targets. Cells obtained by exogenously binding the peptide from HSP105 (HSPA-7) used in peptide vaccines to TAP-deficient cell lines, i.e. T2 cells, were used as positive controls, and cells bound to peptides from HIV (HIVA2) were used as negative controls to evaluate antigen specificity. C. Cancer cell lines endogenously expressing HSP105 were used as targets to evaluate cytotoxic activity. Caski cells and SiHa-A2 cells were used as HSP105 positive cell lines, and SW620 cell lines and HepG2 cell lines were used as HSP105 negative cell lines. The cytotoxic activity of the 11B9 clone was not evaluated.

[0530] <Results>

[0531] T cell clones with HSP105 peptide reactivity were established from patients with HLA-A2 haplotype who received HSP105 peptide vaccine. HLA-A2-HSP105 Dextramer staining is insufficient, and it is difficult to detect reactive clones by Dextramer staining. Therefore, in this study, peripheral blood mononuclear cells of patients that were previously stimulated with peptides were co-cultured with K562 cell lines expressing A2 in the presence of peptides, and T cells that recognized antigens and showed degranulation (in the case of showing degranulation, it is usually based on a degranulation evaluation method that utilizes CD107a molecules present in cells exposed on the cell surface) were collected by sorting and separation, and reactive T cell clones were established. When evaluating the antigen-specific IFN-γ production ability of the five established T cell clones, IFN-γ production was observed in all clones by co-culture with HLA-A2 T2 cells in the presence of peptides, and 7B9, 10G7, and 10C9 showed strong IFN-γ production ability. The 11B9 clone has significantly lower reactivity than other clones, so 7B9, 8D4, 10G7 and 10C9 were then subjected to cytotoxicity tests. These 4 clones all showed toxic activity to T2 cells that were exogenously bound to peptides, and in particular, 7B9 and 8D4 clones showed strong toxic activity. The cytotoxic activity of these clones that endogenously express HSP105 on cancer cell lines was further evaluated, and the result was that 7B9, 10G7 and 10C9 clones all showed significant cytotoxic activity on CasKi cell lines. Regarding the toxic activity to SiHaA2 and SW620 cell lines, no significant cytotoxic activity was observed compared to the HSP105 negative HepG2 cell line. The 8D4 and 11B9 clones were not evaluated and will be evaluated later. The amino acid sequence isolated from each clone and the gene sequence of the TCR gene are shown in the table.

[0532] [Table 8]

[0533] Table 8: Amino acid sequences of HLA-A2 restricted T cell receptors recognizing peptides from HSP105 (RLMNDMTAV or KLMSSNSTDL)

[0534]

[0535]

[0536] [Table 9]

[0537] Table 9: Gene sequences of HLA-A2 restricted T cell receptors recognizing peptides from HSP105 (RLMNDMTAV or KLMSSNSTDL)

[0538]

[0539]

[0540] Fig.61 The identification of peptide-reactive TCR genes using mice into which human HLA genes were introduced was shown.

[0541] Fig.61 Figures A and B show the evaluation of peptide vaccines for mice into which human HLA genes were introduced and the peptide reactivity in the spleen. 50 ug of a peptide derived from a candidate cancer antigen was mixed with polyI:CLC as an adjuvant and administered intradermally to the tail base of the mice three times a week. Afterwards, the spleen was removed, the peptide used for the vaccine was added in vitro, and the cells were cultured for 20 hours, and the production of antigen-specific IFN-γ was detected by the ELISPOT method.

[0542] Fig.61C shows the separation of peptide-reactive TCR genes and the evaluation of antigen reactivity by reporter gene assay. The spleen of mice vaccinated was cultured for 4 hours in the presence of antigen peptides and anti-CD107a antibodies, and single cell sorting was performed to cells (CD107a positive cells) that degranulated, and recovered to 96 well plates. Reverse transcription and TCR-specific primers were used to amplify TCR genes from recovered cells, and the TAP fragments of the respective TCR genes inserted were constructed. The TAP fragments of each constructed TCR gene and the NFAT transcription factor responsive luciferase expression vector were introduced into Jurkat cells. After 16 hours of cultivation, the Jurkat cells introduced with genes and the COS-7 cells expressing HLA-A24 exogenously combined with antigen peptides were co-cultured, and the expression of the stimulation-dependent luciferase from the reconstructed TCR was measured by luciferase assay. Figure is taken from the paper by Hamana et al. from Toyama University (Biochem Biophys Res Commun. 2016, 10; 474(4): 709-714.).

[0543] Fig.61 D shows the reactivity evaluation of EPHB4 peptide-reactive TCR isolated from mice into which human HLA genes were introduced. Using the produced TCR-expressing Jurnakt cells, the reactivity to the peptide from EPHB4 is shown in the form of luciferase activity (RLU). A known NY-ESO-1 peptide-specific TCR (1G4 TCR) was used as a positive control. The co-culture with Cos-7 cells bound to the EPHB4 peptide is shown in white (□), and the co-culture with Cos-7 cells bound to the NY-ESO-1 peptide is shown in black (■).

