Predicting the effectiveness of cancer vaccines

Abstract

Providing cancer prediction technology [Solution] This disclosure provides a method for predicting the effectiveness of a cancer vaccine, which includes confirming the reactivity of immune cells (e.g., peripheral blood mononuclear cells) derived from the patient to stimulation by a cancer antigen corresponding to the cancer vaccine or the antigenic portion of the cancer antigen, and predicting and calculating the effectiveness of the cancer vaccine (e.g., response (response and duration) / determining which patients should receive the vaccine) based on the reactivity.

Description

Technical Field

[0001] The present disclosure relates to predicting the effect of a cancer vaccine.

Background Art

[0002] Cancer is one of the major causes of death worldwide, and the development of its treatment methods has become an important issue in the medical field. There are various treatment methods for cancer, such as surgery, radiotherapy, chemotherapy, and molecular target drugs. However, the treatment effects vary depending on the individual constitution of patients and the type of cancer, and do not show a uniform effect on all patients.

[0003] In recent years, immunotherapy that activates the patient's immune system and attacks cancer cells has attracted attention. Among them, cancer vaccines are expected as innovative treatment methods aimed at inducing an immune response against cancer antigens in the patient's body and suppressing or preventing the progression of cancer. However, it is known that the effects of cancer vaccines vary greatly among patients, and the establishment of a technology for predicting their effects in advance has become an urgent issue.

