Prediction of effects of cancer vaccine

Abstract

The present disclosure provides a feature related to cancer prediction. More specifically, the present disclosure provides a method for predicting effects of a cancer vaccine. The method includes checking reactivity with immune cells (for example, peripheral blood mononuclear cells) derived from the patient caused by stimulation from a cancer antigen corresponding to the cancer vaccine or an antigenic portion of the cancer antigen, and predicting / calculating the effects of the cancer vaccine (for example, efficacy (efficacy / persistence) / determining patients to be vaccinated) on the basis of the reactivity.

Description

Predicting the effectiveness of cancer vaccines

[0001] This disclosure relates to predicting the effectiveness of cancer vaccines.

[0002] Cancer is one of the leading causes of death worldwide, and developing treatments for it is a critical challenge in the medical field. While there are a wide range of cancer treatments, including surgery, radiation therapy, chemotherapy, and molecular targeted drugs, the effectiveness of these treatments varies depending on the individual patient's constitution and the type of cancer, and they do not show uniform results for all patients.

[0003] In recent years, immunotherapy, which activates a patient's immune system to attack cancer cells, has attracted attention. Among these, cancer vaccines are expected to be innovative treatments that aim to suppress or prevent cancer progression by inducing an immune response against cancer antigens in the patient's body. However, it is known that the effectiveness of cancer vaccines varies greatly from patient to patient, and establishing technology to predict their effectiveness in advance is an urgent task.