[0544] <Results>

[0545] So far, this topic has shown that epitope peptides can be identified with high throughput for novel cancer antigens by using mice into which human HLA genes have been introduced. Similarly, in order to study whether TCR genes that recognize novel cancer antigens can be identified by using mice into which human HLA genes have been introduced, HLA-A24Tg mice were repeatedly immunized with peptides derived from EPHB4 and adjuvants, peptide-responsive CD8T cells were induced, and then EPHB4-reactive TCR genes were isolated from the spleen by single cell sorting. Splenocytes of vaccinated mice were restimulated in vitro in the presence of peptides, and T cells that were degranulated and CD107a-positive were recovered by sorting. So far, T cell clones have been established by re-culturing in the presence of feeder cells, but stable proliferation takes time and may not be established from all sorted cells. Therefore, a high-throughput method for detecting and evaluating the function of TCR genes from single cells developed by Hamana et al. at the University of Toyama was introduced. In the method of Hamana et al., instead of re-culturing the sorted cells, the TCR gene is amplified and separated by direct PCR, and the TCR gene separated by Gibson assembly is connected to a linear DNA containing a gene expression promoter to construct a TCR gene expression vector, and the constructed TCR gene expression vector is introduced into the Jurkat cell line from T cell lymphoma to reconstruct the TCR molecule and evaluate its reactivity. Because it is not cultured, the sorted cells will not be lost in a short period of time, and evaluation can be performed. Among the 39 CD107a-positive T cells separated from the spleen cells of vaccinated mice by sorting, the TCR gene of 21 cells can be identified, and the TCR gene of 8 T cells with the same TCR gene omitted is reconstructed to evaluate its antigen reactivity. In the Jurkat cell line with reconstructed TCR, a reporter gene that induces the expression of the luciferase gene by the transcription factor NFAT activated downstream of the TCR signal in advance is inserted. When the reconstructed TCR molecule recognizes the antigen, the luminescence is measured by the luciferase reaction dependent on the intensity of its activation signal, and the reactivity can be quantitatively evaluated. When evaluating the reactivity of Jurkat cells reconstructing TCR in the presence of peptides from EPHB4 or NY-ESO-1, reactivity to EPHB4 peptide was observed in 7 of 8 TCR genes, and the strongest antigen-specific reactivity was confirmed in the TCR_07 gene. The table shows the amino acid sequence isolated from each clone and the gene sequence of the TCR gene.

[0546] [Table 10]

[0547] Table 10: Amino acid sequences of HLA-A2 restricted T cell receptors recognizing peptides from EPHB4 (RLNDGQFTV)

[0548]

[0549]

[0550] [Table 11]

[0551] Table 11: Gene sequences of HLA-A2 restricted T cell receptors recognizing peptides from EPHB4 (RLNDGQFTV)

[0552]

[0553]

[0554] Fig.62 The results show that TCR-T cells that recognize a universal cancer antigen (FOXM1) were produced by electroporation.

[0555] Fig.62 A shows that TCR-T cells were made by electroporation. After culturing peripheral blood mononuclear cells in the presence of anti-CD3 antibodies and IL2, specific sgRNA and Cas9 protein were introduced into the constant region of TCR by electroporation to produce T cells with endogenous TCR deletion (TCR DKO donor cells). After cultivation, the mRNA of the single-chain TCR gene obtained by connecting the TCRα chain gene and the TCRβ chain gene with the P2A sequence was then introduced by electroporation to produce TCR-T cells.

[0556] Fig.62 B shows the expression of TCR molecules on the cell surface of the prepared TCR-T cells. The prepared TCR-T cells were stained with anti-CD3 antibodies and anti-TCRαβ antibodies to evaluate the expression of TCR molecules on the cell surface. The histograms show the expression of CD3 and TCRαβ molecules on the cell surface (red: TCR-T cells, blue: donor cells).

[0557] Fig.62 C shows that the prepared TCR-T cells produce IFN-γ by antigen stimulation. The prepared TCR-T cells were co-cultured with T2-A24 cells to which cancer antigen peptide (FOXM1 pep.) was exogenously bound for 20 hours, and the production of IFN-γ was detected by the ELISPOT method.