Summary of the Invention

Means for Solving the Problems

[0004] [[ID=2)6]]The present disclosure is the result of intensive research by the present inventors, and provides the following as non-limiting examples. (Item 1) A method for predicting the effect of a cancer vaccine, comprising: confirming the reactivity of patient-derived immune cells to stimulation by a cancer antigen corresponding to the cancer vaccine, an antigenic portion of the cancer antigen, or an equivalent thereof, and predicting or calculating the effect of the cancer vaccine based on the reactivity. (Item 2) The method according to any of the above items, wherein the cancer antigen or the antigenic portion of the cancer antigen or equivalent thereof is a partial peptide protein contained in the amino acid sequence of the cancer antigen, a partial peptide protein contained in the amino acid sequence translated from the nucleic acid in the case of a nucleic acid vaccine, etc., or an equivalent thereof. (Item 3) The prediction is made by the method described in any one of the above items, either before administration of the vaccine or during treatment with the vaccine. (Item 4) The method of any one of the above items, which includes obtaining immune cells derived from the patient. (Item 5) The method of any one of the above items, comprising providing a cancer antigen corresponding to the cancer vaccine or an antigenic portion of the cancer antigen. (Item 6) The method described in any one of the above items, performed in vitro. (Item 7) The prediction of the effect described in any one of the above items, performed before administration of the cancer vaccine. (Item 8) The method according to any one of the above items, wherein the immune cells include at least one selected from the group consisting of peripheral blood mononuclear cells, lymphocytes (e.g., T cells, B cells, NK cells), dendritic cells, macrophages, monocytes, and neutrophils. (Item 9) The method according to any one of the above items, wherein the effect includes at least one selected from the group consisting of 1) the efficacy of the cancer vaccine, 2) the persistence of the cancer vaccine, 3) the determination of patients to whom the cancer vaccine should be administered, 4) the determination of the cancer vaccine to be administered, and 5) the determination of the dosage and administration of the cancer vaccine. (Item 10) The method according to any one of the above items, wherein the confirmation of the reactivity is performed using ELISPOT or a functional equivalent thereof (e.g., FluoroSpot, etc.). (Item 11) The method described in any one of the above items, wherein the confirmation of the reactivity is performed by ELISPOT. (Item 12) The method according to any one of the above items, wherein if the measured value (Spot Counts) by ELISPOT is equal to or greater than a predetermined value, the patient is determined to be one for whom the cancer vaccine will be effective / should be administered the vaccine. (Item 13) 1) The efficacy of the cancer vaccine If the measurement value (Spot Counts) obtained by the ELISPOT is above a predetermined value, it is determined that the cancer vaccine is effective. 2) Durability of the cancer vaccine If the measurement value (Spot Counts) obtained by the ELISPOT is above a predetermined value, it is determined that the cancer vaccine is effective. 3) Determining which patients should receive the aforementioned cancer vaccine. If the measurement value (Spot Counts) obtained by the aforementioned ELISPOT is above a predetermined value, the patient is determined to be a patient who should be administered the cancer vaccine. 4) Determining which cancer vaccine to administer If the measurement value (Spot Counts) obtained by the aforementioned ELISPOT exceeds a predetermined value, the patient is determined to be a patient who should receive the target vaccine. The method described in any one of the above items. (Item 14) The method according to any one of the above items, wherein the predetermined value of the measurement value (Spot Counts) by ELISPOT is 7. (Item 15) The method described in any one of the above items, wherein the cancer antigen includes all cancer antigens contained in a cancer vaccine. (Item 16) The method according to any one of the above items, wherein the cancer antigen comprises at least one selected from the group consisting of WT1, AGEA1-4, NY-ESO-1, PRAME, SSX2, CT8, CD19, GP100, MART1, PSA, PSMA, tyrosinase, HER2, MUC1, CEA, survivin, cyclin B1, EGFR, mesothelin, telomerase, and neoantigens resulting from gene mutations in the patient's tumor cells. (Item 17) The method according to any one of the above items, wherein the cancer antigen is WT1. (Item A0) An agent for predicting the effect of a cancer vaccine, wherein the agent comprises a cancer antigen corresponding to the cancer vaccine or an antigenic portion of the cancer antigen or an equivalent thereof, and the agent confirms the reactivity of immune cells derived from the patient in response to stimulation by the cancer antigen corresponding to the cancer vaccine or an antigenic portion of the cancer antigen or an equivalent thereof, and predicts and / or calculates the effect of the cancer vaccine based on the reactivity. (Item A1) The above prediction and / or calculation, To confirm the reactivity of immune cells derived from the patient in response to stimulation with a cancer antigen corresponding to the cancer vaccine, or the antigenic portion of the cancer antigen, or equivalents thereof, and Predicting or calculating the effect of the cancer vaccine based on the aforementioned reactivity. A drug that includes any one of the items listed above. (Item A2) The agent described in any one of the above items, wherein the cancer antigen or the antigenic portion of the cancer antigen or equivalent thereof is a partial peptide protein contained in the amino acid sequence of the cancer antigen, a partial peptide protein contained in the amino acid sequence translated from the nucleic acid in the case of a nucleic acid vaccine, etc., or an equivalent thereof. (Item A3) The prediction is made before administration of the vaccine or during the treatment period with the vaccine, using the agent described in any one of the above items. (Item A4) The agent described in any one of the above items, which includes obtaining immune cells derived from the patient. (Item A5) The agent according to any one of the above items, comprising providing a cancer antigen corresponding to the cancer vaccine or an antigenic portion of the cancer antigen. (Item A6) The method described above is performed in vitro with respect to the agent described in any one of the above items. (Item A7) The prediction of the effect is made before administration of the cancer vaccine with respect to the agent described in any one of the above items. (Item A8) The agent according to any one of the above items, wherein the immune cells include at least one selected from the group consisting of peripheral blood mononuclear cells, lymphocytes (e.g., T cells, B cells, NK cells), dendritic cells, macrophages, monocytes, and neutrophils. (Item A9) The agent according to any one of the above items, wherein the effect includes at least one selected from the group consisting of 1) the efficacy of the cancer vaccine, 2) the persistence of the cancer vaccine, 3) the determination of patients to whom the cancer vaccine should be administered, 4) the determination of the cancer vaccine to be administered, and 5) the determination of the dosage and administration of the cancer vaccine. (Item A10) The confirmation of the reactivity is performed using ELISPOT or a functional equivalent thereof (e.g., FluoroSpot, etc.), and the agent is one of the agents described in any of the above items. (Item A11) The agent described in any one of the above items, the confirmation of the reactivity is performed by ELISPOT. (Item A12) The agent described in any one of the above items, which is determined to be effective in / should be administered to a patient if the measured value (Spot Counts) by ELISPOT is equal to or greater than a predetermined value. (Item A13) 1) The efficacy of the cancer vaccine If the measurement value (Spot Counts) obtained by the ELISPOT is above a predetermined value, it is determined that the cancer vaccine is effective. 2) Durability of the cancer vaccine If the measurement value (Spot Counts) obtained by the ELISPOT is above a predetermined value, it is determined that the cancer vaccine is effective. 3) Determining which patients should receive the aforementioned cancer vaccine. If the measurement value (Spot Counts) obtained by the ELISPOT is above a predetermined value, the patient is determined to be a patient who should be administered the cancer vaccine. 4) Determining which cancer vaccine to administer If the ELISPOT measurement (Spot Counts) is above a predetermined value, the patient is determined to be a patient who should receive the target vaccine. The agent described in any one of the above items. (Item A14) The agent described in any one of the above items, wherein the predetermined value of the measurement value (Spot Counts) by ELISPOT is 7. (Item A15) The cancer antigen is the agent according to any one of the above items, including all cancer antigens contained in the cancer vaccine. (Item A16) The cancer antigen is the agent according to any one of the above items, including at least one selected from the group consisting of WT1, AGEA1-4, NY-ESO-1, PRAME, SSX2, CT8, CD19, GP100, MART1, PSA, PSMA, tyrosinase, HER2, MUC1, CEA, survivin, cyclin B1, EGFR, mesothelin, telomerase, and neoantigens resulting from gene mutations in the patient's tumor cells. (Item A17) The cancer antigen is WT1, and it is the agent according to any one of the above items. (Item A18) The agent according to any one of the above items, further comprising the features described in any one or more of Items 1 to 17. (Item B0) A kit for predicting the effect of a cancer vaccine, the kit comprising a cancer antigen corresponding to the cancer vaccine, an antigenic part of the cancer antigen, or an equivalent thereof, and means for confirming the reactivity of immune cells derived from a patient to stimulation by the cancer antigen corresponding to the cancer vaccine, an antigenic part of the cancer antigen, or an equivalent thereof, and predicting and / or calculating the effect of the cancer vaccine based on the reactivity. (Item B1) The prediction and / or calculation confirming the reactivity of the immune cells derived from the patient to stimulation by the cancer antigen corresponding to the cancer vaccine, an antigenic part of the cancer antigen, or an equivalent thereof, and predicting or calculating the effect of the cancer vaccine based on the reactivity The kit according to any one of the above items, including. (Item B2) The cancer antigen, an antigenic part of the cancer antigen, or an equivalent thereof is a partial peptide protein contained in the amino acid sequence of the cancer antigen, in the case of a nucleic acid vaccine, a partial peptide protein contained in the amino acid sequence translated from the nucleic acid, or an equivalent thereof, etc. The kit according to any one of the above items. (Item B3) The prediction is made before administration of the vaccine or during the treatment period with the vaccine, and the kit according to any one of the above items. (Item B4) The kit according to any one of the above items, which includes obtaining immune cells derived from the patient. (Item B5) The kit according to any one of the above items, which includes providing a cancer antigen or an antigenic portion of the cancer antigen against the cancer vaccine. (Item B6) The method is performed in vitro, and the kit according to any one of the above items. (Item B7) The prediction of the effect is made before administration of the cancer vaccine, and the kit according to any one of the above items. (Item B8) The immune cells include at least one selected from the group consisting of peripheral blood mononuclear cells, lymphocytes (e.g., T cells, B cells, NK cells), dendritic cells, macrophages, monocytes, and neutrophils, and the kit according to any one of the above items. (Item B9) The effect includes at least one selected from the group consisting of 1) the efficacy of the cancer vaccine, 2) the persistence of the cancer vaccine, 3) the determination of the patient to whom the cancer vaccine should be administered, 4) the determination of the cancer vaccine to be administered, and 5) the determination of the change in the dosage and usage of the cancer vaccine, and the kit according to any one of the above items. (Item B10) The means for confirming the reactivity includes the means for performing ELISPOT or its functional equivalent (e.g., FluoroSpot, etc.), and the kit according to any one of the above items. (Item B11) The means for confirming the reactivity includes the means for performing ELISPOT, and the kit according to any one of the above items. (Item B12) When the measured value (Spot Counts) by the ELISPOT is not less than a predetermined value, it is determined that the cancer vaccine is effective / it is determined that the patient is the one to whom the vaccine should be administered, and the kit according to any one of the above items. (Item B13) 1) The efficacy of the cancer vaccine If the measurement value (Spot Counts) obtained by the ELISPOT is above a predetermined value, it is determined that the cancer vaccine is effective. 2) Durability of the cancer vaccine If the measurement value (Spot Counts) obtained by the ELISPOT is above a predetermined value, it is determined that the cancer vaccine is effective. 3) Determining which patients should receive the aforementioned cancer vaccine. If the measurement value (Spot Counts) obtained by the aforementioned ELISPOT is above a predetermined value, the patient is determined to be a patient who should be administered the cancer vaccine. 4) Determining which cancer vaccine to administer If the measurement value (Spot Counts) obtained by the aforementioned ELISPOT exceeds a predetermined value, the patient is determined to be a patient who should receive the target vaccine. A kit as described in any one of the items in item B above. (Item B14) A kit according to any one of the above items, wherein the predetermined value of the measurement (Spot Counts) by ELISPOT is 7. (Item B15) The cancer antigen is a kit according to any one of the above items, which includes all cancer antigens contained in the cancer vaccine. (Item B16) The kit according to any one of the above items, wherein the cancer antigen comprises at least one selected from the group consisting of WT1, AGEA1-4, NY-ESO-1, PRAME, SSX2, CT8, CD19, GP100, MART1, PSA, PSMA, tyrosinase, HER2, MUC1, CEA, survivin, cyclin B1, EGFR, mesothelin, telomerase, and neoantigens resulting from gene mutations in the patient's tumor cells. (Item B17) The kit according to any one of the above items, wherein the cancer antigen is WT1. (Item B18) The kit according to any one of the above items, further comprising the features described in any one or more of the above items 1-17 and A1-A18. (Item C1) A method for predicting the effectiveness of a cancer vaccine, comprising: collecting immune cells from the patient; providing a cancer antigen or an antigenic portion of the cancer antigen or equivalent thereof corresponding to the cancer vaccine; stimulating the immune cells with the cancer antigen or the antigenic portion of the cancer antigen; confirming the responsiveness to the stimulation; and calculating a prediction of the effectiveness of the cancer vaccine based on the responsiveness. (Item C2) The method according to any one of the above items, wherein the cancer antigen or the antigenic portion of the cancer antigen or equivalent thereof is a partial peptide protein contained in the amino acid sequence of the cancer antigen, a partial peptide protein contained in the amino acid sequence translated from the nucleic acid in the case of a nucleic acid vaccine, etc., or an equivalent thereof. (Item C3) The prediction is made by the method described in any one of the above items, either before administration of the vaccine or during treatment with the vaccine. (Item C4) The method of any one of the above items, which includes obtaining immune cells derived from the patient. (Item C5) The method of any one of the above items, comprising providing a cancer antigen corresponding to the cancer vaccine or an antigenic portion of the cancer antigen. (Item C6) The method described above is performed in vitro, according to any one of the above items. (Item C7) The prediction of the effect is made before administration of the cancer vaccine, using the method described in any one of the above items. (Item C8) The method according to any one of the above items, wherein the immune cells include at least one selected from the group consisting of peripheral blood mononuclear cells, lymphocytes (e.g., T cells, B cells, NK cells), dendritic cells, macrophages, monocytes, and neutrophils. (Item C9) The method according to any one of the above items, wherein the effect includes at least one selected from the group consisting of 1) the efficacy of the cancer vaccine, 2) the persistence of the cancer vaccine, 3) the determination of patients to whom the cancer vaccine should be administered, 4) the determination of the cancer vaccine to be administered, and 5) the determination of the dosage and administration of the cancer vaccine. (Item C10) The method according to any one of the above items, wherein the confirmation of the reactivity is performed using ELISPOT or a functional equivalent thereof (e.g., FluoroSpot, etc.). (Item C11) The method according to any one of the above items, wherein the confirmation of the reactivity is performed using ELISPOT. (Item C12) The method according to any one of the above items, wherein if the measured value (Spot Counts) by ELISPOT is equal to or greater than a predetermined value, it is determined that the cancer vaccine will be effective for the patient / that the patient should be administered the vaccine. (Item C13) 1) The efficacy of the cancer vaccine If the measurement value (Spot Counts) obtained by the ELISPOT is above a predetermined value, it is determined that the cancer vaccine is effective. 2) Durability of the cancer vaccine If the measurement value (Spot Counts) obtained by the ELISPOT is above a predetermined value, it is determined that the cancer vaccine is effective. 3) Determining which patients should receive the aforementioned cancer vaccine. If the measurement value (Spot Counts) obtained by the ELISPOT is above a predetermined value, the patient is determined to be a patient who should be administered the cancer vaccine. 4) Determining which cancer vaccine to administer If the ELISPOT measurement (Spot Counts) is above a predetermined value, the patient is determined to be a patient who should receive the target vaccine. The method described in any one of the items C above. (Item C14) The method according to any one of the above items, wherein the predetermined value of the measurement value (Spot Counts) by ELISPOT is 7. (Item C15) The method described in any one of the above items, wherein the cancer antigen includes all cancer antigens contained in a cancer vaccine. (Item C16) The method according to any one of the above items, wherein the cancer antigen comprises at least one selected from the group consisting of WT1, AGEA1-4, NY-ESO-1, PRAME, SSX2, CT8, CD19, GP100, MART1, PSA, PSMA, tyrosinase, HER2, MUC1, CEA, survivin, cyclin B1, EGFR, mesothelin, telomerase, and neoantigens resulting from gene mutations in the patient's tumor cells. (Item C17) The method according to any one of the above items, wherein the cancer antigen is WT1. (Item C18) A method according to any one of the above items, further comprising the features described in any one or more of the above items, items 1 to 17, items A1 to A18, and items B1 to B18. (Item C19) The method of any one of the above items, further comprising identifying the cancer antigen or the antigenic portion of the cancer antigen corresponding to the patient. (Item D0) Use of a cancer antigen or an antigenic portion of the cancer antigen or an equivalent thereof, corresponding to the cancer vaccine, for manufacturing a diagnostic agent for predicting the effectiveness of a cancer vaccine, wherein the diagnostic agent confirms the reactivity of patient-derived immune cells to stimulation with the cancer antigen or an antigenic portion of the cancer antigen or an equivalent thereof, corresponding to the cancer vaccine, and predicts and / or calculates the effectiveness of the cancer vaccine based on the reactivity. (Item D1) The prediction and / or calculation, To confirm the reactivity of immune cells derived from the patient in response to stimulation with a cancer antigen corresponding to the cancer vaccine, or the antigenic portion of the cancer antigen, or equivalents thereof, and To predict or calculate the effect of the cancer vaccine based on the aforementioned reactivity. The use described in any one of the above items, including: (Item D2) The use described in any one of the above items, wherein the cancer antigen or the antigenic portion of the cancer antigen or equivalent thereof is a partial peptide protein contained in the amino acid sequence of the cancer antigen, a partial peptide protein contained in the amino acid sequence translated from the nucleic acid in the case of a nucleic acid vaccine, etc. (Item D3) The prediction is made before administration of the vaccine or during treatment with the vaccine, using any one of the items described above. (Item D4) Use as described in any one of the above items, which includes obtaining immune cells derived from the patient. (Item D5) The use described in any one of the above items, which includes providing a cancer antigen corresponding to the cancer vaccine or an antigenic portion of the cancer antigen. (Item D6) The use described in any one of the above items, performed in vitro. (Item D7) The prediction of the effect is made before administration of the cancer vaccine, using any one of the items described above. (Item D8) The use described in any one of the above items, wherein the immune cells include at least one selected from the group consisting of peripheral blood mononuclear cells, lymphocytes (e.g., T cells, B cells, NK cells), dendritic cells, macrophages, monocytes, and neutrophils. (Item D9) The use described in any one of the above items, wherein the effect includes at least one selected from the group consisting of 1) the efficacy of the cancer vaccine, 2) the persistence of the cancer vaccine, 3) the determination of patients to whom the cancer vaccine should be administered, 4) the determination of which cancer vaccine should be administered, and 5) the determination of changes in the dosage and administration of the cancer vaccine. (Item D10) The confirmation of the reactivity is performed using ELISPOT or its functional equivalent (e.g., FluoroSpot, etc.), as described in any one of the above items. (Item D11) The confirmation of the reactivity is performed using an ELISPOT, as described in any one of the above items. (Item D12) Use as described in any one of the above items, in which, if the measured value (Spot Counts) by ELISPOT is equal to or greater than a predetermined value, it is determined that the cancer vaccine will be effective / that the patient should be administered the vaccine. (Item D13) 1) The efficacy of the cancer vaccine If the measurement value (Spot Counts) obtained by the ELISPOT is above a predetermined value, it is determined that the cancer vaccine is effective. 2) Durability of the cancer vaccine If the measurement value (Spot Counts) obtained by the ELISPOT is above a predetermined value, it is determined that the cancer vaccine is effective. 3) Determining which patients should receive the aforementioned cancer vaccine. If the measurement value (Spot Counts) obtained by the ELISPOT is above a predetermined value, the patient is determined to be a patient who should be administered the cancer vaccine. 4) Determining which cancer vaccine to administer If the ELISPOT measurement (Spot Counts) is above a predetermined value, the patient is determined to be a patient who should receive the target vaccine. Use as described in any one of the items D above. (Item D14) The use described in any one of the above items, wherein the predetermined value of the measurement (Spot Counts) by ELISPOT is 7. (Item D15) The use described in any one of the above items, wherein the cancer antigen includes all cancer antigens contained in the cancer vaccine. (Item D16) The use described in any one of the above items, wherein the cancer antigen comprises at least one selected from the group consisting of WT1, AGEA1-4, NY-ESO-1, PRAME, SSX2, CT8, CD19, GP100, MART1, PSA, PSMA, tyrosinase, HER2, MUC1, CEA, survivin, cyclin B1, EGFR, mesothelin, telomerase, and neoantigens resulting from gene mutations in the patient's tumor cells. (Item D17) The cancer antigen is WT1, as described in any one of the above items. (Item D18) A use as described in any one of the above items, further comprising the characteristics described in any one or more of the above items, including items 1-17, A0-A18, B0-B18, C1-C19, and D0-D18. (Item E0) A pharmaceutical product comprising a cancer vaccine, wherein the vaccine regimen is determined based on the reactivity of patient-derived immune cells (e.g., peripheral blood mononuclear cells) to stimulation with a cancer antigen corresponding to the cancer vaccine or the antigenic portion of the cancer antigen. (Item E1) The above prediction and / or calculation, To confirm the reactivity of immune cells derived from the patient in response to stimulation with a cancer antigen corresponding to the cancer vaccine, or the antigenic portion of the cancer antigen, or equivalents thereof, and To predict or calculate the effect of the cancer vaccine based on the aforementioned reactivity. A pharmaceutical product that includes any one of the items listed above. (Item E2) The pharmaceutical product described in any of the above items, wherein the cancer antigen or the antigenic portion of the cancer antigen or equivalent thereof is a partial peptide protein contained in the amino acid sequence of the cancer antigen, a partial peptide protein contained in the amino acid sequence translated from the nucleic acid in the case of a nucleic acid vaccine, etc., or an equivalent thereof. (Item E3) The prediction is made before administration of the vaccine or during treatment with the vaccine, as described in any one of the above items. (Item E4) A pharmaceutical product as described in any one of the above items, which includes obtaining immune cells derived from the patient. (Item E5) A pharmaceutical product according to any one of the above items, comprising providing a cancer antigen corresponding to the cancer vaccine or an antigenic portion of the cancer antigen. (Item E6) The method described above is performed in vitro, and the pharmaceutical product is as described in any one of the above items. (Item E7) The prediction of the effect is made before administration of the cancer vaccine, using the pharmaceutical product described in any one of the above items. (Item E8) The pharmaceutical product according to any one of the above items, wherein the immune cells include at least one selected from the group consisting of peripheral blood mononuclear cells, lymphocytes (e.g., T cells, B cells, NK cells), dendritic cells, macrophages, monocytes, and neutrophils. (Item E9) The pharmaceutical product according to any one of the above items, wherein the effect includes at least one selected from the group consisting of 1) the efficacy of the cancer vaccine, 2) the persistence of the cancer vaccine, 3) the determination of patients to whom the cancer vaccine should be administered, 4) the determination of the cancer vaccine to be administered, and 5) the determination of the dosage and administration of the cancer vaccine. (Item E10) The confirmation of reactivity is performed using ELISPOT or its functional equivalent (e.g., FluoroSpot, etc.), as described in one of the above items. (Item E11) The confirmation of reactivity is performed using ELISPOT, as described in one of the above items. (Item E12) A pharmaceutical product according to any one of the above items, which is determined to be effective in / should be administered the cancer vaccine to a patient if the measured value (Spot Counts) by ELISPOT is equal to or greater than a predetermined value. (Item E13) 1) The efficacy of the cancer vaccine If the measurement value (Spot Counts) obtained by the ELISPOT is above a predetermined value, it is determined that the cancer vaccine is effective. 2) Durability of the cancer vaccine If the measurement value (Spot Counts) obtained by the ELISPOT is above a predetermined value, it is determined that the cancer vaccine is effective. 3) Determining which patients should receive the aforementioned cancer vaccine. If the measurement value (Spot Counts) obtained by the ELISPOT is above a predetermined value, the patient is determined to be a patient who should be administered the cancer vaccine. 4) Determining which cancer vaccine to administer If the ELISPOT measurement (Spot Counts) is above a predetermined value, the patient is determined to be a patient who should receive the target vaccine. A pharmaceutical product as described in any one of the items E above. (Item E14) A pharmaceutical product according to any one of the above items, wherein the predetermined value of the measurement value (Spot Counts) by ELISPOT is 7. (Item E15) The cancer antigen is a pharmaceutical product as described in any one of the above items, including all cancer antigens contained in cancer vaccines. (Item E16) The pharmaceutical product according to any one of the above items, wherein the cancer antigen comprises at least one selected from the group consisting of WT1, AGEA1-4, NY-ESO-1, PRAME, SSX2, CT8, CD19, GP100, MART1, PSA, PSMA, tyrosinase, HER2, MUC1, CEA, survivin, cyclin B1, EGFR, mesothelin, telomerase, and neoantigens resulting from gene mutations in the patient's tumor cells. (Item E17) The cancer antigen is WT1, as described in any one of the above items. (Item E18) A pharmaceutical product described in any one of the above items, further possessing the characteristics described in any one or more of the above items: items 1-17, items A0-A18, items B0-B18, items C1-C19, and items D0-D18. (Item E19) The pharmaceutical product according to any one of the above items, characterized in that the pharmaceutical product is administered to a subject whose immune cells obtained before administration have a reactivity value to the cancer vaccine to antigen stimulation of the cancer vaccine that is equal to or greater than a predetermined value. (Item E20) The pharmaceutical product described in any one of the above items, which is for the prevention or treatment of cancer. (Item F0) A cancer vaccine for the prevention or treatment of cancer, wherein the vaccine regimen is determined based on the reactivity of patient-derived immune cells (e.g., peripheral blood mononuclear cells) to stimulation with a cancer antigen corresponding to the cancer vaccine or the antigenic portion of the cancer antigen. (Item F1) The above prediction and / or calculation, To confirm the reactivity of immune cells derived from the patient in response to stimulation by the cancer antigen corresponding to the cancer vaccine, the antigenic portion of the cancer antigen, or equivalents thereof, and to predict or calculate the effect of the cancer vaccine based on the reactivity. A cancer vaccine that includes any one of the items listed above. (Item F2) The cancer vaccine described in any of the above items, wherein the cancer antigen or the antigenic portion of the cancer antigen or equivalent thereof is a partial peptide protein contained in the amino acid sequence of the cancer antigen, a partial peptide protein contained in the amino acid sequence translated from the nucleic acid in the case of a nucleic acid vaccine, etc., or an equivalent thereof. (Item F3) The prediction is made before administration of the vaccine or during treatment with the vaccine, for any cancer vaccine as described in any one of the above items. (Item F4) A cancer vaccine according to any one of the above items, which includes obtaining immune cells derived from the patient. (Item F5) A cancer vaccine according to any one of the above items, comprising providing a cancer antigen corresponding to the cancer vaccine or an antigenic portion of the cancer antigen. (Item F6) The method described above is performed in vitro with any of the cancer vaccines described in any one of the above items. (Item F7) The prediction of the effect is made before administration of the cancer vaccine, using any one of the above items. (Item F8) The cancer vaccine according to any one of the above items, wherein the immune cells include at least one selected from the group consisting of peripheral blood mononuclear cells, lymphocytes (e.g., T cells, B cells, NK cells), dendritic cells, macrophages, monocytes, and neutrophils. (Item F9) The cancer vaccine according to any one of the above items, wherein the effect includes at least one selected from the group consisting of 1) the efficacy of the cancer vaccine, 2) the persistence of the cancer vaccine, 3) the determination of patients to whom the cancer vaccine should be administered, 4) the determination of the cancer vaccine to be administered, and 5) the determination of the dosage and administration of the cancer vaccine. (Item F10) The confirmation of reactivity is performed using ELISPOT or a functional equivalent thereof (e.g., FluoroSpot, etc.), and the cancer vaccine is one of the items listed above. (Item F11) The reactivity is confirmed by ELISPOT, and the cancer vaccine is one of the items listed above. (Item F12) A cancer vaccine according to any one of the above items, which is determined to be effective in / should be administered to a patient if the measured value (Spot Counts) by ELISPOT is equal to or greater than a predetermined value. (Item F13) 1) The efficacy of the cancer vaccine If the measurement value (Spot Counts) obtained by the ELISPOT is above a predetermined value, it is determined that the cancer vaccine is effective. 2) Durability of the cancer vaccine If the measurement value (Spot Counts) obtained by the ELISPOT is above a predetermined value, it is determined that the cancer vaccine is effective. 3) Determining which patients should receive the aforementioned cancer vaccine. If the measurement value (Spot Counts) obtained by the aforementioned ELISPOT is above a predetermined value, the patient is determined to be a patient who should be administered the cancer vaccine. 4) Determining which cancer vaccine to administer If the measurement value (Spot Counts) obtained by the aforementioned ELISPOT exceeds a predetermined value, the patient is determined to be a patient who should receive the target vaccine. A cancer vaccine as described in any one of the items F above. (Item F14) A cancer vaccine according to any one of the above items, wherein the specified value of the measurement (Spot Counts) by ELISPOT is 7. (Item F15) The cancer antigen is a cancer vaccine as described in any one of the above items, which includes all cancer antigens contained in the cancer vaccine. (Item F16) A cancer vaccine according to any one of the above items, wherein the cancer antigen comprises at least one selected from the group consisting of WT1, AGEA1-4, NY-ESO-1, PRAME, SSX2, CT8, CD19, GP100, MART1, PSA, PSMA, tyrosinase, HER2, MUC1, CEA, survivin, cyclin B1, EGFR, mesothelin, telomerase, and neoantigens resulting from gene mutations in the patient's tumor cells. (Item F17) The cancer vaccine described in any one of the above items, wherein the cancer antigen is WT1. (Item F18) A cancer vaccine according to any one of the above items, further comprising the characteristics described in any one or more of the items 1-17, A0-A18, B0-B18, C1-C19, D1-D18, and E0-E20. (Item F19) The cancer vaccine according to any one of the above items, characterized in that the cancer vaccine is administered to a subject whose immune cells obtained before administration have a reactivity value to antigen stimulation for the cancer vaccine that is equal to or greater than a predetermined value. (Item G1) A method for preventing or treating a patient using a cancer vaccine, comprising: collecting immune cells from the patient; providing a cancer antigen or an antigenic portion of the cancer antigen or an equivalent thereof corresponding to the cancer vaccine; stimulating the immune cells with the cancer antigen or the antigenic portion of the cancer antigen; confirming the responsiveness to the stimulation and calculating a prediction of the effect of the cancer vaccine based on the responsiveness; and administering the cancer vaccine to the patient based on the prediction of the effect. (Item G2) The method according to any of the above items, wherein the cancer antigen or the antigenic portion of the cancer antigen or equivalent thereof is a partial peptide protein contained in the amino acid sequence of the cancer antigen, a partial peptide protein contained in the amino acid sequence translated from the nucleic acid in the case of a nucleic acid vaccine, etc., or an equivalent thereof. (Item G3) The prediction is made by the method described in any one of the above items, either before administration of the vaccine or during treatment with the vaccine. (Item G4) The method of any one of the above items, which includes obtaining immune cells derived from the patient. (Item G5) The method according to any one of the above items, comprising providing a cancer antigen corresponding to the cancer vaccine or an antigenic portion of the cancer antigen. (Item G6) The method described above is performed in vitro, according to any one of the above items. (Item G7) The prediction of the effect is made before administration of the cancer vaccine, using the method described in any one of the above items. (Item G8) The method according to any one of the above items, wherein the immune cells include at least one selected from the group consisting of peripheral blood mononuclear cells, lymphocytes (e.g., T cells, B cells, NK cells), dendritic cells, macrophages, monocytes, and neutrophils. (Item G9) The method according to any one of the above items, wherein the effect includes at least one selected from the group consisting of 1) the efficacy of the cancer vaccine, 2) the persistence of the cancer vaccine, 3) the determination of patients to whom the cancer vaccine should be administered, 4) the determination of the cancer vaccine to be administered, and 5) the determination of the dosage and administration of the cancer vaccine. (Item G10) The method according to any one of the above items, wherein the confirmation of the reactivity is performed using ELISPOT or a functional equivalent thereof (e.g., FluoroSpot, etc.). (Item G11) The method according to any one of the above items, wherein the confirmation of the reactivity is performed using ELISPOT. (Item G12) The method according to any one of the above items, wherein if the measured value (Spot Counts) by ELISPOT is equal to or greater than a predetermined value, it is determined that the cancer vaccine will be effective for the patient / that the patient should be administered the vaccine. (Item G13) 1) The efficacy of the cancer vaccine If the measurement value (Spot Counts) obtained by the ELISPOT is above a predetermined value, it is determined that the cancer vaccine is effective. 2) Durability of the cancer vaccine If the measurement value (Spot Counts) obtained by the ELISPOT is above a predetermined value, it is determined that the cancer vaccine is effective. 3) Determining which patients should receive the aforementioned cancer vaccine. If the measurement value (Spot Counts) obtained by the ELISPOT is above a predetermined value, the patient is determined to be a patient who should be administered the cancer vaccine. 4) Determining which cancer vaccine to administer If the ELISPOT measurement (Spot Counts) is above a predetermined value, the patient is determined to be a patient who should receive the target vaccine. The method described in any one of the above item G. (Item G14) The method according to any one of the above items, wherein the predetermined value of the measurement value (Spot Counts) by ELISPOT is 7. (Item G15) The method described in any one of the above items, wherein the cancer antigen includes all cancer antigens contained in the cancer vaccine. (Item G16) The method according to any one of the above items, wherein the cancer antigen comprises at least one selected from the group consisting of WT1, AGEA1-4, NY-ESO-1, PRAME, SSX2, CT8, CD19, GP100, MART1, PSA, PSMA, tyrosinase, HER2, MUC1, CEA, survivin, cyclin B1, EGFR, mesothelin, telomerase, and neoantigens resulting from gene mutations in the patient's tumor cells. (Item G17) The method according to any one of the above items, wherein the cancer antigen is WT1. (Item G18) A method according to any one of the above items, further comprising the characteristics described in any one or more of the above items: items 1-17, items A0-A18, items B0-B18, items C1-C19, items D0-D18, items E0-E20, and items F0-F19. (Item G19) The method of any one of the above items, further comprising identifying the cancer antigen or the antigenic portion of the cancer antigen corresponding to the patient. (Item H0) Use of a cancer vaccine for manufacturing a pharmaceutical product for the prevention or treatment of cancer, wherein the vaccine regimen is determined based on the reactivity of patient-derived immune cells (e.g., peripheral blood mononuclear cells) to stimulation with a cancer antigen corresponding to the cancer vaccine or the antigenic portion of the cancer antigen. (Item H1) The above prediction and / or calculation, To confirm the reactivity of immune cells derived from the patient in response to stimulation by the cancer antigen corresponding to the cancer vaccine, the antigenic portion of the cancer antigen, or equivalents thereof, and to predict or calculate the effect of the cancer vaccine based on the reactivity. The use described in any one of the above items, including: (Item H2) The use described in any of the above items, where the cancer antigen or the antigenic portion of the cancer antigen or equivalent thereof is a partial peptide protein contained in the amino acid sequence of the cancer antigen, a partial peptide protein contained in the amino acid sequence translated from the nucleic acid in the case of a nucleic acid vaccine, etc. (Item H3) The prediction is made before administration of the vaccine or during treatment with the vaccine, using any one of the items described above. (Item H4) Use as described in any one of the above items, which includes obtaining immune cells derived from the patient. (Item H5) The use described in any one of the above items, which includes providing a cancer antigen corresponding to the cancer vaccine or an antigenic portion of the cancer antigen. (Item H6) The use described in any one of the above items, performed in vitro. (Item H7) The prediction of the effect is made before administration of the cancer vaccine, using any one of the items described above. (Item H8) The use described in any one of the above items, wherein the immune cells include at least one selected from the group consisting of peripheral blood mononuclear cells, lymphocytes (e.g., T cells, B cells, NK cells), dendritic cells, macrophages, monocytes, and neutrophils. (Item H9) The use described in any one of the above items, wherein the effect includes at least one selected from the group consisting of 1) the efficacy of the cancer vaccine, 2) the persistence of the cancer vaccine, 3) the determination of patients to whom the cancer vaccine should be administered, 4) the determination of which cancer vaccine to administer, and 5) the determination of changes in the dosage and administration of the cancer vaccine. (Item H10) The confirmation of the reactivity is performed using ELISPOT or its functional equivalent (e.g., FluoroSpot, etc.), as described in any one of the above items. (Item H11) The confirmation of the reactivity is performed using ELISPOT, as described in any one of the above items. (Item H12) Use as described in any one of the above items, where the measured value (Spot Counts) by ELISPOT is determined to be equal to or equal to a predetermined value, and the patient is determined to be a patient for whom the cancer vaccine will be effective / should be administered the vaccine. (Item H13) 1) The efficacy of the cancer vaccine If the measurement value (Spot Counts) obtained by the ELISPOT is above a predetermined value, it is determined that the cancer vaccine is effective. 2) Durability of the cancer vaccine If the measurement value (Spot Counts) obtained by the ELISPOT is above a predetermined value, it is determined that the cancer vaccine is effective. 3) Determining which patients should receive the aforementioned cancer vaccine. If the measurement value (Spot Counts) obtained by the aforementioned ELISPOT is above a predetermined value, the patient is determined to be a patient who should be administered the cancer vaccine. 4) Determining which cancer vaccine to administer If the measurement value (Spot Counts) obtained by the aforementioned ELISPOT exceeds a predetermined value, the patient is determined to be a patient who should receive the target vaccine. Use as described in any one of the items H above. (Item H14) Use as described in any one of the above items, wherein the predetermined value of the measurement value (Spot Counts) by ELISPOT is 7. (Item H15) The use described in any one of the above items, wherein the cancer antigen includes all cancer antigens contained in the cancer vaccine. (Item H16) The use described in any one of the above items, wherein the cancer antigen comprises at least one selected from the group consisting of WT1, AGEA1-4, NY-ESO-1, PRAME, SSX2, CT8, CD19, GP100, MART1, PSA, PSMA, tyrosinase, HER2, MUC1, CEA, survivin, cyclin B1, EGFR, mesothelin, telomerase, and neoantigens resulting from gene mutations in the patient's tumor cells. (Item H17) The cancer antigen is WT1, and the use is as described in any one of the above items. (Item H18) Use as described in any one of the above items, further comprising the characteristics described in any one or more of the above items, including (item H18), items 1-17, items A0-A18, items B0-B18, items C1-C19, items D0-D18, items E0-E20, and items F0-F19. (Item H19) The use of the pharmaceutical product as described in any one of the above items, characterized in that the pharmaceutical product is administered to a subject whose immune cells obtained before administration have a reactivity value to the cancer vaccine by antigen stimulation equal to or greater than a predetermined value. (Item I0) A pharmaceutical kit, wherein the pharmaceutical kit is Pharmaceuticals including cancer vaccines and The device comprises a detection agent for detecting stimulation by a cancer antigen corresponding to the cancer vaccine or by the antigenic portion of the cancer antigen, The vaccine regimen is determined based on the responsiveness of patient-derived immune cells to stimulation with a cancer antigen or equivalent corresponding to the cancer vaccine, according to the kit. (Item I1) The above prediction and / or calculation, To confirm the reactivity of immune cells derived from the patient in response to stimulation by the cancer antigen corresponding to the cancer vaccine, the antigenic portion of the cancer antigen, or equivalents thereof, and to predict or calculate the effect of the cancer vaccine based on the reactivity. A kit that includes any one of the items listed above. (Item I2) The kit described in any of the above items, wherein the cancer antigen or the antigenic portion of the cancer antigen or equivalent thereof is a partial peptide protein contained in the amino acid sequence of the cancer antigen, a partial peptide protein contained in the amino acid sequence translated from the nucleic acid in the case of a nucleic acid vaccine, etc., or an equivalent thereof. (Item I3) The prediction is made using the kit described in any one of the above items, either before administration of the vaccine or during treatment with the vaccine. (Item I4) A kit according to any one of the above items, which includes obtaining immune cells derived from the patient. (Item I5) A kit according to any one of the above items, comprising providing a cancer antigen corresponding to the cancer vaccine or an antigenic portion of the cancer antigen. (Item I6) The method described above is performed in vitro using the kit described in any one of the above items. (Item I7) The prediction of the effect is made before administration of the cancer vaccine using the kit described in any one of the above items. (Item I8) The kit according to any one of the above items, wherein the immune cells include at least one selected from the group consisting of peripheral blood mononuclear cells, lymphocytes (e.g., T cells, B cells, NK cells), dendritic cells, macrophages, monocytes, and neutrophils. (Item I9) The kit according to any one of the above items, wherein the effect includes at least one selected from the group consisting of 1) the efficacy of the cancer vaccine, 2) the persistence of the cancer vaccine, 3) the determination of patients to whom the cancer vaccine should be administered, 4) the determination of the cancer vaccine to be administered, and 5) the determination of changes in the dosage and administration of the cancer vaccine. (Item I10) The means for confirming the reactivity includes means for performing ELISPOT or a functional equivalent thereof (e.g., FluoroSpot, etc.), as described in one of the above items. (Item I11) The kit described in any one of the above items, which includes means for performing ELISPOT to confirm the reactivity. (Item I12) A kit according to any one of the above items, which determines that the cancer vaccine will be effective / should be administered to a patient if the measured value (Spot Counts) by ELISPOT is equal to or greater than a predetermined value. (Item I13) 1) The efficacy of the cancer vaccine If the measurement value (Spot Counts) obtained by the ELISPOT is above a predetermined value, it is determined that the cancer vaccine is effective. 2) Durability of the cancer vaccine If the measurement value (Spot Counts) obtained by the ELISPOT is above a predetermined value, it is determined that the cancer vaccine is effective. 3) Determining which patients should receive the aforementioned cancer vaccine. If the measurement value (Spot Counts) obtained by the aforementioned ELISPOT is above a predetermined value, the patient is determined to be a patient who should be administered the cancer vaccine. 4) Determining which cancer vaccine to administer If the measurement value (Spot Counts) obtained by the aforementioned ELISPOT exceeds a predetermined value, the patient is determined to be a patient who should receive the target vaccine. A kit as described in any one of the items in item I above. (Item I14) A kit according to any one of the above items, wherein the predetermined value of the measurement (Spot Counts) by ELISPOT is 7. (Item I15) The cancer antigen is a kit according to any one of the above items, which includes all cancer antigens contained in the cancer vaccine. (Item I16) The kit according to any one of the above items, wherein the cancer antigen comprises at least one selected from the group consisting of WT1, AGEA1-4, NY-ESO-1, PRAME, SSX2, CT8, CD19, GP100, MART1, PSA, PSMA, tyrosinase, HER2, MUC1, CEA, survivin, cyclin B1, EGFR, mesothelin, telomerase, and neoantigens resulting from gene mutations in the patient's tumor cells. (Item I17) The kit according to any one of the above items, wherein the cancer antigen is WT1. (Item I18) The kit according to any one of the above items, further comprising the features described in any one or more of the above items: items 1-17, items A0-A18, items B0-B18, items C1-C19, items D0-D18, items E0-E20, items F0-F19, items G1-G19, and items H0-H19. (Item I19) The kit according to any one of the above items, characterized in that the pharmaceutical is administered to a subject whose immune cells obtained before administration have a value of responsiveness to antigen stimulation for the cancer vaccine that is equal to or greater than a predetermined value. (Item I20) The kit described in any one of the above items, wherein the pharmaceutical product is for the prevention or treatment of cancer. [Effects of the Invention]