[0004] This disclosure is the result of diligent research by the inventors and provides, as non-limiting examples, the following: (Item 1) A method for predicting the effect of a cancer vaccine, comprising confirming the reactivity of patient-derived immune cells to stimulation with a cancer antigen or an antigenic portion of the cancer antigen or 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 method according to any of the above items, wherein the prediction is performed before administration of the vaccine or during the treatment period with the vaccine. (Item 4) The method according to any of the above items, comprising obtaining patient-derived immune cells. (Item 5) The method according to any of the above items, comprising providing a cancer antigen or an antigenic portion of the cancer antigen corresponding to the cancer vaccine. (Item 6) The method according to any one of the above items, wherein the method is performed in vitro. (Item 7) The method according to any one of the above items, wherein the prediction of the effect is made 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 a change in the dosage or 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 by ELISPOT or a functional equivalent thereof (e.g., FluoroSpot, etc.). (Item 11) The method according to any one of the above items, wherein the confirmation of 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, it is determined that the cancer vaccine will be effective / that the patient should be administered the vaccine. (Item 13) 1) Effectiveness of the cancer vaccine: If the measured value (Spot Counts) by ELISPOT is greater than or equal to a predetermined value, it is determined that the cancer vaccine is effective. 2) Durability of the cancer vaccine: If the measured value (Spot Counts) by ELISPOT is greater than or equal to a predetermined value, it is determined that the cancer vaccine is durable. 3) Determination of patients who should receive the cancer vaccine: If the measured value (Spot Counts) by ELISPOT is greater than or equal to a predetermined value, it is determined that the patients should receive the cancer vaccine. 4) Determination of the cancer vaccine to be administered: If the measured value (Spot Counts) by ELISPOT is greater than or equal to a predetermined value, it is determined that the patients should receive the target vaccine. The method according to any one of the above items. (Item 14) The method according to any one of the above items, wherein the predetermined value of the measured value (Spot Counts) by ELISPOT is 7. (Item 15) The method according to any one of the above items, wherein the cancer antigen comprises all cancer antigens contained in the 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, tyrosine, HER2, MUC1, CEA, survivin, cycling 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 agent according to any one of the above items, wherein the prediction and / or calculation comprises confirming the responsiveness of immune cells derived from the patient to stimulation with a cancer antigen or the antigenic portion of the cancer antigen or equivalent thereof, corresponding to the cancer vaccine, and predicting or calculating the effect of the cancer vaccine based on the responsiveness. (Item A2) The agent 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 A3) The agent according to any one of the above items, wherein the prediction is performed before administration of the vaccine or during the treatment period with the vaccine. (Item A4) The agent according to any one of the above items, wherein the prediction comprises obtaining immune cells derived from the patient. (Item A5) The agent according to any one of the above items, wherein the prediction comprises providing a cancer antigen or the antigenic portion of the cancer antigen corresponding to the cancer vaccine. (Item A6) The agent according to any one of the above items, wherein the method is performed in vitro. (Item A7) The agent according to any one of the above items, wherein the prediction of the effect is made before administration of the cancer vaccine. (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 agent according to any one of the above items, wherein the confirmation of the reactivity is performed using ELISPOT or its functional equivalent (e.g., FluoroSpot, etc.). (Item A11) The agent described in any one of the above items, wherein the reactivity is confirmed by ELISPOT.(Item A12) The agent described in any one of the above items, which is determined to be effective in the cancer vaccine / should be administered to a patient when the measured value (Spot Counts) by ELISPOT is equal to or greater than a predetermined value. (Item A13) 1) The effectiveness of the cancer vaccine: If the measured value (Spot Counts) by ELISPOT is greater than or equal to a predetermined value, it is determined that the cancer vaccine is effective. 2) The persistence of the cancer vaccine: If the measured value (Spot Counts) by ELISPOT is greater than or equal to a predetermined value, it is determined that the cancer vaccine is persistent. 3) Determination of patients to whom the cancer vaccine should be administered: If the measured value (Spot Counts) by ELISPOT is greater than or equal to a predetermined value, it is determined that the patient should be administered the cancer vaccine. 4) Determination of the cancer vaccine to be administered: If the measured value (Spot Counts) by ELISPOT is greater than or equal to a predetermined value, it is determined that the patient should be administered the target vaccine. The agent according to any one of the above items. (Item A14) The agent according to any one of the above items, wherein the predetermined value of the measured value (Spot Counts) by ELISPOT is 7. (Item A15) The agent according to any one of the above items, wherein the cancer antigen comprises all cancer antigens contained in a cancer vaccine. (Item A16) The agent 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, tyrosine, HER2, MUC1, CEA, survivin, cycling B1, EGFR, mesothelin, telomerase, and neoantigens resulting from gene mutations in the patient's tumor cells. (Item A17) The agent according to any one of the above items, wherein the cancer antigen is WT1. (Item A18) The agent according to any one of the above items, further comprising the characteristics described in any one or more of items 1 to 17.