[0558] <Results>

[0559] TCR-T cells with reconstructed cancer antigen reactive TCR genes identified were constructed. In this subject, an artificial single-chain TCR gene obtained by connecting the α chain and β chain of TCR via the P2A sequence was synthesized, and the TCR mRNA produced by in vitro transcription of the synthesized artificial gene was introduced into CD8 positive T cells from peripheral blood cultured with anti-CD3 antibody stimulation by electroporation. In addition, the endogenous TCR molecules were knocked out in advance using the CRISPR-Cas9 system, so that only the exogenously introduced and reconstructed TCR molecules were efficiently expressed on the cell surface. When studying the FOXM1-specific TCR genes identified so far, it was confirmed that any of the TCRs in the F5, (21)1D5 and 1F5 clones were highly expressed on the cell surface. In addition, when the antigen reactivity of the produced TCR-T cells was evaluated using the production of IFN-γ as an indicator, as expected, all TCR-T cells showed strong reactivity to peptides from FOXM1. Although the TCR of the 1F5 clone was fully expressed, its reactivity was lower than that of the other two clones, while (21)1D5 showed the highest reactivity, indicating that among these FOXM1-reactive TCRs, the TCR gene of the (21)1D5 clone is the most promising. In the future, this method will be used to evaluate the antigen specificity of the acquired TCR gene or its effectiveness in TCR-T cell therapy.

[0560] Fig.63 A development plan for cocktail TCR-T cells that demonstrates the ability to overcome the diversity of cancer. A library of epitope peptides of 10 cancer antigens was constructed, and HLA-A2 or A24-restricted TCR genes that specifically recognize these peptides were isolated from the peripheral blood of cancer patients who received cancer vaccines using these peptides or from human HLA gene-introduced mice that were repeatedly vaccinated with the peptides, to construct a cancer antigen-specific TCR library. After screening the expression of each cancer antigen and testing the reactivity to the peptide vaccine, specific TCR genes were selected for each of the multiple cancer antigen peptides to produce TCR-T cells. The multiple cancer antigen-specific TCR-T cells produced were mixed and administered in the form of a "cocktail TCR-T cell" preparation, thereby establishing an effective T cell therapy for the diversity of cancer.

[0561] <Results>

[0562] By identifying epitope peptides of 10 cancer antigens and constructing a cancer antigen peptide library, not only does it make ready-made cancer peptide vaccines possible, but by identifying potent TCRs that recognize these peptides and making a cancer antigen-specific TCR library, it is also possible to achieve "cocktail TCR-T cell" therapy, which breaks the immune escape caused by cancer diversity targeting multiple cancer antigens. Example 12: Development of CAR-T cell therapy targeting membrane protein universal cancer antigens and cocktail CAR-T cell therapy

[0563] <Method>

[0564] Fig.64 (a) In the in vitro cytotoxicity evaluation of CAR-T cells, rhabdomyosarcoma cell line Rh30 and intrahepatic cholangiocarcinoma cell line SSP25 were used as target cells. Calcein-AM (Dojindo) was added to the cell line at a concentration of 10 μg / ml, and the cells were left to stand at 37°C for 30 minutes for staining. EPHB4 CAR-T cells as effector cells and stained target cells were co-cultured in a 96-well half-area, flat-bottom plate (Corning) at a ratio of E / T=1, E / T=3, E / T=10 or E / T=30 for 4-6 hours, and the fluorescence values ​​before and after culture were measured using Terascan VPC2 (Minerva Tech). The natural release of calcein is the co-culture of inactivated PBMCs (heated on a heat block set at 80°C for more than 15 minutes) to replace effector cells. Similarly, the maximum release was obtained by adding IGEPAL CA-630 (MP Biomedicals) (Nonidet P-40 equivalent) to inactivated PBMC at a concentration of 6%. The cytotoxicity rate was calculated by the following calculation method.

[0565] Cytotoxicity rate (%) = free amount of experimental group - natural free amount of experimental group) / (maximum free amount of experimental group - natural free amount of experimental group) × 100

[0566] (b) GPC3 CAR-T cells use the produced GC33 CARγδT cells as effector cells. The cells use a retroviral vector expressing CAR, which comes from the specific antibody GC33 against GPC3 developed by the Chugai Roche Group. CAR is introduced into healthy human PBMCs on the 6th day of zoledronic acid stimulation culture, and a clone of Vg9-positive γδT cells expressing CAR is produced by single sorting of positive cells. The target cells use the cell line SK / hGPC3 that forcibly expresses the full-length sequence gene of human GPC3 (hGPC3) in the liver cancer cell line SK-Hep-1 that does not express GPC3, and the cell line (SK / vec) transformed with an empty vector is used as a negative control. Calcein staining of target cells and calculation of toxicity rate are performed in the same manner as above.

[0567] (c) The cells were stained and measured in the same manner as (a) and (b). PDX maintained / swollen in immunodeficient mice were removed as target cells, the tissue was cut as much as possible with scissors, and then dispersed using a human tumor dissociation kit (Miltenyi Biotec), and then used after removing red blood cells.

[0568] (d) PDX-derived cells prepared in the same manner as in (c) were co-cultured with EPHB4 CAR-T cells or mother cells activated by PHA stimulation in advance, and CD107a-positive cells among CD8-positive cells were analyzed by flow cytometry.