[0005] This disclosure provides a technology for predicting the effectiveness of cancer vaccines in advance or during use. Using this disclosure, it is possible to analyze individual patient immune responses and accurately evaluate whether a cancer vaccine is likely to be effective. This technology allows for the selection of patients who are likely to benefit, avoiding ineffective treatments and identifying more effective therapies, thereby reducing the burden on patients and improving treatment efficiency.

[0006] Furthermore, this disclosure provides a process for rapidly and efficiently collecting and analyzing data necessary for predicting the effectiveness of cancer vaccines, thereby contributing to faster diagnosis and personalized treatment in clinical settings. This enables the efficient use of limited medical resources and contributes to an overall improvement in the quality of cancer treatment. [Brief explanation of the drawing]

[0007] [Figure 1]Figure 1 shows an overview of the ELISPOT assay method. Exemplarily, it can be performed by 1) isolating peripheral blood mononuclear cells from a whole blood sample, 2) stimulating them with antigenic moieties such as WT1 antigen protein or peptides, and 3) calculating the number of cells (spots) that respond to cytokines (IFN-γ). [Figure 2] Figure 2 shows an example of a treatment sequence for metastatic urothelial carcinoma. Typically, the first line is cisplatin-based chemotherapy, the second line is a PD-1 / PD-L1 inhibitor (pembrolizumab / avelumab), and the third line is an anti-Nectin-4 antibody-microtubule inhibitor conjugate (enfortumab vedotin). Since 2017, immune checkpoint inhibitors have become available as a second-line treatment, but the PFS is only 3.7 months, which is not sufficient, and even when combined with the third line, sufficient therapeutic efficacy is not achieved. While pembrolizumab + enfortumab vedotin combination therapy received approval as a first-line treatment in September 2024, and its therapeutic effect is expected to improve in the future, there remains a high need for treatments with different mechanisms of action for the second line and beyond. The B440-1 trial evaluated the safety and efficacy of this treatment in urothelial carcinoma patients who had completed up to the third line of treatment, in order to address this still-existing need. [Figure 3] Figure 3 shows the B440 administration schedule, DLT evaluation period, and adverse event / efficacy evaluation period in the B440-1 clinical trial. The WT1-ELISPOT assay was performed multiple times at the timings indicated by the red arrows. The detailed schedule of the assay timings is as follows: Day 1 (before B440 administration), Day 15, Day 28, Day 57, Day 113, Day 169. [Figure 4] Figure 4 shows the tumor reduction rates in five patients who underwent a second attempt with pembrolizumab. Of these, all three ELISPOT-positive patients (green) were able to control their disease progression with pembrolizumab (RECIST evaluation: one each of CR, PR, and SD). On the other hand, both ELISPOT-negative patients (red) were judged to have disease progression (PD). [Figure 5]Figure 5 shows the change in the mean number of spots over time in the ELISPOT-positive and ELISPOT-negative groups. Six cases (four low-dose and two high-dose) were ELISPOT-positive after B440 administration. The ELISPOT-positive group had significantly more spots on Day 0 (before B440 administration) compared to the negative group (P=0.030). This suggests that cases that become ELISPOT-positive after B440 administration have a subtle immune response to WT1 expressed by the tumor even before treatment. [Figure 6] Figure 6 shows the design of a study involving B440 administration to solid tumors expressing WT1. The results in Figure 8 suggest that ELISPOT-positive cases are likely to show a high therapeutic effect with B440, and the results in Figure 4 indicate a good response to subsequent ICI administration. Furthermore, as shown in Figure 5, ELISPOT-positive cases were also ELISPOT-positive before administration (WT1-ELISPOT pre-positive), suggesting that WT1-ELISPOT pre-positive is likely useful as a screening tool for B440 candidates. Figure 6 reflects these findings. Patients who have become resistant to prior treatment and are WT1-ELISPOT pre-positive are recruited as candidates for treatment and B440 administration is initiated. Patients who subsequently become WT1-ELISPOT post-positive are then treated with ICI. For patients who already have weak immune activity against WT1, B440 administration is expected to further stimulate a WT1-specific immune response, leading to a sustained therapeutic effect against the tumor. [Figure 7] Figure 7 shows an overview of the experimental design for Example 3. A total of 15 C57BL / 6 mice were randomly assigned to three groups (n=5), and mouse splenocytes were extracted at three time points: before MBT-2 transplantation (Day 0), after transplantation (Day 7), and after vaccine administration (Day 30), and the WT1-ELISPOT assay was performed. The vaccine was administered orally as B440 at a dose of 1 × 10⁹ CFU / 100 μl on Days 7-11, 14-18, and 21-25. [Figure 8]Figure 8 shows the Kaplan-Meier curves in the examples. Regarding the treatment response to B440, the Pre-ELISPOT positive group showed a significantly longer progression-free survival than the Pre-ELISPOT negative group. This result suggests that the number of IFN-γ producing cells produced by WT1 antigen protein stimulation, as measured by ELISOPT, is a major prognostic factor in predicting the therapeutic effect against B440. [Figure 9] Figure 9 summarizes the trends for ELISPOT (12 cases in total). The horizontal axis shows the passage of time, and the vertical axis shows the average value of ELISPOT. [Figure 10] An example of pre-vaccination stratification using ELISPOT is shown. The criteria for a weak positive were defined as 1) having at least 7 WT1-specific spots, and 2) having more WT1-specific spots than the number of spots in the negative control. [Figure 11] Figure 11 shows the mean test (Mann-Whitney U test) for ELISPOT at the pre-vaccination time point. [Figure 12] Figure 12 shows an example of stratification using ELISPOT after vaccine administration (during the treatment period). The criteria for a positive result after vaccine administration in ELISPOT were: 1) the presence of at least 15 WT1-specific spots, and 2) the presence of WT1-specific spots at least 1.5 times the number of spots in the negative control. [Figure 13] Figure 13 shows the mean test (Mann-Whitney U test) at approximately two months after the start of vaccine administration (during the treatment period). [Figure 14] Figure 14 shows the Kaplan-Meier curves in the examples. Regarding the treatment response to B440, the Post-ELISPOT-positive group showed a significantly longer progression-free survival than the negative group during the treatment period. [Figure 15] Figure 15 shows the pre- and post-administration concordance rates of ELISPOT as a confusion matrix. [Figure 16] Figure 16 shows the progress after retrying with pembrolizumab (n=5). The data is stratified by pre-ELISPOT. [Figure 17] Figure 17 shows the progress after a second attempt with pembrolizumab (n=5). The data is stratified by ELISPOT after administration (during the treatment period). [Modes for carrying out the invention]

[0008] The present disclosure is described below in best form. Throughout this specification, singular expressions should be understood to include the concept of their plural form unless otherwise specified. Accordingly, singular articles (e.g., "a," "an," "the" in English) should be understood to include the concept of their plural form unless otherwise specified. Furthermore, terms used herein should be understood to have the meaning commonly used in the art unless otherwise specified. Accordingly, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains. In case of any conflict, this specification (including definitions) shall prevail.

[0009] The following provides definitions of terms used specifically in this specification and / or basic technical concepts as appropriate.

[0010] In this specification, "approximately" refers to significant figures unless otherwise specified, but is understood to mean an equivalent amount permitted by the pharmacopoeia.

[0011] In this specification, "cancer vaccine" refers to a pharmaceutical or medical technology that aims to prevent or treat cancer by stimulating the host's immune system using antigens specific to cancer cells or cancer tissue. Cancer vaccines include protein vaccines (formulated by purifying cancer-related antigens (e.g., HER2, CEA, MUC1, etc.)); peptide vaccines (specific peptides derived from proteins overexpressed in cancer cells (e.g., WT1 peptide, gp100 peptide, etc.)); DNA vaccines (introducing DNA encoding cancer antigens into the host to promote endogenous expression of the antigens); RNA vaccines (using mRNA encoding cancer antigens to produce the antigens in the body); cell vaccines (dendritic cells derived from the patient or allogeneically loaded with cancer antigens); and viral vector vaccines (viral vectors encoding cancer antigens). (e.g., adenovirus, lentivirus, etc.); polysaccharide vaccines (targeting glycan antigens specifically present on the surface of cancer cells (e.g., GM2, MUC1-related glycans, etc.)); neoantigen vaccines (targeting neoantigens that are expressed in a tumor-specific or tumor-selective manner (neoantigens refer to specific peptide antigens that result from gene mutations occurring in tumor cells and are not present in normal cells, and are designed based on the tumor-specific mutations of each patient. Neoantigen vaccines are expected to be a personalized treatment for each patient, aiming to enhance the immune response to tumors and suppress tumor growth and metastasis)). Each of these cancer vaccines is designed to target different antigens and immune mechanisms and is used as monotherapy or in combination therapy. In this specification, "cancer vaccine" includes, in addition to the above examples, technologies that utilize newly developed peptides, proteins, nucleic acids, cells, or complexes thereof. Furthermore, the term "cancer vaccine" as used herein includes not only those used alone, but also therapies used in combination with adjuvants (e.g., monophosphoryl lipid A, aluminum salts), immunostimulatory cytokines (e.g., IL-2, GM-CSF), and immune checkpoint inhibitors (e.g., anti-PD-1 antibodies, anti-CTLA-4 antibodies).

[0012] More detailed types and specific examples of “cancer vaccines” that may be used in this disclosure include, but are not limited to, the following: 1. Peptide vaccines This vaccine uses peptides derived from specific cancer antigens, which activate T cells to attack tumors. Specific example: WT1 peptide vaccine (for leukemia, pancreatic cancer, etc.) NY-ESO-1 peptide vaccine (for multiple myeloma, melanoma, etc.) MAGEA3 peptide vaccine (for lung cancer, melanoma, etc.) PRAME peptide vaccine GP100 peptide vaccine (melanoma) HER2 peptide vaccine (breast cancer, stomach cancer) 2. Protein vaccines Purified, full-length proteins are used and processed by antigen-presenting cells to induce an immune response. Specific example: CEA (Cell-Embryonic Antigen) Vaccine (Colorectal Cancer, Pancreatic Cancer) HER2 protein vaccine (breast cancer) MUC1 protein vaccine (pancreatic cancer, breast cancer) 3. DNA vaccines A vaccine that administers DNA encoding cancer antigens to patients, inducing an immune response by producing the antigens in the body. Specific example: HPV (Human Papillomavirus) DNA Vaccine (for cervical cancer and head and neck cancer) PSA (Prostate-Specific Antigen) DNA Vaccine (for Prostate Cancer) WT1 DNA vaccine (leukemia, pancreatic cancer) 4. RNA vaccines This method uses mRNA encoding cancer antigens, which are translated in the body to express the antigens and induce an immune response. Specific example: Neoantigen RNA vaccine (personalized vaccine based on the gene mutations of the patient's tumor) KRAS mutant RNA vaccine (lung cancer, pancreatic cancer) 5. Cell-based vaccines This method utilizes patient-derived or allogeneic dendritic cells or tumor cells to present cancer antigens and stimulate the immune system. Specific example: Sipuleucel-T (prostate cancer) Dendritic cell vaccine (WT1 peptide load, NY-ESO-1 load) 6. Viral vector vaccines A vaccine that uses genetically modified viruses to express cancer antigens in the patient's body. Specific example: ProstVac-VF (prostate cancer) TG4010 (Vaccine encoding MUC1, for non-small cell lung cancer) OncoVEX (T-VEC, melanoma) 7. Polysaccharide vaccines A vaccine that targets abnormal sugar chain structures on the surface of cancer cells. Specific example: Glob H polysaccharide vaccine (for breast cancer and ovarian cancer) GM2 vaccine (melanoma) 8. Neoantigen vaccine A personalized vaccine that targets neoantigens derived from gene mutations in the patient's tumor. Specific example: NeoVax (Personalized Neo-Antigen Peptide Vaccine) Personalized Neoantigen RNA Vaccines 9. Viral vaccines A vaccine to prevent viral infections that cause tumors. Specific example: HPV vaccine (for cervical cancer and head and neck cancer) HBV vaccine (hepatocellular carcinoma) 10. Combination vaccines The vaccine is used in combination with other immunotherapies (immune checkpoint inhibitors, adjuvants, etc.) to enhance its effects. Specific example: CEA vaccine + anti-PD-1 antibody (pancreatic cancer) HER2 vaccine + anti-CTLA-4 antibody (for breast cancer).

[0013] In this specification, “cancer antigen” is interchangeably referred to as “tumor-specific antigen” or “tumor-associated antigen,” and refers to a molecule that is specifically or preferentially expressed in cancer cells or cancer tissue, and is a target substance that stimulates the host immune system to recognize and eliminate cancer cells. Once a cancer vaccine is identified, those skilled in the art can identify the corresponding cancer antigen, and in this specification, this cancer antigen is referred to as “cancer antigen for cancer vaccine” or “cancer antigen corresponding to cancer vaccine,” and these terms are used interchangeably herein. Such specific methods are described by Cheever MA, Allison JP, and Ferris. AS, Finn OJ, Hastings BM, Hecht TT, Mellman I, Prindiville SA, Viner JL, Weiner LM, Matrisian LM. The prioritization of cancer antigens: a national cancer institute pilot project for the acceleration This can be carried out based on translational research. Clin Cancer Res. 2009 Sep 1;15(17):5323-37. doi: 10.1158 / 1078-0432.CCR-09-0737. PMID: 19723653; PMCID: PMC5779623. etc.

[0014] Cancer antigens include, but are not limited to, the following types. First, tumor-specific antigens (TSAs) are molecules that are specifically expressed in cancer cells and refer to proteins newly created by gene mutations or viral infections. Specific examples include Ras mutant proteins, p53 mutant proteins, and E6 / E7 proteins derived from human papillomavirus (HPV).

[0015] In this specification, “cancer antigen” refers to a molecule that is specifically or preferentially expressed in cancer cells or cancer tissue, and is a target substance that stimulates the host’s immune system to recognize and eliminate cancer cells. Examples of cancer antigens include, but are not limited to, the following:

[0016] In other words, cancer antigens can be broadly categorized into the following types: 1. Cancer / Testis Antigens (CTAs) These are molecules that are normally expressed only in limited areas of testicular tissue and the placenta, but are abnormally expressed in cancer cells. Specific examples include MAGEA1, MAGEA2, MAGEA3, MAGEA4, NY-ESO-1, PRAME, SSX2, and CT8. 2. Tumor-associated antigens (TAAs) This refers to molecules that are expressed in normal cells but whose expression levels are abnormally increased or altered in cancer cells. Specific examples include CD19, GP100, MART1, PSA (prostate-specific antigen), PSMA (prostate-specific membrane antigen), tyrosinase, HER2, MUC1 (mucin 1), CEA (carcinoembryonic antigen), survivin, cyclin B1, EGFR (epidermal growth factor receptor), and mesothelin. 3.Tumor-Specific Antigens (TSAs) Antigens are antigens specifically expressed on cancer cells and include new proteins resulting from gene mutations or viral infections. Neoantigens, particularly those resulting from gene mutations in the patient's tumor cells, are an important example. These neoantigens are peptides derived from tumor cell-specific mutant proteins and are not present in normal cells, making them selective targets for the immune system.

[0017] These cancer antigens play a crucial role in the design and development of cancer immunotherapy, particularly cancer vaccines. For example, peptide vaccines that induce tumor antigen-specific T-cell responses, and DNA or RNA vaccines that encode tumor antigens, are used. They are also utilized as targets for tumor antigen-specific antibody therapies and CAR-T cell therapies.

[0018] In this specification, "cancer antigens" include, in addition to the examples above, peptide fragments, mutant epitopes, or nucleic acid molecules (DNA or RNA) that encode them. These antigens are intended to be used alone or in combination with adjuvants, immune checkpoint inhibitors, cytokine therapies, etc. Cancer antigens also play an important role as the foundation for personalized medicine based on individual tumor characteristics and genetic mutations of each patient.

[0019] In this specification, "cancer antigen" includes not only the molecules classified above, but also peptide fragments, mutant epitopes, or nucleic acid molecules (DNA and mRNA) that encode them. Furthermore, cancer antigens are used not only alone, but also in combination with adjuvants, immune checkpoint inhibitors, etc.

[0020] In this specification, the "antigenic portion of a cancer antigen" refers to a region of a structure called an epitope contained in a cancer antigen that is directly recognized by the host immune system and specifically binds to an antibody or T cell receptor (TCR). Antigenic portions mainly exist as proteins, peptides, glycans, or complexes thereof. Antigenic portions of cancer antigens include T cell epitopes and B cell epitopes. T cell epitopes are short peptides that are presented when a cancer antigen is processed by an antigen-presenting cell (APC) and bound to a major histocompatibility complex (MHC) class I or class II molecule. B cell epitopes, on the other hand, are three-dimensional structures or linear arrangements present on the surface of a cancer antigen and can directly bind to antibodies. For example, the WT1 peptide (a 9-11 amino acid peptide derived from WT1) is recognized as a T cell epitope. In addition, specific structural portions of the glycan antigens GM2 and GD2 are B cell epitopes recognized by antibodies. Furthermore, techniques may be used to enhance the immunogenicity of the antigenic moiety by conjugating it with adjuvants (e.g., aluminum salts or monophosphoryl lipid A) or carrier proteins (e.g., KLH, BSA). In this specification, "antigenic moiety of cancer antigen" includes not only naturally occurring structures but also synthetic peptides, modified glycans, or genetically engineered epitopes. These antigenic moieties are intended for use in therapy, as well as for predicting and diagnosing the efficacy of the therapeutic agents disclosed herein.

[0021] In this specification, "immune cells" refers to a group of cells belonging to the host's immune system that are responsible for recognizing and eliminating foreign substances, pathogens, tumor cells, and other foreign objects. Immune cells can be broadly divided into cells involved in innate immunity and cells involved in adaptive immunity. Specifically, these include peripheral blood mononuclear cells, T cells, B cells, natural killer (NK) cells, macrophages, dendritic cells, neutrophils, eosinophils, mast cells, regulatory T cells (Treg cells), and tumor-associated macrophages (TAMs). Immune cells involved in innate immunity mainly include the following cells. First, macrophages engulf and break down foreign substances and present this information to other immune cells. Next, dendritic cells function as antigen-presenting cells and play an important role in activating the adaptive immune system. Natural killer (NK) cells are cells that directly attack and eliminate infected cells and tumor cells. In addition, neutrophils, eosinophils, and mast cells are also involved in controlling inflammatory responses and parasitic infections. Immune cells involved in adaptive immunity primarily include T cells and B cells. T cells include helper T cells (CD4-positive) and killer T cells (CD8-positive), which perform regulatory and cytotoxic functions of the immune response, respectively. B cells produce antibodies and play a role in humoral immunity. Memory T cells and memory B cells retain memories of past antigens, enabling a rapid response during reinfection. Furthermore, recent research has shown they play an immunosuppressive role. The roles of specialized immune cells such as regulatory T cells (Treg cells) and tumor-associated macrophages (TAMs), which play a crucial role in the tumor microenvironment, are also attracting attention. In this specification, "immune cells" include, in addition to the above, genetically modified immune cells (e.g., CAR-T cells and CAR-NK cells). These immune cells are used alone or in combination with other therapies to treat infectious diseases, cancer, autoimmune diseases, and other conditions, as well as for predicting and diagnosing the effectiveness of the therapeutic agents disclosed herein.

[0022] In this specification, "Peripheral Blood Mononuclear Cells (PBMCs)" refers to mononuclear white blood cells present in peripheral blood that play a central role in the host's immune and inflammatory responses. Peripheral blood mononuclear cells mainly include, but are not limited to, T cells, B cells, natural killer (NK) cells, and monocytes. T cells are lymphocytes belonging to the adaptive immune system and are further classified into helper T cells (CD4-positive) and cytotoxic T cells (CD8-positive). They recognize antigens presented by antigen-presenting cells and activate the immune response or eliminate infected or tumor cells. B cells are lymphocytes that produce antibodies and play an important role in humoral immunity. Memory B cells also retain memories of past antigens, enabling a rapid immune response in the event of reinfection. NK cells are lymphocytes belonging to the innate immune system and can directly attack cells with reduced expression of major histocompatibility complex (MHC) molecules, as well as infected and tumor cells. Monocytes function as progenitor cells that differentiate into macrophages and dendritic cells, regulating immune responses through phagocytosis, antigen presentation, and cytokine production. Furthermore, peripheral blood mononuclear cells are used as important research materials in cancer immunotherapy, infectious disease research, and the development of treatments for autoimmune diseases. For example, dendritic cells and T cells isolated from PBMCs are used as the basis for cancer vaccines and T cell therapies (e.g., CAR-T cells). In addition, ELISPOT and FluoroSpot assays using PBMCs are standard methods for evaluating antigen-specific immune responses. In this specification, "peripheral blood mononuclear cells" includes not only the above-mentioned cell groups, but also genetically modified cells and cells prepared by culture or differentiation induction.

[0023] In this specification, “stimulation” (by a cancer antigen or antigenic moiety) refers to the operation or action of exposing an antigen to the host’s immune system in order to induce or enhance an antigen-specific immune response. This stimulation may occur in vivo or extracellularly and activates immune cells such as antigen-presenting cells, T cells, B cells, and natural killer (NK) cells, triggering an adaptive or innate immune response. Embodiments of this specification typically assume in vitro stimulation. Stimulation of an antigen involves the following processes: First, the antigen is taken up by antigen-presenting cells (APCs), such as dendritic cells and macrophages, processed, and then bound to major histocompatibility complex (MHC) molecules and presented on the cell surface. This presented antigen fragment (epitope) is recognized by helper T cells (CD4-positive) or cytotoxic T cells (CD8-positive), initiating an immune response. In stimulation of B cells, the antigen binds to the B cell receptor (BCR), triggering antibody production or class switching. Antigen stimulation is performed using externally added synthetic antigens, peptide antigens, glycosylated antigens, or viral vectors. It is also common to enhance the immune response by using adjuvants (e.g., aluminum salts, monophosphoryl lipid A) or cytokines (e.g., interleukin-2, interferon-γ) in combination with this stimulation. In vitro, immune cells can be isolated and stimulated with antigens to proliferate or activate antigen-specific T cells and B cells. For example, in ELISPOT and FluoroSpot assays using peripheral blood mononuclear cells (PBMCs), antigen stimulation is performed to evaluate the cellular immune response to an antigen. Furthermore, the creation of genetically modified immune cells such as CAR-T cells also includes a step of activating target T cells using antigen stimulation. In this specification, "stimulation" includes not only naturally occurring antigen stimulation but also stimulation intentionally induced using artificially designed antigens or adjuvants.

[0024] In this specification, "reactivity to immune cells (e.g., peripheral blood mononuclear cells)" refers to the effect or responsiveness of an antigen or stimuli on immune cells, including changes in cell activation, differentiation, proliferation, cytokine secretion, or attack ability against target cells. Peripheral blood mononuclear cells (PBMCs) are particularly used as a standard model for evaluating reactivity to antigens and stimuli because they are composed of a diverse population of immune cells. The main cell groups that make up peripheral blood mononuclear cells include T cells, B cells, natural killer (NK) cells, and monocytes. Reactivity to antigenic stimulation is measured by evaluating the processes by which these cell groups respond specifically or nonspecifically. For example, T cells recognize specific antigens, become activated, and exhibit responsiveness by secreting cytokines (e.g., interferon-γ, interleukin-2). Monocytes, on the other hand, play a role in the initial stages of the immune response through phagocytosis and antigen presentation. One representative method for measuring reactivity to immune cells is the ELISPOT assay. The ELISPOT assay is a technique for detecting single immune cells that secrete cytokines or antibodies in response to specific antigens. In this method, privately-mediated cell mass cells (PBMCs) are stimulated with the antigen, and the secreted molecules (e.g., interferon-γ, IL-2) are captured by immobilized antibodies. These are then visualized as spots using detection antibodies and enzymatic reactions, and the number of secreting cells is quantitatively evaluated. The number of spots corresponds to the number of cells exhibiting an antigen-specific response.

[0025] In this specification, the following are examples of indicators and measures of responsiveness: 1. Number of spots: In ELISPOT, the number of spots generated after antigen stimulation is a direct indicator of reactivity. For example, 50 to 500 spots per 100,000 PBMCs is a common range, and varies depending on the type and concentration of the antigen. Example 1: In the response of PBMCs to tumor antigens (cancer antigens), there may be 5 or more spots, 7 or more spots, 10 or more spots, 15 or more spots, 75 or more spots per 100,000 PBMCs, and 5 to 500 spots per 100,000 PBMCs is usually adopted in the response to tumor antigens (cancer antigens). If there are 5 to 15 or more spots, preferably 7 or more spots, it can be used for pre-treatment prediction, and if there are 10 or more spots, preferably 15 or more spots, it can be used for prognosis prediction during the treatment period. 2. Stimulation Index (SI): The number of spots after antigen stimulation divided by the number of spots in the unstimulated control (background response). Example: If the number of spots in the unstimulated control is 10 and the number of spots after antigen stimulation is 200, the SI is 20. Generally, an SI of 2 or higher is considered a significant response. An SI < 1 can be used for pre-treatment prediction, and an SI of 1.5 or higher can be used for prediction during treatment. 3. Cytokine levels: Responsiveness is supplemented by measuring the amount of secreted cytokines using ELISA or flow cytometry in conjunction with ELISPOT. Example 1: The concentration of interferon-γ secreted by PBMCs in response to antigen stimulation increases from 0.1 ng / mL without stimulation to 0.2 ng / mL (the stimulated value is used as the relative value to the unstimulated value). Example 2: The concentration of IL-2 may increase from 100 pg / mL without stimulation to 150 pg / mL with stimulation. As a relative value (stimulated / unstimulated), for example, if it is 1.1 or higher, preferably 1.5 or higher, it can be judged that there is responsiveness.

[0026] In this specification, "responsiveness to immune cells (e.g., peripheral blood mononuclear cells)" includes not only evaluation using the indicators and scales described above, but also the effects of drugs, adjuvants, immune checkpoint inhibitors, or genetic modification technologies on immune cells. Furthermore, this evaluation of responsiveness is intended to be applied to infectious diseases, cancer, autoimmune diseases, transplant medicine, vaccine development, and the development of novel immunotherapies.

[0027] In this specification, "prediction of cancer vaccine effectiveness" refers to quantitatively or qualitatively evaluating the expected immune response or therapeutic effect of administering a cancer vaccine and optimizing the treatment plan based on the results. This prediction of effectiveness is performed by comprehensively analyzing the patient's immune profile, responsiveness to cancer antigens, the presence of immunosuppressive factors, and the characteristics of the cancer vaccine itself.

[0028] The following are specific examples of "predictions of the effectiveness of cancer vaccines." 1. Efficacy of cancer vaccines This method predicts the efficacy (treatment success rate) of cancer vaccines. For example, by measuring the frequency and intensity of the patient's immune cells' response to specific cancer antigens using methods such as the ELISPOT assay or flow cytometry, efficacy can be predicted. 2. Durability of cancer vaccines This involves predicting the duration of the therapeutic effect of cancer vaccines. This includes evaluating the level of induction of immune memory cells (memory T cells or memory B cells). For example, measuring the immune response at 6 or 12 months after vaccination is used to assess the duration of the effect. 3. Determining which patients should receive cancer vaccines. Patients who are likely to respond to cancer vaccines are selected. This is done by analyzing the patient's HLA type, cancer antigen expression levels, and the presence of immunosuppressive factors (e.g., Treg cells and MDSCs) in the tumor microenvironment. 4. Determining which cancer vaccine to administer. The optimal cancer vaccine is selected based on the patient's cancer type, stage, and specific antigens expressed in the tumor. For example, a HER2 peptide vaccine is recommended for HER2-positive breast cancer patients, and a WT1 peptide vaccine is recommended for WT1-high-expressing leukemia patients. 5. Determining other medications and / or nutritional supplements to be administered. To enhance the effectiveness of cancer vaccines, the administration of immune checkpoint inhibitors (e.g., anti-PD-1 antibodies, anti-CTLA-4 antibodies), immunostimulatory cytokines (e.g., IL-2, GM-CSF), and nutritional supplements (e.g., vitamin D and amino acid supplements) may be considered. 6. Decision on changes to the dosage and administration of cancer vaccines

[0029] Adjust the vaccine dosage and administration schedule. For example, continuously monitor the patient's immune response and, if the initial immune response is insufficient, increase the vaccine dosage or shorten the administration interval.

[0030] These "predictions of the effectiveness of cancer vaccines" are based on scientific evidence, using methods such as immune monitoring and biomarker analysis, taking into account the individual patient's condition and responsiveness. The predictions of effectiveness described herein contribute to the optimization of treatment plans, the streamlining of the cancer vaccine development process, and the improvement of treatment outcomes.

[0031] In this specification, "before or during vaccine administration / treatment (also referred to as after the first dose)" refers to the timing for evaluating or predicting the effectiveness of a cancer vaccine, and specifically includes the condition before vaccine administration and the period after the first dose of the vaccine during which its effectiveness and immune response are monitored. Evaluation during this period is important for comprehensively analyzing the patient's immune status and treatment response and determining the optimal treatment plan. In the pre-vaccination stage, the likelihood of vaccine efficacy is predicted by analyzing the patient's cancer antigen expression level, HLA type, profile of immune cells in peripheral blood (e.g., proportion of T cells, B cells, natural killer cells, and monocytes), and the state of immunosuppressive factors in the tumor microenvironment (e.g., Treg cells and MDSCs). In addition, a safe and effective vaccine administration plan is formulated by confirming the presence or absence of underlying diseases and concomitant medications. In the vaccine administration / treatment stage (also referred to as after the first dose), the patient's immune response after the first dose is evaluated, and the treatment plan is adjusted based on that response. During this period, antigen-specific immune responses (e.g., increased interferon-gamma secreting T cells) and changes in immunosuppressive factors are monitored using ELISPOT assays, flow cytometry, or cytokine assays. Furthermore, the patient's clinical improvement and the presence of side effects are assessed, and adjustments to the dosage, schedule, or the addition of combination therapies are considered. This detailed evaluation and monitoring before, during, or after vaccine administration (also known as post-first dose) enables the optimization of treatment plans tailored to each individual patient's condition. This process is also a crucial element in maximizing the effectiveness of cancer vaccines and improving patient outcomes.

[0032] In this specification, "ELISPOT (Enzyme-Linked ImmunoSpot)" refers to a technology for detecting cytokines and antibodies secreted by immune cells at the single-cell level. It is used to quantitatively evaluate the function of immune cells that respond to specific antigens and plays a particularly important role in cancer immunotherapy, infectious disease research, vaccine development, and the analysis of autoimmune diseases. ELISPOT is performed using the following procedure: First, a capture antibody specific to the target of detection (e.g., cytokine or antibody) is immobilized on the bottom of a plate. Then, peripheral blood mononuclear cells (PBMCs) and other immune cells are cultured in the plate with the antigen or stimulant to capture secretions secreted by the immune cells. Subsequently, a detection antibody that binds to the secretions and enzyme-labeled streptavidin are added, and the sites where the secretions are present are visualized as spots using an enzymatic reaction. Finally, the number of antigen-specific secreting cells is quantitatively evaluated by counting the number of visualized spots. The following are some of the characteristics of ELISPOT: This technology can detect antigen-specific immune responses at the single-cell level and has high sensitivity and specificity. In addition, quantitative evaluation is possible because the number of secreting cells is directly counted. Specific applications of ELISPOT include evaluating T cells that respond to tumor antigens in cancer immunotherapy, measuring pathogen-specific immune responses in infectious diseases, monitoring immune responses after vaccination, analyzing abnormal autoantigen responses in autoimmune diseases, and evaluating immune responses to donor antigens in transplant medicine.

[0033] Furthermore, ELISPOT is also useful in evaluating the effectiveness of cancer vaccines. For example, by measuring the frequency with which antigen-specific T cells secrete cytokines such as interferon-γ using patient PBMCs before or during vaccine administration, it is possible to predict vaccine efficacy and the duration of the immune response.

[0034] In this specification, "ELISPOT" includes all of the above technologies, as well as their variations and improvements (for example, FluoroSpot technology). Furthermore, the measurement of immune responses using ELISPOT contributes to the optimization of cancer immunotherapy, vaccine development, and treatment strategies for autoimmune diseases.

[0035] In this specification, "FluoroSpot" refers to a technology based on ELISPOT (Enzyme-Linked ImmunoSpot) technology that uses fluorescent labeling to simultaneously detect multiple cytokines and antibodies in a single assay. FluoroSpot is used to comprehensively evaluate the function of immune cells responding to specific antigens and plays a crucial role in cancer immunotherapy, infectious disease research, vaccine development, and the analysis of autoimmune diseases. FluoroSpot analysis is performed using the following procedure: First, multiple capture antibodies that specifically detect different targets (e.g., multiple cytokines and antibodies) are immobilized on the bottom of a multi-well plate. Then, peripheral blood mononuclear cells (PBMCs) and other immune cells are cultured with specific antigens or stimulants to capture multiple secretions released by the cells. Subsequently, fluorescently labeled detection antibodies corresponding to the secretions are added, visualizing each target molecule with a different fluorescence spectrum. Spots are detected using a fluorescence plate reader, and the number of cells secreting each cytokine or antibody is quantified.

[0036] The most significant feature of FluoroSpot is its ability to simultaneously measure multiple molecules secreted from a single cell (e.g., interferon-γ and IL-2), enabling detailed evaluation of cellular versatility and the quality of the immune response. This feature allows for efficient analysis of complex immune responses in a single assay. Specific applications include: for example, in cancer immunotherapy, evaluating the multiple functions of T cells responding to tumor antigens (e.g., diverse cytokine secretion) to predict the effectiveness of immunotherapy. FluoroSpot overcomes the limitations of conventional ELISPOT, which can only detect a single target molecule, by simultaneously evaluating multiple targets, thus more accurately reflecting the quality and intensity of the immune response. Furthermore, in cancer vaccine and immunotherapy research, detailed profiling of patient-specific immune responses enables the design of personalized treatments and prediction of efficacy. In this specification, "FluoroSpot" refers to the entire technology described above, including its variations and improvements. Analysis of versatility immune responses using FluoroSpot contributes to cancer treatment, infectious disease research, vaccine development, and the optimization of treatment for autoimmune diseases.

[0037] In addition to ELISPOT and FluoroSpot, the following can also be listed.

[0038] In this specification, techniques for single-cell secretion analysis include many similar techniques and methods in addition to ELISPOT and FluoroSpot. These techniques are used according to different analytical objectives and applications, enabling detailed evaluation of the characteristics and diversity of immune responses.