[0569] Fig.65 (a) The PDX model was created by transplanting each PDX adjusted to 5 mm square into SCID / Beige mice (colorectal cancer liver metastasis PDX) and NSG mice (head and neck cancer PDX model). The size of the tumor was measured by using a caliper to measure the long and short diameters and calculated as the area (b) or volume (d). (c) and (e) For immunohistological analysis, FFPE thin-section specimens were prepared as described above. In short, after collecting each organ, it was immersed in 4% PFA for 18-24 hours, and then the PFA was washed with PBS (-). After soaking in 70% ethanol for more than one night, it was embedded in paraffin to prepare paraffin blocks. The block was thinly sliced ​​to 4 μm and attached to a slide, and after drying, various stainings were performed. DAB staining used a polymer reagent suitable for the primary antibody (polymer reagent: EnVision+System-HRP labeled polymer anti-rabbit or EnVision+System-HRP labeled polymer anti-mouse) and a colorimetric reagent (liquid DAB+substrate colorimetric system). After contrast staining with hematoxylin, the specimens were made into virtual slides using NanoZoomer (Hamamatsu Photonics KK). Multiple fluorescent immunohistochemical staining was also performed according to the above method, using antibodies against each antigen EPHB4 (Cell Signaling Technology), human CD8 (Leica Biosystems), CD3 (abcam), Cleaved Caspase-3 (Cell Signaling Technology), polymer reagents (EnVision + System-HRP labeled polymer anti-rabbit or EnVision + System-HRP labeled polymer anti-mouse) as secondary antibodies, and fluorescent dyes Opal 520, 540, 570, 620, 650, 690 (AKOYA BIOSCIENCES), and observation was performed using Vectra3 (PerkinElmer).

[0570] <Results>

[0571] Fig.64 Shows in vitro cytotoxicity evaluation of CAR-T cells.

[0572] (a) Cytotoxicity of EPHB4 CAR-T cells against EPHB4-positive tumor cell lines. It was confirmed that the cytotoxicity of EPHB4-expressing rhabdomyosarcoma cell line (Rh30) and intrahepatic cholangiocarcinoma cell line (SSP25) increased in a manner dependent on the E:T ratio.

[0573] (b) Evaluation of GPC3 CAR-T cytotoxicity. For the cell line SK / hGPC3 that forcibly expresses the full-length human GPC3 gene (hGPC3), the cytotoxicity of GPC3 CAR-T cells increased depending on the E:T ratio.

[0574] (c) Cytotoxicity of EPHB4 CAR-T cells against EPHB4-positive PDX cells. Using EPHB4 CAR-T cells as effector cells and PDX cells as target cells, the cytotoxicity rates after 8 hours of reaction at various E:T ratios were compared. By increasing the E:T ratio, the cytotoxicity rate increased, and the strongest cytotoxicity rate was shown at E / T=10. The figure on the right shows an endoscopic photograph at E / T=10.

[0575] (d) Confirmation of the cytotoxic response of EPHB4 CAR-T cells to EPHB4-positive PDX cells. EPHB4 CAR-T cells and mother cells activated by PHA stimulation were co-cultured with EPHB4-positive PDX cells, and the exposure of CD107a molecules that recognize antigens and show degranulation on the cell surface was measured. CD107a exposure was observed only in EPHB4 CAR-T cells, so it was found that the antigen specifically induced cytotoxic activity.

[0576] Fig.65 Shows in vivo cytotoxicity evaluation of CAR-T cells.

[0577] (a) Model animals (colorectal cancer liver metastasis / head and neck cancer) were prepared by transplanting EPHB4-positive PDX. EPHB4 CAR-T cells were transplanted 7 days after transplantation of each model PDX, and additional transplantation was performed on the second day (colorectal cancer liver metastasis model) / third day (head and neck cancer model). The control group was transplanted with physiological saline.

[0578] (b) Changes in tumor size (area) of the colorectal cancer liver metastasis PDX model. The enlargement of the tumor was suppressed in the CAR-T cell transplantation group. (c) Confirmation of EPHB4 expression in the PDX region of the colorectal cancer liver metastasis PDX model. The tumor part (PDX) was collected 6 days after the CAR-T cells were transplanted, and then the expression of EPHB4 was confirmed by DAB staining in the FFPE thin-section sample. The disappearance of EPHB4 was observed in the CAR-T cell transplantation group. (d) Changes in tumor size (area) of the head and neck cancer PDX model. The enlargement of the tumor was suppressed in the CAR-T cell transplantation group. (e) Immunohistological analysis of the PDX region in the head and neck cancer PDX model. Except for the lower right figure, evaluation was performed by DAB staining and hematoxylin staining. hCD3 and hCD8 were still confirmed on the 15th day of the CAR-T cell transplantation group, suggesting the presence of CAR-T cells. In addition, the expression of EPHB4 disappeared in the transplanted group, and the expression of activated Caspase 3 (C-Caspase 3), a marker of apoptosis, was strongly confirmed compared with the control (upper figure). In the lower left figure, the expression of EPHB4 decreased in the area where hCD8 was confirmed, and the expression of C-Caspase3 was also confirmed in the lower right figure after multiple immunohistochemical staining (*).

[0579] Fig.66 Showing the development of cocktail CAR-T therapy.

[0580] Taking advantage of the fact that any one of the five membrane protein universal cancer antigens is expressed in most cancer tissues, we have created cocktail CAR-T cells expressing multiple CAR molecules, aiming to develop cocktail CAR-T therapy with the optimal combination or ratio. This will allow us to address issues such as cancer heterogeneity and differences between patients in multiple ways, effectively and efficiently inhibiting cancer growth or inducing T cell infiltration, etc.

[0581] Example 13: Analysis of the expression of membrane protein universal cancer antigens in various cancer cell lines / Development of a method for diagnosing cancer risk by detecting circulating cancer cells in the blood using antibodies against membrane protein universal cancer antigens

[0582] <Method>

[0583] Hepatocellular carcinoma cell lines (HepG2, Hep3B, HuH-7, PLC / PRF / 5, JHH-2, JHH-4, JHH-5, JHH-6, JHH-7), intrahepatic cholangiocarcinoma cell lines (RBE, SSP-25), colorectal cancer cell lines (HCT116, Lovo, colo201, HT29, WiDr, SW480, CaCO2), pancreatic cancer cell lines (Panc1, AsPC1, PK1, PK9), lung cancer cell lines (RERF-LC-AI, LK-2, II-18, A549, LU99, H1975, Lu-135, NCI-H446), breast cancer cell lines (MDA-MB-231, MCF7), cervical cancer cell lines (HeLa), ovarian cancer cell lines (KOC-7C, NOY1, NOY2, RMG-II, RMUG-S), renal cell carcinoma cell lines (Caki-1, ACHN, RCC7), malignant melanoma cell lines (A375ml, SKmel23), oral cancer cell lines (SAS, Ca9-22, HSC2, HSC3), gastric cancer cell lines (MKN74, MKN45, KATO-3), lymphoma cell lines (Raji, K562), and then Fc in FACS buffer (PBS + 2% FCS). The cells were treated with a PE-labeled anti-EpCAM antibody (Miltenyi, 100-fold dilution), a PE-labeled anti-cell surface vimentin (CSV) antibody (Abnova, clone: ​​84-1, 100-fold dilution), an APC-labeled anti-human GPC3 antibody (Sino Biological, clone: ​​024, 10-fold dilution), an AF647-labeled anti-human ROBO1 antibody (R&D, clone: ​​770502, 50-fold dilution), an AF647-labeled anti-human Claudin-1 antibody (R&D, clone: ​​421203, 25-fold dilution), and an AF647-labeled anti-human EphB4 antibody (Miltenyi, clone: ​​REA923, 50-fold dilution) for 20 minutes, respectively, and analyzed using FACS Canto (BD Biosciences).

[0584] <Results>

[0585] The above results can be seen in Fig.67 and Fig.68 .

[0586] In addition to EpCAM and CSV, the hepatocellular carcinoma cell line HepG2 also expresses all four membrane proteins, the universal cancer antigens GPC3, ROBO1, CLDN1, and EPHB4.

[0587] When 50 cell lines from various cancer types were stained with antibodies against these antigens, 7 cell lines that barely expressed EpCAM or CSV, which are existing CTC markers, expressed any one of the four membrane protein universal cancer antigens GPC3, ROBO1, CLDN1, and EPHB4 on their cell membranes. This suggests the possibility of capturing CTCs that have escaped from existing CTC markers by using these four membrane protein universal cancer antigens in combination.

[0588] <Method>

[0589] About 5 mL of peripheral blood was collected from a patient with hepatocellular carcinoma 3 days after tumor removal surgery using an EDTA-2K blood collection tube. Calcein-AM (DOJINDO) adjusted to 0.1 ug / mL with PBS was added to 1 / 2 volume of the hepatocellular carcinoma cell line HepG2 and the colorectal cancer cell line HCT116 suspended in PBS, and labeled at 37°C for 15 minutes. 1×10 3 Calcein-labeled HepG2 and HCT116. Add 3 times the volume of RBC lysis buffer (GBIOSCIENCES) to the peripheral blood with cancer cell lines added and the peripheral blood without adding, and hemolyze for 10 minutes at room temperature. Suspend the remaining cells in 1mL of MACS buffer (PBS+0.5% BSA+2mM EDTA), add 250uL of CD45Dynabeads and 250uL of CD15Dynabeads, stir at 4℃ for 1 hour, and remove CD45-positive cells and CD15-positive cells using a magnet. The remaining cells were stained with zombie-NIR diluted 1200-fold with PBS at room temperature for 15 minutes, treated with Fc block (BD Biosciences, 10-fold dilution) in FACS buffer for 20 minutes, and then stained with PerCP-labeled anti-CD45 antibody (Miltenyi, 50-fold dilution), PE-labeled anti-EpCAM antibody (Miltenyi, 100-fold dilution), PE-labeled anti-cell surface Vimentin (CSV) antibody (Abnova, 100-fold dilution), APC-labeled anti-human GPC3 antibody (Sino Biological, 10-fold dilution), AF647-labeled anti-human ROBO1 antibody (R&D, 50-fold dilution), AF647-labeled anti-human Claudin-1 antibody (R&D, 50-fold dilution), AF647-labeled anti-human EphB4 antibody (Miltenyi, 50-fold dilution), and Hoechst33342 (DOJINDO, 30 ug / mL) for 20 minutes, and analyzed using FACS Melody (BD Biosciences). Biosciences) for analysis.