[0039] 1. ISOCODE (IsoPlexis) ISOCODE is an assay developed by IsoPlexis that utilizes microchip technology. This technology can simultaneously detect multiple cytokines and secretory factors at the single-cell level and is used to analyze the diversity of immune responses and the multifunctionality of cells. 2. ELISA (Enzyme-Linked Immunosorbent Assay) ELISA is positioned as a foundational technology for ELISPOT, but it differs in that it measures the total amount of secretions from the entire cell population. ELISA is widely used as a simple and highly sensitive method. 3. CBA (Cytokine Bead Array) CBA is an analytical technique that uses antibodies bound to beads. This method utilizes fluorescent labeling, allowing for the simultaneous measurement of multiple cytokines and chemokines in a single sample, making it suitable for cytokine profile analysis. 4. MACSima TM Imaging MACSima TM This technology enables high-resolution spatial distribution analysis of proteins and intracellular factors secreted by specific cells, making multi-omics analysis possible. 5. FACS-based secretion assay This method utilizes flow cytometry to detect secretions (e.g., cytokines) captured on the cell surface using antibodies. This allows for the evaluation of the functional characteristics of single cells. 6. Microfluidic Single-Cell Analysis This technology utilizes microfluidic chips to analyze secretions from single cells. By individually isolating cells, the detection sensitivity of secretions is improved, enabling high-throughput analysis. 7. DropSeq (Droplet Technology) Microdroplet technology is used to isolate single cells into each droplet, allowing for analysis of secretions and gene expression within them. This enables highly accurate evaluation of immune responses and secretion profiles. 8. Mes Scale Discovery (MSD) This is a multiplexing analysis technique using electrochemical luminescence, which allows for highly sensitive measurement of multiple types of cytokines within a single well. 9. Imaging Mass Cytometry(IMC) This imaging method utilizes mass spectrometry to spatially detect cellular secretions and simultaneously analyze multiple factors. It is used for high-resolution analysis of tissue slices and the immune environment. 10. Luminex assay This method utilizes multiplex suspension array technology, employing beads to simultaneously detect multiple cytokines and secreted proteins. It is suitable for high-throughput cytokine profiling.

[0040] These technologies are optimized and selected according to their respective applications and analytical objectives. For example, ELISPOT and FluoroSpot are suitable for highly sensitive detection of specific cytokines at the single-cell level, while CBA and Luminex excel at analyzing secretion profiles at the population level. The selection of these technologies must be appropriate depending on the research objectives, the characteristics of the secretions to be analyzed, and the cells or tissues being analyzed. This specification includes all of these technologies, as well as their applications and improvements.

[0041] In this specification, “vaccine regimen” refers to a treatment plan that includes the administration schedule, dosage, number of doses, and adjuvant therapies (such as adjuvants and immune checkpoint inhibitors) used in combination with a cancer vaccine. Vaccine regimens are designed to maximize the patient’s immune response and minimize side effects.

[0042] Specifically, the following elements are included in the vaccine regimen: 1. Dosage schedule This refers to the interval and duration of vaccine administration. Generally, multiple booster doses are given after the initial dose (priming). For example, a schedule is set where booster doses are given every two or four weeks after the initial dose, and treatment continues for several months. 2. Dosage This refers to the amount of vaccine administered (e.g., in μg or mg units of peptides or proteins, or in μg units of DNA / RNA). The dosage is adjusted based on the patient's weight, age, and immune status. 3. Number of administrations This refers to the total number of doses of the vaccine administered. For example, it may be 3, 5, or adjusted based on the patient's immune response. 4. Route of administration This refers to the method by which a vaccine is administered, which includes subcutaneous, intramuscular, intravenous, or oral administration. For example, peptide vaccines are usually administered subcutaneously or intramuscularly, while DNA / RNA vaccines are commonly administered intramuscularly. 5. Adjuvants for concomitant use These are adjuvants used to enhance the effectiveness of vaccines and play a role in optimizing the immune response. For example, monophosphoryl lipid A (MPL), aluminum salts, or GM-CSF are used. 6. Combination therapy These include immune checkpoint inhibitors (e.g., anti-PD-1 antibodies, anti-CTLA-4 antibodies), cytokines (e.g., IL-2), or other immunomodulatory agents used in combination with vaccines. Combination therapy is used to enhance the effects of cancer vaccines and overcome the immunosuppressive environment. 7. Monitoring and Evaluation The regimen includes regular monitoring of the patient's immune response and clinical response to evaluate the vaccine's effectiveness. This may involve ELISPOT assays, flow cytometry, cytokine measurements, or imaging studies of tumor reduction. Example: Peptide vaccine regimen: 20 μg of peptide is administered subcutaneously with an adjuvant (e.g., montmilron ITE) every two weeks for six doses. The immune response is monitored, and boost doses are added as needed. DNA vaccine regimen: 100 μg of DNA is administered intramuscularly, followed by three boost doses at 4-week intervals after the initial dose. Anti-PD-1 antibody is administered concurrently during the treatment period. RNA vaccine regimen: 50 μg of mRNA is encapsulated in lipid nanoparticles (LNPs) and administered intramuscularly every three weeks for five doses. In this specification, "vaccine regimen" refers to a treatment plan that includes the above elements and is adjusted according to the patient's immune status and tumor characteristics in order to realize personalized medicine for the patient. Furthermore, regimen optimization is important to maximize vaccine efficacy and improve treatment outcomes.

[0043] In this specification, “vaccine kit” refers to a package containing the components and associated auxiliary items necessary for the proper administration or use of a cancer vaccine. Vaccine kits are designed for accurate and effective administration to patients and are supplied as a single unit. The kit includes, but is not limited to, the following components: 1. Vaccine components It is a central component of the kit and is used for therapeutic or preventative purposes. Peptide vaccines (e.g., WT1 peptide, MAGEA3 peptide, etc.) Protein vaccines (e.g., HER2 protein, MUC1 protein, etc.) DNA / RNA vaccines (e.g., DNA or mRNA encoding cancer antigens) Cell-based vaccines (e.g., dendritic cell vaccines, vaccines using patient-derived tumor cells) Viral vector vaccines (e.g., adenovirus vectors encoding tumor antigens) 2. Adjuvants (immune support agents) Ingredients included to enhance the immune response to vaccines. Monophosphoryl Lipid A (MPL) Aluminum salt GM-CSF (Granulocyte-Macrophage Colony-Stimulating Factor) CpG oligonucleotides 3. Diluent or solvent for administration It contains solvents and diluents used in preparing vaccines. Examples: Sterile saline, phosphate buffer (PBS), sterile water 4. Administration device Items necessary for administering vaccines to patients. Syringe: Used to accurately measure and administer the required dose. Needle: Suitable for subcutaneous, intramuscular, or intravenous administration of vaccine. Patch delivery system: When transdermal administration is possible. 5. Instructions for Use A detailed instruction manual outlining how to use all the ingredients included in the kit. Vaccine preparation procedure Route of administration (e.g., subcutaneous, intramuscular, intravenous, etc.) Dosage and schedule Storage conditions and expiration date 6. Packaging for storage and transport A container for storing and transporting vaccine components at appropriate temperatures and conditions. Cooling box: A device for maintaining a refrigerated temperature of 2-8°C. Lyophilized vials: Lyophilized vials for vaccines that can be stored for a long period of time. 7. Additional auxiliary items Auxiliary items necessary for smooth administration. Disinfectant alcohol swabs sterile gloves Disposal containers (e.g., Sharps containers for hypodermic needles) Uses and Benefits Precise Dosage: Because it contains all the necessary ingredients, it allows for accurate and effective administration to the patient. Convenience: The kit is all in one package, making preparation in medical settings easy. Safety: All ingredients are properly sterilized and packaged, reducing the risk of infection.

[0044] In this specification, "vaccine kit" includes the above-mentioned components, as well as individualized combinations tailored to specific cancer types and patient conditions. Furthermore, cancer vaccine kits are intended to be used alone or in combination with therapies (e.g., immune checkpoint inhibitors or chemotherapy drugs), and are an important tool for realizing personalized medicine.

[0045] In this specification, "level" refers to the quantitative or qualitative state of a specific measurement target (e.g., immune response, molecular concentration, cell percentage, etc.) and means an indicator that serves as a standard for determining treatment strategies and evaluations based on the results. Levels are used as an important factor in determining the effectiveness of cancer vaccines, the patient's immune response, or optimizing treatment plans. Specifically, the term "level" applies in the following situations: 1. Evaluation of the immune response: For example, the amount of cytokines (e.g., interferon-γ) secreted when patient-derived immune cells (e.g., peripheral blood mononuclear cells) are stimulated with cancer antigens or their antigenic regions is measured, and it is evaluated whether the value is above a certain standard. In this case, "level" refers to the magnitude of the secretion (e.g., pg / mL or number of spots), and is used as an indicator to predict the efficacy of cancer vaccines. 2. Monitoring of treatment effectiveness: After administration of the cancer vaccine, the patient's immune response is measured regularly, and the changes are evaluated as a "level." For example, the percentage of specific T cells and the induction rate of memory T cells after vaccine administration are measured using flow cytometry, and the extent to which these have increased compared to the pre-treatment state is indicated. Based on this "level," the need for additional vaccine administration or combination therapy is determined. 3. Application as a Reference Value: "Level" is also used as a reference value in selecting treatment targets and adjusting treatment plans. For example, in the ELISPOT assay, if the number of specific cytokine-secreting cells is 200 spots or more per 100,000 PBMCs, the patient is considered to meet the criteria for cancer vaccine application. "Level" may be set as a quantitative indicator or classified as a qualitative category representing the patient's immune status (e.g., high level, medium level, low level). In this specification, "Level" comprehensively refers to these situations and functions as a scientific and practical standard for evaluating the applicability and effectiveness of treatment. This makes it an important indicator for achieving precision medicine and improved treatment outcomes based on individual patient immune response data.

[0046] In this specification, "level information" refers to quantitative or qualitative data concerning a specific measure (e.g., immune response, molecular concentration, cell percentage, gene expression, etc.), and is fundamental information used to assess a patient's condition and determine the appropriateness and strategy for treatment. "Level information" plays a crucial role in planning cancer vaccine administration, evaluating treatment efficacy, and developing treatment strategies tailored to individual patient characteristics. 1. Specific examples of level information The "level information" includes the following data: Cytokine secretion: For example, the concentration of cytokines such as interferon-γ (IFN-γ) and IL-2 secreted after stimulation by a cancer antigen or its antigenic moiety (unit: pg / mL or number of spots). Cell percentage: For example, the percentage of CD4-positive T cells, CD8-positive T cells, or memory T cells (CD45RO-positive) obtained by flow cytometry analysis. Molecular marker expression level: The expression level of molecular markers (e.g., PD-L1, HER2, MAGEA3) in tumor cells and immune cells. Gene expression profile: Expression levels of genes related to cancer antigens or neoantigens (e.g., RNA sequencing data). 2. Uses of Level Information "Level information" is used for the following purposes: Assessment of therapeutic eligibility: This is used to select patients who should receive the cancer vaccine. For example, if the cytokine secretion level of specific T cells is above a certain threshold, the patient is considered eligible for vaccine administration. Monitoring of treatment effectiveness: Compare "level information" before and after vaccine administration to evaluate changes in the immune response and efficacy due to treatment. Developing an individualized treatment plan: Design the optimal administration regimen (dosage, schedule, combination therapy, etc.) based on "level information." 3. How to obtain level information "Level information" is obtained using various measurement techniques. For example: ELISPOT assay: Quantifies the number of cytokine-secreting cells and evaluates antigen-specific T cell responses. Flow cytometry (FACS): Analyzes activation markers (e.g., CD69, CD25) and the proportion of memory T cells. CBA or Luminex assay: Simultaneously measures the concentrations of multiple cytokines. Genetic analysis: Next-generation sequencing (NGS) is used to obtain gene expression data related to neoantigens and tumor antigens. 4. The significance of level information "Level-based information" functions as the foundation for personalized medicine and contributes to the refinement of treatment strategies. This enables treatment plans that take into account the differences in immune responses among patients, leading to improved treatment efficacy and minimized side effects. This information is also used for selecting patient groups for treatment and for objectively evaluating treatment efficacy in clinical trials. In this specification, "level-based information" refers to information that is essential for quantitatively or qualitatively evaluating immune responses and molecular data to maximize the effectiveness of cancer vaccine therapy and other immunotherapies. Based on this, it is possible to formulate an optimal treatment plan for each patient and realize personalized medicine.

[0047] In this specification, "subject" or "subject(s)" means an entity that is the subject of diagnosis, detection, or treatment, etc., as disclosed herein (for example, a living organism such as a human, or cells, blood, serum, etc., extracted from a living organism).

[0048] In this specification, "treatment" means, with respect to a disease or disorder (e.g., cancer, allergy), preventing the worsening of such disease or disorder when such a condition occurs, preferably maintaining the current state, more preferably reducing it, and even more preferably eliminating it, thereby exhibiting a symptom-improving or preventive effect on the patient's disease or one or more symptoms associated with the disease. This includes the possibility of doing so. Providing a diagnosis in advance and then taking appropriate treatment is called "companion therapy," and the diagnostic drugs used for this purpose are sometimes called "companion diagnostic drugs."

[0049] In this specification, “therapeutic agent” broadly refers to any agent capable of treating a target condition (e.g., diseases such as cancer or allergies). In one embodiment of this disclosure, “therapeutic agent” comprises an active ingredient and one or more pharmacologically acceptable carriers. It may also be a pharmaceutical composition. A pharmaceutical composition can be manufactured, for example, by mixing an active ingredient with the carrier and using any method known in the field of pharmaceutical technology. Furthermore, the therapeutic agent is not limited in its form of use as long as it is used for treatment, and may be an active ingredient alone or a mixture of the active ingredient with any other ingredient. Furthermore, the shape of the carrier is not particularly limited, and may be a solid or a liquid (for example, a buffer solution). The therapeutic agents for cancer, allergies, etc. include drugs used for the prevention of cancer, allergies, etc. (preventive drugs), or inhibitors of cancer, allergies, etc.

[0050] In this specification, “prevention” means preventing a disease or disorder (e.g., allergy) from occurring before it occurs. The agents disclosed herein can be used to make a diagnosis and, if necessary, to prevent allergies or other conditions, or to take preventive measures.

[0051] In this specification, "preventive medicine" broadly refers to any medicine that can prevent a desired condition (for example, diseases such as allergies).

[0052] In this specification, "kit" means the parts that are to be provided, usually divided into two or more sections. This refers to a unit that provides (for example, a test reagent, diagnostic reagent, therapeutic agent, antibody, label, instructions, etc.). This kit form is preferred when the aim is to provide a composition that should not be provided mixed for stability or other reasons, but is preferably mixed immediately before use. Such a kit is preferably advantageous to include instructions or instructions describing how to use the provided parts (for example, how to use the test reagent, diagnostic reagent, therapeutic agent, or how to handle the reagents). When a kit is used as a reagent kit in this specification, the kit usually includes instructions describing how to use the test reagent, diagnostic reagent, therapeutic agent, antibody, etc.

[0053] In this specification, “Instructions” are instructions for a physician or other user on how to use the Disclosure. These instructions include language instructing the administration of the detection method, diagnostic agent, or drug of the Disclosure. The instructions may also include language instructing the administration site, such as oral or esophageal administration (e.g., by injection). These instructions are prepared in accordance with the format prescribed by the supervisory authority of the country where the Disclosure is implemented (e.g., the Ministry of Health, Labour and Welfare in Japan, or the Food and Drug Administration (FDA) in the United States) and are clearly indicated as having been approved by that supervisory authority. The instructions are a package insert and are usually provided in paper format, but are not limited to that, and may also be provided in electronic format (e.g., a homepage provided on the Internet, email).

[0054] In this specification, "agent," "agent," or "factor" (all equivalent to "agent" in English) may broadly refer to any substance or other element (e.g., energy such as light, radioactivity, heat, or electricity) that can be used interchangeably and achieve the intended purpose. Such substances include, but are not limited to, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (e.g., DNA such as cDNA and genomic DNA, RNA such as mRNA), polysaccharides, oligosaccharides, lipids, small organic molecules (e.g., hormones, ligands, signaling molecules, small organic molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (e.g., small molecule ligands, etc.)), and complex molecules thereof. Typical examples of factors specific to polynucleotides include, but are not limited to, polynucleotides that complement the sequence of the polynucleotide with a certain degree of sequence homology (e.g., 70% or more sequence identity), and polypeptides such as transcription factors that bind to promoter regions. Examples of polypeptide-specific factors include, but are not limited to, antibodies or derivatives or analogues specifically targeted to that polypeptide (e.g., single-chain antibodies), specific ligands or receptors when the polypeptide is a receptor or ligand, and substrates when the polypeptide is an enzyme. The antigenic portion of the cancer antigen used in this disclosure may be used as an agent, in which case it refers to a partial peptide protein contained in the amino acid sequence of the cancer antigen, or in the case of a nucleic acid vaccine, a partial peptide protein contained in the amino acid sequence translated from the nucleic acid. In this specification, "functional equivalent" is also simply referred to as "equivalent," and refers to any entity that has the same intended function as the original entity being referred to, but differs in structure. This includes the entity itself (e.g., cancer antigen or the antigenic portion of said cancer antigen), as well as variants or modifiers of the entity (e.g., amino acid sequence modifiers in the case of proteins) that have the same effect as the entity, and entities that can be transformed into the entity itself or its variants or modifiers at the time of action (e.g., nucleic acids encoding the entity itself or variants or modifiers of entity S, vectors containing such nucleic acids, cells, etc.). In this disclosure, it is understood that functional equivalents of the entity can be used in the same way as the entity, even if not specifically mentioned. An equivalent of a cancer antigen or the antigenic portion of said cancer antigen may be a functional equivalent of this disclosure if it has the same function as the cancer antigen or the antigenic portion of said cancer antigen (which may be used as a cancer vaccine or for diagnostic purposes as described in this disclosure).

[0055] (Preferred embodiment) Preferred embodiments of the Disclosure are described below. The embodiments provided below are provided for a better understanding of the Disclosure, and the scope of the Disclosure should not be limited to the descriptions below. It will be apparent that those skilled in the art can make appropriate modifications within the scope of the Disclosure, taking into consideration the descriptions herein. Furthermore, the embodiments of the Disclosure below can be used individually or in combination.

[0056] (Technologies for predicting the effectiveness of cancer vaccines and related technologies) In the context of this disclosure, the disclosure provides a method for predicting the efficacy of a cancer vaccine, which includes confirming the reactivity of patient-derived immune cells (e.g., peripheral blood mononuclear cells) to stimulation with a cancer antigen corresponding to the cancer vaccine or an antigenic portion of the cancer antigen or an equivalent thereof, and predicting and / or calculating the efficacy of the cancer vaccine (e.g., response (response and duration) / determining which patients should receive the vaccine) based on the reactivity.

[0057] In one aspect, the present disclosure provides an agent for predicting the efficacy of a cancer vaccine, wherein the agent comprises a cancer antigen corresponding to the cancer vaccine or an antigenic portion of the cancer antigen or an equivalent thereof, and the agent confirms the reactivity of patient-derived immune cells (e.g., peripheral blood mononuclear cells) to stimulation by the cancer antigen corresponding to the cancer vaccine or an antigenic portion of the cancer antigen or an equivalent thereof, and predicts and / or calculates the efficacy of the cancer vaccine (e.g., response (response and duration) / determining which patients should receive the vaccine) based on the reactivity.

[0058] In another aspect, the present disclosure provides a kit for predicting the efficacy of a cancer vaccine, the kit comprising a cancer antigen or an antigenic portion of the cancer antigen or an equivalent thereof corresponding to the cancer vaccine, and means (e.g., an ELISPOT assay kit) for confirming the responsiveness of patient-derived immune cells (e.g., peripheral blood mononuclear cells) to stimulation by the cancer antigen or the antigenic portion of the cancer antigen or an equivalent thereof corresponding to the cancer vaccine, and for predicting and / or calculating the efficacy of the cancer vaccine (e.g., response (response and duration) / determining which patients should receive the vaccine) based on the responsiveness.