[0590] <Results>

[0591] The above results can be seen in Fig.69 .

[0592] In multiple cases of hepatocellular carcinoma or colorectal cancer, CTC-like cells that did not express the existing CTC markers EpCAM and CSV but expressed any one of the four membrane protein universal cancer antigens GPC3, ROBO1, CLDN1, and EPHB4 on the cell membrane were detected in 5-10 mL of blood samples collected before or after resection surgery (on the 1st or 3rd day). This showed that the detection sensitivity of CTC-like cells was increased by adding GPC3, ROBO1, CLDN1, and EPHB4 in addition to the existing CTC markers.

Claims

1. A cancer vaccine comprising: (1) containing three or more universal cancer antigens selected from the group consisting of GPC3, ROBO1, EPHB4, CLDN1 and LAT1; (2) partial peptides of the above three or more universal cancer antigens, which have CTL inducibility; (3) dendritic cells stimulated with the above partial peptides; or (4) mRNA encoding the above-mentioned universal cancer antigen or the above-mentioned partial peptide.

2. The cancer vaccine according to claim 1, wherein The universal cancer antigen further includes one or more of AFP, TGFBI, SPARC, HSP105α, and FOXM1.

3. The cancer vaccine according to claim 1, wherein The above-mentioned universal cancer antigens include all of GPC3, ROBO1, EPHB4, CLDN1, and LAT1.

4. The cancer vaccine according to claim 1, wherein The above-mentioned universal cancer antigens include all of GPC3, ROBO1, EPHB4, CLDN1, LAT1, AFP, TGFBI, SPARC, HSP105α and FOXM1.

5. The cancer vaccine according to any one of claims 1 to 4, wherein The partial peptide is a peptide having an amino acid sequence described in any one of SEQ ID NOs: 1 to 80.

6. A cancer vaccine comprising: (1) comprising three or more universal cancer antigens selected from the group consisting of GPC3, ROBO1, EPHB4, CLDN1, LAT1, AFP, TGFBI, SPARC, HSP105α, and FOXM1; (2) partial peptides of the above three or more universal cancer antigens, which have CTL inducibility; (3) dendritic cells stimulated with the above partial peptides; or (4) mRNA encoding the above-mentioned universal cancer antigen or the above-mentioned partial peptide.

7. A peptide having an amino acid sequence as set forth in any one of SEQ ID NOs: 5, 7 to 10, 14 to 22, 24 to 38, 40, 42, 48, 49, 52 to 80.

8. A CAR-T cell therapy comprising a mixture of T cells having a chimeric antigen receptor (CAR) against a universal cancer antigen, wherein the universal cancer antigen comprises three or more selected from GPC3, ROBO1, EPHB4, CLDN1 and LAT1.

9. The CAR-T cell therapeutic agent according to claim 8, wherein The universal cancer antigen further includes one or more of AFP, TGFBI, SPARC, HSP105ɑ, and FOXM1.

10. The CAR-T cell therapeutic agent according to claim 8, wherein The above-mentioned universal cancer antigens include all of GPC3, ROBO1, EPHB4, CLDN1, and LAT1.

11. The CAR-T cell therapeutic agent according to claim 8, wherein The above-mentioned universal cancer antigens include all of GPC3, ROBO1, EPHB4, CLDN1, LAT1, AFP, TGFBI, SPARC, HSP105α and FOXM1.

12. A CAR-T cell therapy comprising a mixture of T cells having a chimeric antigen receptor (CAR) against a universal cancer antigen, wherein the universal cancer antigen comprises three or more selected from GPC3, ROBO1, EPHB4, CLDN1, LAT1, AFP, TGFBI, SPARC, HSP105α and FOXM1.

13. A TCR-T cell therapeutic agent comprising a mixture of T cells having a T cell receptor (TCR) capable of recognizing MHC class I binding antigen peptides derived from universal cancer antigens, wherein the universal cancer antigens comprise three or more of GPC3, ROBO1, EPHB4, CLDN1 and LAT1.