[0059] In one embodiment, "response to patient-derived immune cells (e.g., peripheral blood mononuclear cells) stimulated by a cancer antigen corresponding to the cancer vaccine or the antigenic portion of the cancer antigen" refers to the immune response observed when patient-derived immune cells are stimulated with a cancer antigen or its antigenic portion. This responsiveness is an important indicator for predicting the effectiveness of cancer immunotherapy and optimizing the treatment plan. Various embodiments are described below.

[0060] In one embodiment, the present disclosure can be carried out by direct stimulation of immune cells (e.g., peripheral blood mononuclear cells (PBMCs)). In this embodiment, immune cells such as patient-derived peripheral blood mononuclear cells (PBMCs) are isolated and directly stimulated with a cancer antigen or its antigenic moiety. For example, PBMCs are stimulated using MAGEA3, NY-ESO-1, HER2, or neoantigen peptides, and their immune response is evaluated. In this process, the number of cells secreting specific cytokines (e.g., interferon-γ, IL-2) can be measured using an ELISPOT assay or a FluoroSpot assay. This method makes it possible to evaluate the function of antigen-specific T cells and B cells.

[0061] In one embodiment, the present disclosure can be carried out by antigen presentation via dendritic cells. In this embodiment, dendritic cells (DCs) differentiated from patient-derived monocytes are loaded with a cancer antigen or its antigenic moiety. Subsequently, the antigen-presenting dendritic cells are co-cultured with patient-derived PBMCs, and T cell activation is evaluated. At this time, the expression of activation markers (e.g., CD69, CD25), cell proliferation, and cytokine production can be analyzed by flow cytometry. This method is useful for evaluating the antigen-presenting ability of cancer vaccines and the immune response of patients.

[0062] In one embodiment, the present disclosure can be carried out by stimulation using neoantigens. In this embodiment, gene mutations derived from the patient's tumor cells are analyzed, and neoantigen peptides designed based on these mutations are used. These neoantigens are loaded into PBMCs or dendritic cells for stimulation, and a specific immune response is evaluated. This method allows for the prediction of the effectiveness of a personalized cancer vaccine in advance.

[0063] In one embodiment, the present disclosure can be carried out by stimulation with an RNA / DNA vaccine component. In this embodiment, patient-derived PBMCs are stimulated using a nucleic acid molecule encoding a cancer antigen contained in the RNA or DNA vaccine. In this embodiment, the RNA or DNA is introduced into the cells by electroporation or lipofection. After stimulation, the effect of the vaccine component is evaluated by measuring the proliferation and secretion of cytokines by activated T cells and B cells.

[0064] In one embodiment, the present disclosure can be carried out by reactivity assessment using multiplex cytokine analysis. In this embodiment, a CBA (Cytokine Bead Array) or Luminex assay is used to simultaneously measure multiple cytokines (e.g., IFN-γ, IL-2, TNF-α) secreted by PBMCs stimulated by a cancer antigen or its antigenic moiety. This method allows for the assessment of the multifaceted nature of the immune response to the antigen.

[0065] In one embodiment, this disclosure can be carried out by analyzing the immune response using FACS (flow cytometry). In this embodiment, PBMCs stimulated with cancer antigens are analyzed by flow cytometry to evaluate the proportion of cells showing a specific immune response. For example, responsiveness to vaccines can be evaluated by analyzing the proportion of CD4-positive T cells, CD8-positive T cells, or memory T cells. Furthermore, the function of cellular immunity can be evaluated in detail by detecting the production of antigen-specific intracellular cytokines (e.g., IFN-γ, IL-2).

[0066] In one embodiment, the present disclosure can be carried out by analyzing the antibody response using ELISA. In this embodiment, antibodies secreted by PBMC-derived B cells stimulated with a cancer antigen or its antigenic moiety are measured using ELISA (Enzyme-Linked Immunosorbent Assay). By evaluating the presence and amount of antibody production, the humoral immune response can be assessed.

[0067] In one embodiment, the disclosure can be carried out by comparative evaluation of vaccine candidates. In this embodiment, PBMCs are stimulated using different cancer antigens or antigenic moieties (e.g., MAGEA3, NY-ESO-1, HER2), and the immune responses of each are compared and evaluated. This comparison makes it possible to determine the optimal cancer vaccine component and administration regimen.

[0068] In this specification, "the responsiveness of patient-derived immune cells (e.g., peripheral blood mononuclear cells) to stimulation by cancer antigens or their antigenic moieties corresponding to cancer vaccines" is applied to predicting the efficacy of cancer immunotherapy, selecting vaccine components, optimizing administration schedules, and designing personalized therapies. Furthermore, these embodiments can be further enhanced by combining them with technologies such as ELISTOT, flow cytometry, CBA, ELISA, and RNA sequencing to enable more precise analysis.

[0069] In this specification, "predicting or calculating the effectiveness of a cancer vaccine based on reactivity" means analyzing reactivity data of patient-derived immune cells (e.g., peripheral blood mononuclear cells) observed in response to stimulation with a cancer antigen or its antigenic moiety corresponding to the cancer vaccine, and predicting the effectiveness of the cancer vaccine quantitatively or qualitatively. This prediction of effectiveness includes response (treatment success rate), duration, selection of patients to whom the vaccine should be administered, and development of an optimal treatment plan. Specific embodiments are described below.

[0070] In one embodiment, the cancer antigen or the antigenic portion of the cancer antigen or equivalent thereof is a partial peptide protein contained in the amino acid sequence of the cancer antigen, a partial peptide protein contained in the amino acid sequence translated from the nucleic acid in the case of a nucleic acid vaccine, or equivalent thereof.

[0071] In one embodiment, the prediction is made before administration of the vaccine or during treatment with the vaccine.

[0072] In one embodiment, this disclosure encompasses obtaining patient-derived immune cells. This acquisition of immune cells is performed by taking samples from the patient's peripheral blood or tumor tissue and separating mononuclear cells (PBMCs) and lymphocytes therefrom. Specifically, the target immune cells are purified using centrifugation or density gradient separation. The collected immune cells are then stored under appropriate storage conditions (e.g., below -80°C) and used for subsequent immune response analysis.

[0073] In one embodiment, the disclosure includes providing a cancer antigen or an antigenic moiety of the cancer antigen for a cancer vaccine.

[0074] In one embodiment, the method of the present disclosure is performed in vitro.

[0075] In one embodiment, the prediction of the present disclosure is made before vaccine administration or during the course of treatment with the vaccine; in another embodiment, the prediction is made before vaccine administration, and in yet another embodiment, the prediction is made during the course of treatment with the vaccine.

[0076] In one embodiment, the immune cells include at least one selected from the group consisting of peripheral blood mononuclear cells, lymphocytes (e.g., T cells, B cells, NK cells), dendritic cells, macrophages, monocytes, and neutrophils. Lymphocytes may include, for example, T cells, B cells, NK cells, etc.

[0077] In one embodiment, the effects predicted by this disclosure include at least one selected from the group consisting of 1) the efficacy of the cancer vaccine, 2) the persistence of the cancer vaccine, 3) the determination of patients to whom the cancer vaccine should be administered, 4) the determination of which cancer vaccine to administer, and 5) the determination of changes in the dosage and administration of the cancer vaccine.

[0078] In one embodiment, the following prediction may be made. The predetermined values ​​described below may be predetermined or may be set in preliminary tests for each individual vaccine. 1) The efficacy of the cancer vaccine If the measurement value (Spot Counts) obtained by the ELISPOT is above a predetermined value, it is determined that the cancer vaccine is effective. 2) Durability of the cancer vaccine If the measurement value (Spot Counts) obtained by the ELISPOT is above a predetermined value, it is determined that the cancer vaccine is effective. • Determining which patients should receive the aforementioned cancer vaccine. If the measurement value (Spot Counts) obtained by the aforementioned ELISPOT is above a predetermined value, the patient is determined to be a patient who should be administered the cancer vaccine. • Determining which cancer vaccines should be administered. If the ELISPOT measurement (Spot Counts) is above a predetermined value, the patient is determined to be a patient who should receive the target vaccine. • Decision on changing the dosage and administration of the aforementioned cancer vaccine. If the measured value (Spot Counts) from the ELISPOT is below or above a predetermined value, it is decided to increase the dosage or frequency of use; if it is above or below a predetermined value, it is decided to increase the dosage or frequency of use.

[0079] Agents for predicting the effectiveness of cancer vaccines include a cancer antigen corresponding to the cancer vaccine, or the antigenic portion of the said cancer antigen, or an equivalent thereof. Such agents are diagnostic and evaluation compositions used to predict the effectiveness of cancer vaccines, and include a cancer antigen corresponding to the cancer vaccine, the antigenic portion of the said cancer antigen, or equivalents thereof. These components are used to predict the therapeutic effect of cancer vaccines by stimulating patient-derived immune cells and measuring their reactivity. Specifically, the evaluation is based on indicators such as the amount of cytokine secretion induced by stimulation, the activation state of T cells, or cytotoxic activity against tumor cells. This agent plays an important role as a tool for clarifying the characteristics of the immune response in each patient and formulating the optimal treatment strategy. Furthermore, by evaluating the effectiveness of cancer vaccines in advance, this agent can simultaneously improve the success rate of treatment and reduce the risk of side effects. Accordingly, in the agent of this disclosure, the reactivity of immune cells derived from the patient in response to stimulation with a cancer antigen corresponding to the cancer vaccine, or the antigenic portion of the cancer antigen or its equivalent is confirmed, and the effect of the cancer vaccine is predicted and / or calculated based on the reactivity.

[0080] In one embodiment, the means for confirming the responsiveness of patient-derived immune cells to stimulation with a cancer antigen corresponding to a cancer vaccine, or the antigenic portion of said cancer antigen, or its equivalent, provides an assay kit or evaluation system for evaluating the responsiveness of patient-derived immune cells in order to predict the effectiveness of the cancer vaccine. This includes reagents containing cancer antigens, their antigenic portions, or equivalents, auxiliary reagents that promote the immune response, and reagents for detecting responsiveness (e.g., enzyme-labeled antibodies or ELISA plates). Dedicated devices such as ELISPOT readers and flow cytometers are also included in this means, and these can be used to measure cytokine secretion levels, T cell activation status, cytotoxicity, etc., with high precision. The evaluation process involves using peripheral blood mononuclear cells (PBMCs) and T cells collected from patients as antigen reagents. Cells are stimulated, and then cytokine secretion, cell surface marker expression, and cytotoxic activity against target cells are measured. The reliability of the measurement results can be ensured by using positive and negative control peptides. This method is used as an important tool to clarify the characteristics of the immune response in each patient and to improve the success rate of cancer vaccine treatment. Furthermore, based on the obtained data, it becomes possible to design an optimal vaccine administration plan and additional treatment as needed. This is expected to contribute to the realization of personalized medicine and the construction of effective treatment strategies tailored to each patient. Such methods may include, but are not limited to, ELISPOT assay kits and their equivalents (such as FLUOROSPOT).

[0081] 1. Effect prediction using ELISPOT data Embodiment: Patient-derived PBMCs stimulated with cancer antigens (e.g., NY-ESO-1, MAGEA3, WT1, etc.) are used, and the number of cytokine-secreting cells (e.g., interferon-γ, IL-2) is measured using the Erispot assay. Based on this data, the activation level of antigen-specific T cells is evaluated, and the following is predicted: 1-1. Response: A high number of spots (e.g., 300 or more spots per 100,000 PBMCs) suggests a high response rate to cancer vaccines. 1-2. Selection of patients to administer the drug: For patients who do not show an antigen-specific response, consider other treatment options. 2. Functional analysis of T cells by flow cytometry Embodiment: PBMCs stimulated with cancer antigens are analyzed by flow cytometry to measure the expression of activation markers (e.g., CD69, CD25) and intracellular cytokine production (e.g., IFN-γ, TNF-α, IL-2) in T cells (CD4-positive and CD8-positive). Using this data, the following is calculated: 2-1. Response: If the percentage of activated T cells is 50% or higher, the vaccine is likely to exhibit a high therapeutic effect. 2-2. Persistence: The persistence of the immune response is predicted by evaluating the frequency of memory T cells (CD45RO positive) and central memory T cells (CCR7 positive CD45RO positive). 3. Multicytokine profile analysis using CBA or Luminex Embodiment: Multiple cytokines (e.g., IFN-γ, IL-2, IL-6, TNF-α) secreted from PBMCs stimulated with cancer antigens are measured using a CBA (Cytokine Bead Array) or Luminex assay. This data is then integrated and analyzed to predict the following: 3-1. Response: A high proportion of cells secreting multiple cytokines (multifunctional responses) is predicted to indicate a high response rate to the vaccine. 3-2. Selection of patients to whom the drug should be administered: Other treatments are recommended for patients with a poor cytokine response. 4. Predicting the effectiveness of personalized vaccines using neoantigens Embodiment: PBMCs are stimulated using neoantigen peptides designed based on genetic data obtained from patient tumors. The response after stimulation is evaluated using Erispot or flow cytometry, and the following is predicted: 4-1. Determining which patients should receive the treatment: Select patients who show a strong response to neoantigens. 4-2. Efficacy and Sustained Efficacy: When neoantigen-specific T cells exhibit a multifunctional response (e.g., simultaneous secretion of IFN-γ, IL-2, and TNF-α), high efficacy and sustained efficacy of the vaccine are predicted. 5. Model-based prediction algorithms Embodiments: Reactivity data (e.g., number of spots, cytokine profile, percentage of activated T cells) is input into a statistical or machine learning model to build an algorithm that predicts vaccine effectiveness. This model predicts the following: 5-1. Response: The probability of response for each patient is calculated to optimize treatment selection. 5-2. Persistence: Quantitatively predicts the time until the immune response disappears. 5-3. Individualized treatment plan: Determination of antigens to be administered and concomitant medications. 6. Predicting the effectiveness of RNA / DNA vaccines Embodiments: We stimulate PBMCs using RNA or DNA vaccines and evaluate secreted cytokines and intracellular responses. Based on this, we predict the following: 6-1. Efficacy: Vaccine efficacy is evaluated based on the intensity of the antigen-specific response induced after RNA / DNA introduction. 6-2. Optimal Dosage Plan: Calculate the optimal dosage and schedule. 7. Prediction of the combined effect with immune checkpoint inhibitors Embodiments: We will analyze changes in the immune response by using anti-PD-1 antibodies or anti-CTLA-4 antibodies in combination with PBMCs stimulated with cancer antigens. This will predict the following: 7-1. Response: If the immune response is enhanced by the addition of a checkpoint inhibitor, a high therapeutic effect when used in combination with a vaccine is predicted. 7-2. Selection of patients to administer the drug: Select patients who respond to checkpoint inhibitors.

[0082] The method described herein for "predicting and calculating the effectiveness of cancer vaccines based on reactivity" contributes to the individualization of cancer immunotherapy, optimization of treatment selection, and improvement of treatment outcomes. These specific embodiments are applied to the efficacy and persistence of cancer vaccines, patient selection for administration, and adjustment of treatment plans. These analytical methods are expected to be performed by combining technologies such as ELISTOP, flow cytometry, CBA, RNA sequencing, and machine learning models.

[0083] In certain embodiments, various predictions and calculations can be performed, such as 1) the efficacy of the cancer vaccine, 2) the persistence of the cancer vaccine, 3) determining which patients should receive the cancer vaccine, 4) determining which cancer vaccine to administer, 5) determining other drugs and / or nutrition to be administered in addition, and 6) determining whether the dosage and administration of the cancer vaccine should be changed.

[0084] In this specification, specific embodiments will be described for each of the following items. 1. Efficacy of cancer vaccines Responsiveness refers to the likelihood that a cancer vaccine will exert a therapeutic effect in patients. In this embodiment, response is evaluated using immune response data based on cancer antigens. For example, the secretion of cytokines (e.g., IFN-γ, IL-2) from peripheral blood mononuclear cells (PBMCs) of patients stimulated with cancer antigens (e.g., MAGEA3, NY-ESO-1) is measured using the ELISPOT assay. If the number of spots is above a certain level (e.g., 200 spots or more per 100,000 PBMCs), a high response rate can be expected. Auxiliary techniques: Combining this with the analysis of T cell activation markers (e.g., CD69, CD25) using flow cytometry improves the accuracy of response assessments. 2. Durability of cancer vaccines Durability is an indicator used to evaluate how long the therapeutic effect of a cancer vaccine is maintained. Example: Evaluate the induction of immune memory cells (memory T cells) after cancer vaccine administration. For example, measure the percentage of CD45RO-positive T cells by flow cytometry at 6 and 12 months after administration to assess the duration of the immune response. Auxiliary techniques: Use the Tetramer assay or ELISPOT assay to measure the presence of neoantigen-specific memory T cells. 3. Determining which patients should receive cancer vaccines. In this embodiment, patients who are likely to respond well to the cancer vaccine are selected based on their immune profile and tumor characteristics. Example: The cancer antigen expression levels of patient tumors are evaluated by RT-PCR or immunohistochemistry (IHC), and patients with high expression of antigens such as MAGEA3 and NY-ESO-1 are selected. In addition, the patient's HLA type (e.g., HLA-A*02:01) is identified by genetic analysis, and a peptide vaccine compatible with that HLA is selected. Supporting techniques: Measure the proportion of immunosuppressive factors (e.g., Treg cells, MDSCs) in the tumor microenvironment using flow cytometry and prioritize patients with low immunosuppression. 4. Determining which cancer vaccine to administer. The optimal cancer vaccine is selected based on the patient's tumor characteristics and immune status. For example, a peptide vaccine, protein vaccine, or RNA vaccine is selected based on the type of cancer antigen (e.g., MAGEA3, WT1, HER2) and the patient's HLA type. For instance, a HER2 protein vaccine is recommended for HER2-positive breast cancer patients, and a WT1 peptide vaccine is recommended for WT1-high-expression leukemia patients. Supporting technology: In the case of neoantigen vaccines, personalized vaccines are designed based on next-generation sequencing (NGS) data of the patient's tumor. 5. Determining other medications and / or nutritional supplements to be administered. To enhance the effectiveness of the cancer vaccine, we will determine the adjunctive therapies that should be used in combination. Example: Select an immune checkpoint inhibitor (e.g., anti-PD-1 antibody, anti-CTLA-4 antibody) to be used in combination with a cancer vaccine. For example, in patients with confirmed immunosuppression, the immune response can be enhanced by using an anti-PD-1 antibody in combination. Nutritional examples: Administering nutritional supplements that promote the immune response, such as vitamin D and omega-3 fatty acids, is recommended. Auxiliary techniques: Use cytokine profiling (e.g., CBA or Luminex assay) to determine which cytokine therapy (e.g., IL-2, GM-CSF) should be administered. 6. Determining changes to the dosage and administration of cancer vaccines. Based on the patient's immune response data, the dosage and administration schedule of the cancer vaccine will be adjusted. For example, if the immune response is insufficient in the initial stages of administration, increase the vaccine dose (e.g., increase the peptide amount from 50 μg to 100 μg). Also, if the immune response decreases rapidly, shorten the administration interval from 4 weeks to 2 weeks. Supporting techniques: Monitor the number of spots or the percentage of activated T cells after the initial dose and modify the subsequent dosing plan based on this.

[0085] As described above, each embodiment in this specification is an important element for the individualization of cancer vaccine therapy, maximization of therapeutic efficacy, and ensuring patient safety. By performing detailed analysis of each of these items based on patient-derived immune response data and tumor characteristics, it becomes possible to optimize the regimen (treatment plan).

[0086] In certain embodiments, "a pharmaceutical in which the vaccine regimen is determined based on the responsiveness of patient-derived immune cells (e.g., peripheral blood mononuclear cells) to stimulation by a cancer antigen or antigenic portion of a cancer antigen corresponding to the cancer vaccine" refers to a pharmaceutical in which an optimal vaccine administration schedule (regimen) is designed based on individual patient immune response data to effectively implement cancer immunotherapy. This pharmaceutical incorporation aims to maximize the success rate of treatment by taking into account the different immune profiles and tumor characteristics of each patient.

[0087] First, the reactivity of patient-derived immune cells to cancer antigens or their antigenic moieties is a crucial indicator underlying vaccine regimen determination. For example, patient-derived peripheral blood mononuclear cells (PBMCs) stimulated with cancer antigens (such as MAGEA3, NY-ESO-1, and WT1) are analyzed using ELISPOT or flow cytometry to assess the activation levels of specific T cells and B cells. This reactivity data (e.g., number of interferon-gamma secreting cells, expression rate of activation markers) provides important information for predicting how well an individual patient's immune system will respond to a cancer vaccine.

[0088] Next, the specific design of the vaccine regimen is based on response data. For example, for highly responsive patients, a standard dose and administration interval (e.g., every two weeks) is adopted, while for less responsive patients, an individualized schedule is set, such as increasing the dose (e.g., increasing the peptide amount from 50 μg to 100 μg) or shortening the administration interval (e.g., changing from every four weeks to every two weeks). In addition, the need for and optimal timing of an additional booster vaccine is determined based on the level of induction of immune memory cells (memory T cells).

[0089] Furthermore, vaccine regimens may include adjunctive therapeutic elements other than cancer vaccines. Based on response data, the combination of immune checkpoint inhibitors (e.g., anti-PD-1 antibodies, anti-CTLA-4 antibodies) or cytokine therapy (e.g., IL-2, GM-CSF) can be decided upon. In addition, considering the patient's nutritional status and comorbidities, the regimen may include the addition of vitamin D or amino acid supplements to enhance the immune response. This is expected to further improve the effectiveness of cancer vaccine therapy.

[0090] In this specification, "vaccine regimens" are characterized by designs based on individual patient immune profiles and responsiveness, enabling a therapeutic approach that maximizes the efficacy and persistence of cancer vaccines while minimizing side effects. Such regimens contribute to the realization of personalized medicine in cancer immunotherapy, improving patient outcomes and optimizing treatment plans.

[0091] In one embodiment, administering the cancer vaccine to a subject whose reactivity value is above a predetermined value refers to a treatment method in which the cancer vaccine is administered to a subject patient whose immune response (reactivity) based on stimulation by the cancer antigen or its antigenic moiety corresponding to the cancer vaccine is above a predetermined standard value. This embodiment aims to enhance the effectiveness of vaccine therapy by evaluating the patient's immune response in advance. Specifically, immune cells derived from the patient (e.g., peripheral blood mononuclear cells (PBMCs)) are stimulated with cancer antigens or their antigenic regions, and the immune response is evaluated using the ELISPOT assay or flow cytometry. For example, when stimulated with cancer antigens such as MAGEA3 or NY-ESO-1, if the number of interferon-γ secreting cells is 200 or more spots per 100,000 PBMCs, or if the proportion of activated T cells (CD69-positive T cells) is 50% or more, the patient is judged to have a "response value above a predetermined value." Patients who meet this criterion are expected to have a high response rate to cancer vaccine administration.

[0092] In one embodiment, administration based on responsiveness can not only enhance therapeutic effects but also reduce the burden on patients by avoiding ineffective treatments. For patients with responsiveness below a predetermined value, it may be considered to administer the vaccine after enhancing the immune response by considering combination therapy with immunosuppressive factors (e.g., Treg cells, MDSCs) or immune checkpoint inhibitors (e.g., anti-PD-1 antibodies). In addition, for patients with low responsiveness, personalized neoantigen vaccines or increased-dose administration schedules can be applied instead of standard cancer vaccines.

[0093] One embodiment features the selection of cancer vaccine recipients based on individual patient immune response data. This approach improves the success rate of cancer vaccine therapy and enables the optimal allocation of limited medical resources. Furthermore, by clearly defining criteria for evaluating immune responses, it contributes to the standardization of treatment processes in clinical trials and real-world clinical practice.

[0094] In one embodiment, a detection agent for detecting stimulation by a cancer antigen or its antigenic moiety comprises a reagent or tool for detecting an immune response induced by the cancer antigen or its antigenic moiety. This detection agent is used to evaluate the reactivity of patient-derived immune cells (e.g., peripheral blood mononuclear cells (PBMCs)) after stimulation. This detection agent plays an important role in predicting the effectiveness of cancer vaccines, monitoring a patient's immune status, or evaluating the specificity of cancer antigens.

[0095] Specific examples of detection agents used in this disclosure include interferon-γ (IFN-γ) capture antibodies and detection antibodies used in the ELISPOT assay. These antibodies allow for the visualization of IFN-γ secreted by PBMCs stimulated by cancer antigens (e.g., MAGEA3, NY-ESO-1) as spots. The detection antibody consists of a capture antibody immobilized on the bottom of the plate and a biotinylated detection antibody. By using enzyme-labeled streptavidin in combination, the amount of secreted cytokines can be measured with high sensitivity. This method allows for the quantitative evaluation of the presence of antigen-specific T cells.

[0096] In further embodiments, detection using flow cytometry (FACS) may also be considered. In this case, specific detection agents are used. For example, fluorescently labeled antibodies (e.g., FITC or PE-labeled anti-CD69 antibodies) are used to detect activation markers (e.g., CD69, CD25) expressed on the surface of T cells after stimulation with cancer antigens. In addition, to detect intracellular cytokines (e.g., IFN-γ, TNF-α), antibodies are introduced into cells using cell-permeable reagents, and the fluorescence signal is analyzed. Using these detection agents makes it possible to evaluate antigen-specific responses at the single-cell level in detail. These detection agents play an important role in the development of cancer vaccines and personalized medicine. For example, they are used for screening to identify patients who respond to specific cancer antigens, and also to monitor the immune response after vaccine administration and evaluate its effectiveness. Furthermore, by appropriately combining detection agents, it is possible to analyze multiple cytokines and activation markers simultaneously, making it a powerful tool for comprehensively evaluating the immunogenicity of cancer antigens.

[0097] (Regarding specific examples) A specific example is given below. This example is from Higuchi, Y., Koya, T., Yuzawa, M., Yamaoka, N., Mizuno, Y., Yoshizawa, K., & Shimodaira, S. (2015). Enzyme-Linked Immunosorbent Spot Assay for the Detection of Wilms’ Tumor 1-Specific T Cells Induced by Dendritic Cell Vaccination. Biomedicines, 3(4), 304-315. Refer to https: / / doi.org / 10.3390 / biomedicines3040304 This can be done. The evaluation criteria of the ELISPOT (Enzyme-Linked Immunosorbent Spot) assay are very important elements in the measurement of immune responses. In this study, the ELISPOT assay was used to measure the immune response after dendritic cell (DC) vaccination targeting WT1 (Wilms’ Tumor 1)-specific T cells.

[0098] Examples of evaluation criteria are as follows: Quantitativeness: In the ELISPOT assay, the number of specific IFN-γ-producing cells against the WT1 peptide is measured, and a quantitative criterion for the positive reaction is set. Specifically, at least 15 spots are observed per 1×10 6 PBMCs, and the number of spots needs to be 1.5 times or more compared to the negative control. Reproducibility and accuracy: The reproducibility of the assay is evaluated by the CV (coefficient of variation) under different conditions, and it has been reported in the study to be in the range of 7.4% to 16.3%. Also, the daily accuracy is evaluated, and the CV is in the range of 5.0% to 17.3%. Linearity: The ELISPOT assay shows a linear reactivity according to the cell number, and a correlation coefficient of r = 0.96 - 0.98 was obtained in the dilution experiment. This confirms the reliability of the quantitative evaluation of the assay. Specificity: The response of WT1-specific T cells is compared with the number of spots when using the negative control peptide. It is determined to be positive only when a specific response is observed. Clinical utility: Detection of IFN-γ-producing cells after WT1 peptide stimulation showed a positive response in 34 / 46 cases (73.9%) of patients, indicating clinical efficacy. This result shows that the ELISPOT assay is a useful method for monitoring the immune response in WT1-targeted DC vaccine therapy. Thus, the ELISPOT assay is a reliable method for highly accurate and quantitative evaluation of the functional response of WT1-specific T cells and is an important tool for evaluating the effect of cancer immunotherapy.

[0099] In a specific embodiment of the present disclosure, if the number of spots is 7 or more and the SI is less than 1, it can be determined that the subject is reactive (highly likely to be effective) to the cancer vaccine. That is, an example of pre-vaccination (before vaccine administration) stratification of ELISPOT is shown. As the weak positive criteria, 1) there are at least 7 WT1-specific spot numbers, and 2) the WT1-specific spots are more than the number of spots in the negative control. In this case, 1) the efficacy of the cancer vaccine, 2) the persistence of the cancer vaccine, 3) the determination of the patient to whom the cancer vaccine should be administered, 4) the determination of the cancer vaccine to be administered, etc. can be predicted.

[0100] The positive criteria for ELISPOT after vaccine administration can be determined that the subject is reactive (highly likely to be effective) to the cancer vaccine if the number of spots is 15 or more and the SI is greater than 1.5. 1) There are at least 15 WT1-specific spot numbers, and 2) there are 1.5 times or more WT1-specific spots than the number of spots in the negative control. In this case, 1) the efficacy of the cancer vaccine, 2) the persistence of the cancer vaccine, 3) the determination of the patient to whom the cancer vaccine should be administered, 4) the determination of the cancer vaccine to be administered, etc. can be performed.

[0101] An example of pre-vaccination stratification using ELISPOT is shown. The weak positive criterion is 1) the presence of at least 7 WT1-specific spots, and 2) the number of WT1-specific spots may be greater than the number of spots in the negative control. In this case, it is possible to determine 1) the efficacy of the cancer vaccine, 2) the persistence of the cancer vaccine, 3) which patients should receive the cancer vaccine, and 4) which cancer vaccine should be administered.

[0102] The ELISPOT post-vaccination positive criteria may be: 1) having at least 15 WT1-specific spots, and 2) having WT1-specific spots at least 1.5 times the number of spots in the negative control. In this case, it is possible to determine: 1) the efficacy of the cancer vaccine, 2) the persistence of the cancer vaccine, 3) which patients should receive the cancer vaccine, and 4) which cancer vaccine should be administered.

[0103] (Companion Diagnosis) In another context, this disclosure relates to a method of preventing or treating a patient using a cancer vaccine, To collect immune cells from the aforementioned patient, To provide a cancer antigen corresponding to the cancer vaccine, or the antigenic portion of the cancer antigen, or equivalents thereof, Stimulating the immune cells with the cancer antigen or the antigenic portion of the cancer antigen, The response to the aforementioned stimulus is confirmed, and the effect of the cancer vaccine is predicted based on the aforementioned response. Based on the prediction of the aforementioned effects, the cancer vaccine is administered to the patient. The present invention provides a method that encompasses the following: This method may further comprise any of the features or combinations described herein with respect to the diagnosis of the present disclosure.

[0104] In another aspect, the present disclosure provides a medicament comprising a cancer vaccine, wherein the regimen of the vaccine is determined based on the reactivity of immune cells (e.g., peripheral blood mononuclear cells) derived from the patient to stimulation by a cancer antigen corresponding to the cancer vaccine or an antigenic portion of the cancer antigen. The medicament of the present disclosure may further comprise any feature or combination thereof described herein with respect to the diagnosis of the present disclosure.

[0105] In one embodiment, the present disclosure further includes identifying the cancer antigen corresponding to the patient or an antigenic portion of the cancer antigen.

[0106] In one embodiment, the medicament of the present disclosure may be characterized in that it is administered to a subject in which the value of the reactivity to antigenic stimulation by the cancer vaccine in immune cells obtained before administration from the subject to which the medicament is administered is not less than a predetermined value.

[0107] The present disclosure provides a pharmaceutical kit comprising a medicament containing a cancer vaccine and a detection agent for detecting stimulation by a cancer antigen corresponding to the cancer vaccine or an antigenic portion of the cancer antigen, wherein the regimen of the vaccine is determined based on the reactivity of immune cells derived from the patient to stimulation by the cancer antigen corresponding to the cancer vaccine or an equivalent thereof. The kit of the present disclosure may further comprise any feature or combination thereof described herein with respect to the diagnosis of the present disclosure. In one embodiment, regarding the treatment regimen, the determination formulas for pre-treatment prediction and during-treatment prediction are exemplified as follows, but are not limited thereto. Pre-treatment prediction · WT1≧7 ∩ WT1≧NC → Positive · WT1<7 ∪ WT1<NC → Negative During-treatment prediction · WT1≧15 ∩ WT1≧NC×1.5 → Positive · WT1<15 ∪ WT1<NC×1.5 → Negative In the above, WT1 is the number of specific spots, and NC is the number of spots of the negative control.

[0108] (General technology) The molecular biological, biochemical, and microbiological methods used herein are well-known and commonly used in the field, for example, Sambrook J. et al. al.(1989).Molecular Cloning: A Laboratory Manual,Cold Spring Harbor and its 3rd Ed.(2001);Ausubel,FM(1987).Current Protocols in Molecular Biology,Greene Pub.AssociatESand Wiley-Interscience;Ausubel,FM(1989).Short Protocols in Molecular Biology:A Compendium of Methods from Current Protocols in Molecular Biology,Greene Pub.Associates and Wiley-Interscience;Innis, MA(1990).PCR Protocols: A Guide to Methods and Applications, Academic Press;Ausubel,FM(1992).Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology,Greene Pub.Associates;Ausubel,FM(1995).Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology,Greene Pub.Associates;Innis,MAet al.(1995).PCR Strategies,Academic Press;Ausubel,FM(1999).Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology,Wiley,and annual updates;Sninsky,JJet al.(1999).PCR Applications: Protocols for Functional Genomics,Academic This information is described in Press, Separate Volume of Experimental Medicine "Experimental Methods for Gene Transfer & Expression Analysis," Yodosha, 1997, and relevant parts (possibly all) of these are referenced herein.

[0109] In this specification, "or" is used when "at least one" of the items listed in the text can be adopted. The same applies to "or else". In this specification, when it is specified that "within the range" of "two values", that range includes the two values ​​themselves.

[0110] References such as scientific literature, patents, and patent applications cited herein are incorporated herein by reference to the same extent as they are specifically described herein.

[0111] As described above, the present invention has been described by showing preferred embodiments for easy understanding. Hereinafter, the present invention will be described based on examples. However, the above description and the following examples are provided only for illustrative purposes and not for the purpose of limiting the present invention. Therefore, the scope of the present invention is not limited to the embodiments or examples specifically described in this specification, but is limited only by the claims.

Example

[0112] Examples are described below. This example complied with all the regulations regarding clinical and non-clinical trials defined by Kobe University and was conducted in compliance with the rules regarding clinical ethics such as the Helsinki Declaration. Also, the drugs and the like used were obtained from the suppliers indicated in each description, but they are also available from other sources (for example, Diaclone, R&D Systems, etc.).

[0113] (Example 1) In this example, a test was conducted on B440, an oral cancer vaccine targeting WT1 protein (tumor-associated antigen).

[0114] (Methods and Materials) A phase I clinical trial of B440, an oral cancer vaccine targeting WT1 protein (tumor-associated antigen) for advanced urothelial cancer, was conducted, and the induction of WT1-specific cellular immunity before and after cancer vaccine administration was measured by an ELISPOT assay. As a result, the treatment outcomes of patients with positive WT1-specific cellular immunity after vaccine administration were significantly more effective compared to those with negative results. Also, it was found that the positive / negative of the ELISPOT assay and the treatment effect after vaccine administration could be predicted by setting certain criteria for the ELISPOT assay before vaccine administration and making a diagnosis of weakly positive / negative.

[0115] (Overview of Clinical Trial) In this example, an open-label single-group trial was conducted to evaluate the safety and efficacy of the oral cancer vaccine B440 for advanced urothelial cancer resistant to PD-1 / PD-L1 inhibitors (corresponding to phase 1 of the trial).

[0116] This example evaluates the safety and efficacy of oral cancer vaccine B440 administered orally at a dose of 800 mg or 1,600 mg once daily, five times a week for four weeks, in patients with unresectable advanced urothelial carcinoma who are resistant to or intolerant of PD-1 / PD-L1 inhibitors and have no standard therapy.

[0117] (Evaluation of WT1 heterocellular immune response using the ELISPOT assay) T cells are isolated from peripheral blood mononuclear cells (PBMCs) of subjects and stimulated with WT1 antigen protein culture. Subsequently, the number of IFN-γ producing cells is measured using the ELISPOT method to confirm the WT1-specific immune response.

[0118] The assay was performed at the following points: before vaccine administration (also known as W0), 2 weeks after the start of administration (also known as W2), 4 weeks after the start of administration (also known as W4), and at W8, W16, and W24 (see Figure 3).

[0119] The protocol is described in detail below with reference to Figure 1. It can be carried out by 1) isolating peripheral blood mononuclear cells from a whole blood sample, 2) stimulating culture them with antigenic moieties such as WT1 antigen protein and peptides, and 3) calculating the number of cells (spot number) that respond to cytokines (IFN-γ).

[0120] (Assay method) The ELISPOT test was performed using the hIFNGp-2M / 2 ELISPOT kit (CTL, Cat. No. hIFNG-2M / 2). First, L-glutamine (200mM, Nacalai Tesque, Cat. No. 16948-04) was added to the medium (CTL Test Medium) to adjust the final concentration to 2mM, and then the mixture was heated at 37°C. Warm the cells. After washing the patient's PBMCs collected at each time point with X-VIV 15 medium (Lonza, Cat. No. 04-418Q), suspend them in CTL Test Medium supplemented with L-glutamine and adjust the cell concentration to 1.0 × 10⁻⁶.6 Adjust to cells / mL.

[0121] (Preparation of irritating antigen and control solution) Human WT1 protein was used as the stimulating antigen, adjusted to a final concentration of 1 μg / mL. Concanavalin A (Invitrogen, Cat. No. 00-4978-03, 500-fold dilution) was used as the positive control, and L-glutamine supplemented medium was used as the negative control.

[0122] (stimulated culture) Dispense 100 μL of the prepared PBMC into 96-well plates, and add the stimulating antigen, positive control, or negative control solution to each well. Incubate the wells in a 5% CO2 environment at 37°C for approximately 24 hours.

[0123] (Well cleaning) After incubation, wash the wells with PBS(-) and 0.05% Tween-PBS to remove impurities and nonspecific substances.

[0124] Addition of IFN-γ detection antibody After washing, add 80 μL of h-IFNγ Detection Solution to each well and incubate at room temperature for 2 hours. Then, wash the wells again with Tween-PBS.

[0125] Addition of Strep-AP Next, add 80 μL of Strep-AP Solution to each well and incubate at room temperature for 30 minutes. Wash the wells again with Tween-PBS.

[0126] Color reaction Add 80 μL of Blue Developer Solution to each well and allow the reaction to proceed at room temperature for 15 minutes. Then, rinse the wells with tap water to stop the color development reaction.

[0127] Plate drying and analysis After drying the plate, the wells are scanned using ImmunoSpot® Analyzers (manufactured by MS Techno Systems Co., Ltd.), and the number of spots is counted to evaluate the WT1-specific immunoassay.

[0128] (Judgment criteria) The number of IFN-γ-producing cells stimulated with each antigen is counted, and the number of spots per well is calculated. The positive criteria are as follows:

[0129] ELISPOT (post-vaccine administration) positive criteria (1) There are at least 15 WT1-specific spots, and, (2) The number of WT1-specific spots is 1.5 times or more than the number of spots in the negative control.

[0130] Pre-ELISPOT (pre-vaccine administration) weakly positive criteria (1) There are at least 7 WT1-specific spots. and (2) The number of WT1-specific spots is greater than the number of spots in the negative control.

[0131] (result) The results of this example are shown below. The results shown in Tables 1 to 12 are interpreted as follows. Tables 1 to 12 show the number of positive spots in the ELISPOT assay for each patient. Day 1 is defined as the day vaccine administration started. NC is the Negative Control without antigen stimulation, hWT1 is the number of positive spots stimulated with human WT1 protein (antigen), and ConA is the number of positive spots stimulated with Concanavalin A as the Positive Control. The Pre-ELISPOT weak positivity criterion is that on Day 0, the number of hWT1 spots is 7 or more and the number of hWT1 spots is greater than the number of NC spots. ELISPOT positivity is defined as a positive result if, from Day 14 onwards, the number of hWT1 spots is 15 or more and the number of hWT1 spots is 1.5 times or more than the number of NC spots. In the table below, Pre-ELISPOT positive results are shown in light gray, and ELISPOT positive results are shown in dark gray.