14. The TCR-T cell therapeutic agent according to claim 13, wherein The universal cancer antigen further includes one or more of AFP, TGFBI, SPARC, HSP105α, and FOXM1.

15. The TCR-T cell therapeutic agent according to claim 13, wherein The above-mentioned universal cancer antigens include all of GPC3, ROBO1, EPHB4, CLDN1, and LAT1.

16. The TCR-T cell therapeutic agent according to claim 13, wherein The above-mentioned universal cancer antigens include all of GPC3, ROBO1, EPHB4, CLDN1, LAT1, AFP, TGFBI, SPARC, HSP105α and FOXM1.

17. A TCR-T cell therapeutic agent comprising a mixture of T cells having a T cell receptor (TCR) that can recognize MHC class I binding antigen peptides from a universal cancer antigen, wherein the universal cancer antigen comprises three or more of GPC3, ROBO1, EPHB4, CLDN1, LAT1, AFP, TGFBI, SPARC, HSP105α and FOXM1.

18. The TCR-T cell therapeutic agent according to any one of claims 13 to 17, wherein The T cell receptor (TCR) is a heterodimer of any of the following: A heterodimer composed of a combination of the protein of SEQ ID NO: 103 and the protein of SEQ ID NO: 104; A heterodimer composed of a combination of the protein of SEQ ID NO: 105 and the protein of SEQ ID NO: 106; A heterodimer composed of a combination of the protein of SEQ ID NO: 107 and the protein of SEQ ID NO: 108; a heterodimer composed of a combination of the protein of SEQ ID NO: 109 and the protein of SEQ ID NO: 110; A heterodimer composed of a combination of the protein of SEQ ID NO: 111 and the protein of SEQ ID NO: 112; A heterodimer composed of a combination of the protein of SEQ ID NO: 123 and the protein of SEQ ID NO: 124; A heterodimer composed of a combination of the protein of SEQ ID NO: 125 and the protein of SEQ ID NO: 126; a heterodimer composed of a combination of the protein of SEQ ID NO: 127 and the protein of SEQ ID NO: 128; A heterodimer composed of a combination of the protein of SEQ ID NO: 129 or 130 and the protein of SEQ ID NO: 131; A heterodimer composed of a combination of the protein of SEQ ID NO: 132 and the protein of SEQ ID NO: 133; a heterodimer composed of a combination of the protein of SEQ ID NO: 134 and the protein of SEQ ID NO: 135; a heterodimer composed of a combination of the protein of SEQ ID NO: 136 and the protein of SEQ ID NO: 137; A heterodimer composed of a combination of the protein of SEQ ID NO: 138 or 139 and the protein of SEQ ID NO: 140; A heterodimer composed of a combination of the protein of SEQ ID NO: 141 and the protein of SEQ ID NO: 142; A heterodimer composed of a combination of the protein of SEQ ID NO: 143 and the protein of SEQ ID NO: 144; a heterodimer composed of a combination of the protein of SEQ ID NO: 145 and the protein of SEQ ID NO: 146; a heterodimer composed of a combination of the protein of SEQ ID NO: 147 and the protein of SEQ ID NO: 148; A heterodimer composed of a combination of the protein of SEQ ID NO: 177 and the protein of SEQ ID NO: 178; a heterodimer composed of a combination of the protein of SEQ ID NO: 179 and the protein of SEQ ID NO: 180; A heterodimer composed of a combination of the protein of SEQ ID NO: 181 and the protein of SEQ ID NO: 182; a heterodimer composed of a combination of the protein of SEQ ID NO: 183 and the protein of SEQ ID NO: 184; A heterodimer composed of a combination of the protein of SEQ ID NO: 193 and the protein of SEQ ID NO: 194; A heterodimer composed of a combination of the protein of SEQ ID NO: 195 and the protein of SEQ ID NO: 196; A heterodimer composed of a combination of the protein of SEQ ID NO: 197 and the protein of SEQ ID NO: 198; A heterodimer composed of a combination of the protein of SEQ ID NO: 199 or 200 and the protein of SEQ ID NO: 201; A heterodimer composed of a combination of the protein of SEQ ID NO: 202 and the protein of SEQ ID NO: 203; A heterodimer composed of a combination of the protein of SEQ ID NO: 216 and the protein of SEQ ID NO: 217; or A heterodimer composed of a combination of the protein of SEQ ID NO: 218 and the protein of SEQ ID NO:

219.

19. A protein having an amino acid sequence as recorded in any one of SEQ ID NOs: 103 to 112, 123 to 148, 177 to 184, 193 to 203, and 215 to 219.

20. A T cell receptor (TCR) which is a heterodimer of any of the following: A heterodimer composed of a combination of the protein of SEQ ID NO: 103 and the protein of SEQ ID NO: 104; A heterodimer composed of a combination of the protein of SEQ ID NO: 105 and the protein of SEQ ID NO: 106; A heterodimer composed of a combination of the protein of SEQ ID NO: 107 and the protein of SEQ ID NO: 108; a heterodimer composed of a combination of the protein of SEQ ID NO: 109 and the protein of SEQ ID NO: 110; A heterodimer composed of a combination of the protein of SEQ ID NO: 111 and the protein of SEQ ID NO: 112; A heterodimer composed of a combination of the protein of SEQ ID NO: 123 and the protein of SEQ ID NO: 124; A heterodimer composed of a combination of the protein of SEQ ID NO: 125 and the protein of SEQ ID NO: 126; a heterodimer composed of a combination of the protein of SEQ ID NO: 127 and the protein of SEQ ID NO: 128; A heterodimer composed of a combination of the protein of SEQ ID NO: 129 or 130 and the protein of SEQ ID NO: 131; A heterodimer composed of a combination of the protein of SEQ ID NO: 132 and the protein of SEQ ID NO: 133; a heterodimer composed of a combination of the protein of SEQ ID NO: 134 and the protein of SEQ ID NO: 135; a heterodimer composed of a combination of the protein of SEQ ID NO: 136 and the protein of SEQ ID NO: 137; A heterodimer composed of a combination of the protein of SEQ ID NO: 138 or 139 and the protein of SEQ ID NO: 140; A heterodimer composed of a combination of the protein of SEQ ID NO: 141 and the protein of SEQ ID NO: 142; A heterodimer composed of a combination of the protein of SEQ ID NO: 143 and the protein of SEQ ID NO: 144; a heterodimer composed of a combination of the protein of SEQ ID NO: 145 and the protein of SEQ ID NO: 146; a heterodimer composed of a combination of the protein of SEQ ID NO: 147 and the protein of SEQ ID NO: 148; A heterodimer composed of a combination of the protein of SEQ ID NO: 177 and the protein of SEQ ID NO: 178; a heterodimer composed of a combination of the protein of SEQ ID NO: 179 and the protein of SEQ ID NO: 180; A heterodimer composed of a combination of the protein of SEQ ID NO: 181 and the protein of SEQ ID NO: 182; a heterodimer composed of a combination of the protein of SEQ ID NO: 183 and the protein of SEQ ID NO: 184; A heterodimer composed of a combination of the protein of SEQ ID NO: 193 and the protein of SEQ ID NO: 194; A heterodimer composed of a combination of the protein of SEQ ID NO: 195 and the protein of SEQ ID NO: 196; A heterodimer composed of a combination of the protein of SEQ ID NO: 197 and the protein of SEQ ID NO: 198; A heterodimer composed of a combination of the protein of SEQ ID NO: 199 or 200 and the protein of SEQ ID NO: 201; A heterodimer composed of a combination of the protein of SEQ ID NO: 202 and the protein of SEQ ID NO: 203; A heterodimer composed of a combination of the protein of SEQ ID NO: 216 and the protein of SEQ ID NO: 217; or A heterodimer composed of a combination of the protein of SEQ ID NO: 218 and the protein of SEQ ID NO:

219.

21. A gene having a nucleotide sequence of any one of SEQ ID NOs: 113 to 122, 149 to 176, 185 to 192, 204 to 214, and 220 to 224.

22. A companion diagnostic method comprising the following steps: Step 1, simultaneously determining the presence or absence of expression of three or more of GPC3, ROBO1, EPHB4, CLDN1 and LAT1 and the cell membrane expression of HLA class I in a sample from a subject by multiple fluorescent immunostaining; and step 2, determining the applicability of cancer immunotherapy based on the presence or absence of the above expression.

23. The companion diagnostic method of claim 22, wherein: In step 1, the presence or absence of expression of one or more of AFP, TGFBI, SPARC, HSP105α and FOXM1 is further determined.

24. The companion diagnostic method of claim 22, wherein: In step 1, the presence or absence of expression of all of GPC3, ROBO1, EPHB4, CLDN1 and LAT1 is determined.

25. The companion diagnostic method of claim 22, wherein: In step 1, the presence or absence of expression of all of GPC3, ROBO1, EPHB4, CLDN1, LAT1, AFP, TGFBI, SPARC, HSP105α, and FOXM1 is determined.

26. A method for diagnosing cancer risk, comprising: The expression of three or more of GPC3, ROBO1, EPHB4, CLDN1, and LAT1 is analyzed in cells in a blood sample derived from a subject.

27. The method for diagnosing cancer risk according to claim 26, further comprising: The expression of one or more of AFP, TGFBI, SPARC, HSP105α, and FOXM1 was analyzed.

28. The method for diagnosing cancer risk according to claim 26, wherein: The overall expression of GPC3, ROBO1, EPHB4, CLDN1 and LAT1 was analyzed.

29. The method for diagnosing cancer risk according to claim 26, wherein: The overall expression of GPC3, ROBO1, EPHB4, CLDN1, LAT1, AFP, TGFBI, SPARC, HSP105α and FOXM1 was analyzed.

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