[0132] [Table 1]

[0133] [Table 2]

[0134] [Table 3]

[0135] [Table 4]

[0136] [Table 5]

[0137] [Table 6]

[0138] [Table 7]

[0139] [Table 8]

[0140] [Table 9]

[0141] [Table 10]

[0142] [Table 11]

[0143] [Table 12]

[0144] Figure 5 shows the temporal changes in the mean number of spots in the ELISPOT-positive and ELISPOT-negative groups. Six cases (four low-dose and two high-dose) were ELISPOT-positive after B440 administration. The ELISPOT-positive group had significantly more spots on Day 0 (before B440 administration) compared to the negative group (P=0.030). This suggests that cases that become ELISPOT-positive after B440 administration have a subtle immune response to WT1 expressed by the tumor even before treatment. ELISPOT-positive patients and Pre-ELISPOT weakly positive patients were in complete agreement during the observation period.

[0145] [Table 13]

[0146] The best overall response among Pre-ELISPOT and ELISPOT-positive and negative patients was SD (83.3%) in 5 out of 6 positive patients and SD (20%) in 1 out of 5 evaluable negative patients. All patients without SD experienced PD (progressive disease). Table 14 below summarizes the safety and efficacy of the subjects.

[0147] (B440-1 Clinical Trial: Safety and Efficacy of Subjects - List) (Note: The shortest SD period for the best overall effect is 42 days)

[0148] [Table 14]

[0149] As shown in Table 14, the B440-1 trial evaluated safety and efficacy in a total of 12 patients: 6 in the low-dose group and 6 in the high-dose group. Pre-ELISPOT was positive in 4 patients in the low-dose group and 2 patients in the high-dose group. No cases showed DLT, and no patients terminated the trial due to adverse events. The overall disease control rate (DCR) was 54.5% (6 / 11 patients). Of the 6 ELISPOT-positive patients, 5 were SD (83.3%), and of the 5 evaluable ELISPOT-negative patients, 1 was SD (20%), while all patients other than those with SD were PD (Figures 16 and 17).

[0150] Five patients underwent a second attempt with pembrolizumab after the follow-up observation period. The treatment showed good disease control, with one case achieving complete response (CR) and one each achieving partial response (PR) and stable disease (SD).

[0151] As shown in Figures 8 and 14, the stratification by Pre-ELISPOT and ELISPOT in the results of this example is shown. Patients were divided into two groups based on ELISPOT and Pre-ELISPOT positive / negative results, and Kaplan-Meier curves were created for progression-free survival and a log-rank test was performed (Figure 8 (before administration = Pre) and Figure 14 (Post = during treatment), P-value: 0.0033).

[0152] As shown above, progression-free survival for B440 can be stratified based on results from Pre-ELISPOT and during the observation period (treatment period) (Figure 8, log-rank test, P=0.0033). (Figures 10, 12, 16, and 17)

[0153] Figure 4 shows the treatment with immune checkpoint inhibitors and anti-PD-1 antibodies after vaccine administration.

[0154] In this example, all patients who participated in the trial were those who had developed resistance to anti-PD-1 antibodies. However, some patients received anti-PD-1 antibodies (pembrolizumab) after receiving the vaccine in the clinical trial. The response to this re-administration of anti-PD-1 antibodies was clearly better in ELISPOT-positive patients, and the therapeutic effect of anti-PD-1 antibodies administered after or in combination with the vaccine can be predicted in the pre-ELISPOT setting.

[0155] Figure 4 shows the reduction rate of indicator tumor size after re-administration of anti-PD-1 antibody.

[0156] As can be seen in Figure 4, all three ELISPOT-positive patients who received readmission of anti-PD-1 antibodies responded to the antibodies (RECIST evaluation: CR, PR, and SD in one case each), while both ELISPOT-negative patients were diagnosed with PD.

[0157] (Companion drugs used to determine the suitability of cancer vaccines) In this embodiment, the ELISPOT (post-vaccine administration) results in the 12 cases in the clinical trial were in complete agreement with the Pre-ELISPOT (pre-vaccine administration) results (see Figure 15; also see the comparisons in Figures 8 and 14, and Figures 16-17), and ELISPOT-positive patients had a significantly longer PFS compared to negative patients (P=0.0033). Therefore, it is possible to pre-select patients who will respond to cancer vaccines or cancer vaccines in combination with immune checkpoint inhibitors using the Pre-ELISPOT assay before cancer vaccine administration.

[0158] (judgment formula) The pre-treatment and treatment-period assessment formulas can be summarized as follows.

[0159] Figure 9 summarizes the progress of ELISPOT (12 cases in total). The horizontal axis shows the passage of time, and the vertical axis shows the average value of ELISPOT. As shown in Figure 10, pre-vaccination stratification of ELISPOT is possible. The criteria for a weak positive can be defined as 1) having at least 7 WT1-specific spots, and 2) having more WT1-specific spots than the number of spots in the negative control.

[0160] Figure 11 shows the mean test (Mann-Whitney U test) for ELISPOT at the pre-vaccination (before vaccine administration) time point. As shown in Figure 12, stratification is possible with ELISPOT after vaccine administration (during the treatment period). The post-vaccination positive criteria for ELISPOT can be defined as 1) having at least 15 WT1-specific spots, and 2) having WT1-specific spots at least 1.5 times the number of spots in the negative control.

[0161] (Example 2) In this example, the same tests as in Example 1 will be performed with AGEA1-4, NY-ESO-1, PRAME, SSX2, CT8, CD19, GP100, MART1, PSA, PSMA, tyrosinase, HER2, MUC1, CEA, survivin, cyclin B1, EGFR, mesothelin, telomerase, and neoantigens. The procedure will be the same as in Example 1, except that the cancer peptides will be changed and the corresponding targets will be used.

[0162] (Example 3: Example using animals) This example demonstrates experiments using mouse bladder cancer cells, specifically MBT-2 cells (which express WT1, with over 95% homology between human and mouse WT1 amino acid sequences).

[0163] (Experiment Overview) In this example, mouse splenocytes were extracted at three time points: before MBT-2 transplantation (day 0), after transplantation (day 7), and after vaccine administration (day 30), and the WT1-ELISPOT assay was performed. The ELISPOT kit used was Mouse IFN-γ Single-Color ELISPOT.

[0164] More specifically, a total of 15 C57BL / 6 mice were randomly assigned to three groups (n=5), and mouse spleen cells were extracted at three time points: before MBT-2 transplantation (day 0), after transplantation (day 7), and after vaccination (day 30). The WT1-ELISPOT assay was performed. The ELISPOT kit used was the Mouse IFN-γ Single-Color ELISPOT. In group 1, the spleen of C57BL / 6 mice was collected on Day 0 and the ELISPOT assay was performed. In group 2, 1 × 10⁶ cells were extracted from the C57BL / 6 mice on Day 3. 6 In the third group, 1 x 10⁶ MBT-2 cells were subcutaneously transplanted, and spleen cells were extracted on day 7 and ELISPOT was performed. In the third group, B440 was administered 1 x 10⁶ times on days 7-11, 14-18, and 21-25. 9 CFU / 100μl is administered orally, and spleen cells are extracted on day 30 and ELISPOT is performed.

[0165] (ELISPOT procedure) (Isolation of spleen cells and ELISPOT assay) To isolate spleen cells, mice are euthanized under anesthesia by cervical dislocation, and the spleen is removed using aseptic techniques. The collected spleen is stored in ice-cold RPMI-1640 medium and finely ground using a cell scraper. After filtration using a 70 μm cell strainer, the suspension is centrifuged at 4°C and 2,000 rpm for 5 minutes, and the supernatant is removed. 1 mL of hemolytic agent is added to the pellet, suspended using a 1,000 μL micropipette, and incubated at room temperature for 2 minutes. Then, 5 mL of ice-cold RPMI-1640 medium is added and mixed, and the mixture is centrifuged at 4°C and 2,000 rpm for 5 minutes. This washing process is repeated two more times, and finally, the CTL test for culture is performed. Suspend the cells in 1 mL of Medium. In the ELISPOT assay, wash each well twice with 200 μL / well of PBS, then wash twice more with Tween-PBS. Next, add 80 μL / well of m-IFNγ detection solution and incubate at room temperature for 2 hours. After washing, add Strep-AP solution (80 μL / well) and incubate at room temperature for 30 minutes. Then, add 80 μL / well of Blue Developer solution and incubate for 15 minutes, and wash the plate with running water to stop the color reaction. After drying the plate, measure the spots in each well using an ImmunoSpot® analyzer.

[0166] As shown in Figure 7, the following is expected. Before MBT-2 transplantation → WT1-ELISPOT negative After MBT-2 transplantation → WT1-ELISPOT weakly positive (positive cells 5 or more, less than 15, WT1 stimulation > no stimulation) After MBT-2 transplantation and B440 administration, WT1-ELISPOT positive (more than 15 positive cells, WT1 stimulation was more than 1.5 times more than unstimulated).

[0167] (Example 4: A hypothetical example of a clinical trial (other than WT1 = unlikely to be performed)) We will perform PAP (prostatic acid phosphatase)-ELISPOT before and after administration of the prostate cancer vaccine Provenge® (sipuleucel-T) to confirm the versatility and effectiveness of companion diagnostics using the ELISPOT method.

[0168] (Clinical trial procedure) In 12 patients with castration-resistant prostate cancer, prostatic microcosmocytes (PBMCs) were collected before and 30 days after administration of the prostate cancer vaccine. PAP (prostatic acid phosphatase)-specific immune responses were confirmed using ELISPOT. Progression-free survival was calculated to confirm the versatility and effectiveness of ELISPOT as a companion diagnostic tool.

[0169] (ELISPOT procedure) The ELISPOT test is performed using the hIFNGp-2M / 2 ELISPOT kit (manufactured by CTL). First, L-glutamine is added to the culture medium (CTL Test Medium) to adjust the final concentration to 2 mM, and then the medium is warmed at 37°C. Patient PBMCs collected at each time point are washed with X-VIV 15 medium, then suspended in CTL Test Medium with added L-glutamine, and the cell concentration is adjusted to 1.0 × 10⁻⁶. 6 Adjust to cells / mL.

[0170] (Preparation of irritating antigen and control solution) Human PAP protein was used as the stimulating antigen, adjusted to a final concentration of 1 μg / mL. Concanavalin A (500-fold dilution to final concentration) was used as the positive control, and L-glutamine supplemented medium was used as the negative control.

[0171] (stimulated culture) Dispense 100 μL of the prepared PBMC into 96-well plates, and add the stimulating antigen, positive control, or negative control solution to each well. Incubate the wells in a 5% CO2 environment at 37°C for approximately 24 hours.

[0172] (Well cleaning) After incubation, wash the wells with PBS(-) and 0.05% Tween-PBS to remove impurities and nonspecific substances.

[0173] (Addition of IFN-γ detection antibody) After washing, add 80 μL of h-IFNγ Detection Solution to each well and incubate at room temperature for 2 hours. Then, wash the wells again with Tween-PBS.

[0174] (Addition of Strep-AP) Next, add 80 μL of Strep-AP Solution to each well and incubate at room temperature for 30 minutes. Wash the wells again with Tween-PBS.

[0175] (Color reaction) Add 80 μL of Blue Developer Solution to each well and allow the reaction to proceed at room temperature for 15 minutes. Then, rinse the wells with tap water to stop the color development reaction.

[0176] (Plate drying and analysis) After drying the plate, the wells are scanned using ImmunoSpot® Analyzers (manufactured by MS Techno Systems Co., Ltd.), and the number of spots is counted to evaluate the PAP-specific immune response.

[0177] (Judgment criteria) The number of IFN-γ-producing cells stimulated with each antigen is counted, and the number of spots per well is calculated. The positive criteria are as follows:

[0178] (Pre-ELISPOT (pre-vaccine administration) weakly positive criteria) (1) There must be at least 7 PAP-specific spots. and (2) The number of PAP-specific spots is greater than the number of spots in the negative control.

[0179] (analysis) Patients will be divided into two groups based on whether their ELISPOT test results are positive or negative before and after vaccine administration. Kaplan-Meier curves will be created for progression-free survival and a log-rank test will be performed for each group. If a high concordance rate is observed between the ELISPOT results before and after vaccine administration in prostate cancer, and if the positive group is shown to have a longer progression-free survival, it will demonstrate the versatility and effectiveness of companion diagnostics using ELISPOT.

[0180] (Example 5: Example of kit packaging) This embodiment describes the configuration when provided as a diagnostic kit.

[0181] General ELISPOT assay kit (see below) Pre-coated 96 well PDVF bottomed plates Biotinylated Detection antibody Streptavidin-Alkaline Phosphatase conjugate Bovine Serum Albumin (BSA)-2g Ready t use BCIP / NBT-(Substrate buffer)

[0182] A peptide protein that partially contains the amino acid sequence of a cancer antigen protein or cancer antigen protein (including cancer antigen proteins translated by nucleic acids) corresponding to a cancer vaccine.

[0183] (Example 6: Treatment example) The following provides an explanation of how this embodiment can be implemented as a treatment method.

[0184] For patients being considered for cancer vaccine treatment, PBMCs will be collected via blood sample before the start of treatment.

[0185] Subsequently, ELISPOT assay: PBMCs are cultured in vitro and stimulated with a cancer antigen protein or a partial peptide protein of a cancer antigen protein corresponding to the cancer vaccine.

[0186] Subsequently, only those who test positive in the ELISPOT assay will be deemed eligible for the cancer vaccine treatment and will receive the treatment. Those who test negative will be deemed ineligible and will not receive the cancer vaccine treatment.

[0187] (Note) As described above, while the present disclosure has been illustrated using preferred embodiments thereof, it is understood that the scope of this disclosure should be interpreted solely by the claims. Patents, patent applications and other documents cited herein should be incorporated herein by reference as if their contents were specifically described herein. [Industrial applicability]

[0188] This disclosure provides a novel therapeutic or preventive agent.

Claims

1. A method for predicting the effectiveness of cancer vaccines in vitro, The method includes a step of confirming the reactivity of peripheral blood mononuclear cells taken from a patient to stimulation with a cancer antigen corresponding to the cancer vaccine using ELISPOT or FluoroSpot, and predicting or calculating the effect of the cancer vaccine based on the reactivity, wherein the prediction and / or calculation are performed To confirm the reactivity of peripheral blood mononuclear cells derived from the patient to stimulation with the cancer antigen corresponding to the cancer vaccine, and Predicting or calculating the effect of the cancer vaccine based on the aforementioned reactivity. It includes, The cancer antigen corresponding to the cancer vaccine is a protein composed of all or part of the amino acid sequence contained in the cancer vaccine. method.

2. The method according to claim 1, wherein the prediction is made before administration of the cancer vaccine or during treatment with the cancer vaccine.

3. The method according to claim 1, wherein the prediction includes obtaining peripheral blood mononuclear cells derived from the patient.

4. The method according to claim 1, wherein the prediction includes providing the cancer antigen corresponding to the cancer vaccine.

5. The method according to claim 1, wherein the prediction of the effect is made before the administration of the cancer vaccine.

6. The method according to claim 1, wherein the effect includes at least one selected from the group consisting of 1) the efficacy of the cancer vaccine, 2) the persistence of the cancer vaccine, 3) the determination of patients to whom the cancer vaccine should be administered, 4) the determination of the cancer vaccine to be administered, and 5) the determination of the dosage and administration of the cancer vaccine.

7. The method according to claim 1, wherein the confirmation of the reactivity is performed by ELISPOT.

8. The method according to claim 7, wherein, in the prediction, if the measurement value by ELISPOT is above a predetermined value, it is determined that the cancer vaccine will be effective / that the patient should be administered the vaccine.

9. In the above prediction, 1) Regarding the efficacy of the aforementioned cancer vaccine, If the measurement value obtained by the ELISPOT is above a predetermined value, it is determined that the cancer vaccine is effective. 2) Regarding the persistence of the aforementioned cancer vaccine, If the measurement value obtained by the ELISPOT is above a predetermined value, it is determined that the cancer vaccine is effective. 3) Regarding the determination of which patients should be administered the aforementioned cancer vaccine, If the measurement value obtained by the ELISPOT is above a predetermined value, the patient is determined to be a patient who should be administered the cancer vaccine, and / or, 4) Regarding the decision on which cancer vaccine to administer, If the measurement value obtained by the aforementioned ELISPOT is above a predetermined value, the patient is determined to be a patient who should be administered the target vaccine. The method according to claim 7.

10. The method according to claim 7, wherein the predetermined value of Spot Counts by the ELISPOT is 7.

11. The method according to claim 1, wherein the cancer antigen includes all cancer antigens contained in a cancer vaccine.

12. The method according to claim 1, wherein the cancer antigen comprises at least one selected from the group consisting of WT1, AGEA1-4, NY-ESO-1, PRAME, SSX2, CT8, CD19, GP100, MART1, PSA, PSMA, tyrosine, HER2, MUC1, CEA, survivin, cycling B1, EGFR, mesothelin, telomerase, and neoantigens resulting from gene mutations in the patient's tumor cells.

13. The method according to claim 1, wherein the cancer antigen is the WT1 protein.

14. A kit for predicting the effectiveness of a cancer vaccine in vitro, wherein the kit is The cancer antigen corresponding to the aforementioned cancer vaccine, A kit comprising means for confirming the reactivity of peripheral blood mononuclear cells taken from a patient to stimulation with the cancer antigen corresponding to the cancer vaccine, and for predicting and / or calculating the effect of the cancer vaccine based on the reactivity, wherein the prediction and / or calculation is To confirm the reactivity of peripheral blood mononuclear cells derived from the patient to stimulation with the cancer antigen corresponding to the cancer vaccine, and Predicting or calculating the effect of the cancer vaccine based on the aforementioned reactivity. It includes, Here, the means includes an ELISPOT or a FluoroSpot kit. The cancer antigen corresponding to the cancer vaccine is a protein composed of all or part of the amino acid sequence contained in the cancer vaccine. kit.

15. A pharmaceutical product comprising a cancer vaccine, wherein the regimen of the vaccine is determined based on the reactivity of patient-derived peripheral blood mononuclear cells to stimulation with a cancer antigen corresponding to the cancer vaccine, wherein the regimen is determined by confirming the reactivity of the patient-derived peripheral blood mononuclear cells to stimulation with the cancer antigen corresponding to the cancer vaccine using ELISPOT or FluoroSpot, and Predicting or calculating the effect of the cancer vaccine based on the aforementioned reactivity. It is determined based on, The cancer antigen corresponding to the cancer vaccine is a protein composed of all or part of the amino acid sequence contained in the cancer vaccine. Pharmaceuticals.

16. The pharmaceutical product according to claim 15, characterized in that the pharmaceutical product is administered to a subject whose peripheral blood mononuclear cells obtained from the subject to which the pharmaceutical product is administered before administration have a reactivity value to the antigen for the cancer vaccine that is equal to or greater than a predetermined value.

17. A pharmaceutical kit, wherein the pharmaceutical kit is Pharmaceuticals including cancer vaccines, A detection agent for detecting stimulation by cancer antigens corresponding to the aforementioned cancer vaccine, This includes instructions describing how to use the kit, The aforementioned instructions indicate that the vaccine regimen shall be determined based on the responsiveness of patient-derived immune cells to stimulation with the cancer antigen corresponding to the cancer vaccine, and that the regimen shall confirm the responsiveness of patient-derived peripheral blood mononuclear cells to stimulation with the cancer antigen corresponding to the cancer vaccine, and Predicting or calculating the effect of the cancer vaccine based on the aforementioned reactivity. It is instructed that the decision be made based on the following: Here, the detection agent includes an ELISPOT or a FluoroSpot kit. A kit in which the cancer antigen corresponding to the cancer vaccine is a protein composed of all or part of the amino acid sequence contained in the cancer vaccine.

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