(Item B0) A kit for predicting the effectiveness of a cancer vaccine, the kit comprising a cancer antigen corresponding to the cancer vaccine or an antigenic portion of the cancer antigen or an equivalent thereof, and means for confirming the responsiveness of patient-derived immune cells to stimulation by the cancer antigen corresponding to the cancer vaccine or the antigenic portion of the cancer antigen or an equivalent thereof, and predicting and / or calculating the effectiveness of the cancer vaccine based on the responsiveness. (Item B1) The kit according to any one of the above items, wherein the prediction and / or calculation includes confirming the responsiveness of patient-derived immune cells to stimulation by the cancer antigen corresponding to the cancer vaccine or the antigenic portion of the cancer antigen or an equivalent thereof, and predicting or calculating the effectiveness of the cancer vaccine based on the responsiveness. (Item B2) The kit according to any one of the above items, wherein the cancer antigen or the antigenic portion of the cancer antigen or an 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 B3) The kit according to any one of the above items, wherein the prediction is made before administration of the vaccine or during the treatment period with the vaccine. (Item B4) The kit according to any one of the above items, comprising obtaining immune cells derived from the patient. (Item B5) The kit according to any one of the above items, comprising providing a cancer antigen or an antigenic portion of the cancer antigen for the cancer vaccine. (Item B6) The kit according to any one of the above items, wherein the method is carried out in vitro. (Item B7) The kit according to any one of the above items, wherein the prediction of the effect is made before administration of the cancer vaccine. (Item B8) 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 B9) 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 who should be administered the cancer vaccine, 4) the determination of the cancer vaccine to be administered, and 5) the determination of the change in the dosage and administration of the cancer vaccine. (Item B10) The kit according to any one of the above items, wherein the means for confirming the reactivity includes means for performing ELISPOT or a functional equivalent thereof (e.g., FluoroSpot). (Item B11) The kit according to any one of the above items, wherein the means for confirming the reactivity includes means for performing ELISPOT. (Item B12) The kit according to any one of the above items, wherein if the measured value (Spot Counts) 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. (Item B13) 1) Effectiveness of the cancer vaccine: If the measured value (Spot Counts) by ELISPOT is greater than or equal to a predetermined value, it is determined that the cancer vaccine is effective. 2) Durability of the cancer vaccine: If the measured value (Spot Counts) by ELISPOT is greater than or equal to a predetermined value, it is determined that the cancer vaccine is durable. 3) Determination of patients to whom the cancer vaccine should be administered: If the measured value (Spot Counts) by ELISPOT is greater than or equal to a predetermined value, it is determined that the patients should be administered the cancer vaccine. 4) Determination of the cancer vaccine to be administered: If the measured value (Spot Counts) by ELISPOT is greater than or equal to a predetermined value, it is determined that the patients should be administered the target vaccine. The kit described in any one of the above items of Item B. (Item B14) The kit described in any one of the above items, wherein the predetermined value of the measured value (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 a 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, tyrosine, HER2, MUC1, CEA, survivin, cycling 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 items 1 to 17 and items A1 to A18. (Item C1) A method for predicting the effect of a cancer vaccine, comprising: collecting immune cells from the patient; providing a cancer antigen or an antigenic portion of the cancer antigen corresponding to the cancer vaccine, or equivalents thereof; stimulating the immune cells with the cancer antigen or an 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. (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 equivalents 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 equivalents thereof. (Item C3) The method according to any one of the above items, wherein the prediction is made before administration of the vaccine or during the treatment period with the vaccine. (Item C4) The method according to any one of the above items, comprising obtaining immune cells derived from the patient. (Item C5) The method according to any one of the above items, comprising providing a cancer antigen or an antigenic portion of the cancer antigen corresponding to the cancer vaccine. (Item C6) The method described in any one of the above items, performed in vitro. (Item C7) The prediction of the effect described in any one of the above items, performed before administration of the cancer vaccine.(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 changes in the dosage and administration of the cancer vaccine. (Item C10) The method according to any one of the above items, wherein the confirmation of reactivity is performed by 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 reactivity is performed by 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 / that the patient should be administered the vaccine. (Item C13) The method according to any one of the above items C, wherein the predetermined value of the measurement value of the measurement value of any one of the above items C, wherein the predetermined value of the measurement value of any one of the above items C, wherein the predetermined value of the measurement value of any one of the above items C, wherein the predetermined value of the measurement value of any one of the above items C, wherein the predetermined value of the measurement value of any one of the above items C, wherein the predetermined value of the measurement value of any one of the above items C, wherein the predetermined value of the measurement value of any one of the above items C, wherein the method according to any one of the above items C, wherein the method according to any one of the above items C, wherein the method according to any one of the above items C, wherein the predetermined value of the (Item C15) The method according to 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, tyrosine, HER2, MUC1, CEA, survivin, cycling 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) The method according to any one of the above items, further comprising the features described in any one or more of 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 antigenic portion of the cancer antigen corresponding to the patient. (Item D0) Use of a cancer antigen or 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 responsiveness of patient-derived immune cells to stimulation with the cancer antigen or 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 responsiveness. (Item D1) The use of any one of the above items, wherein the prediction and / or calculation comprises confirming the responsiveness of patient-derived immune cells to stimulation with the cancer antigen or antigenic portion of the cancer antigen or an equivalent thereof corresponding to the cancer vaccine, and predicting or calculating the effectiveness of the cancer vaccine based on the responsiveness. (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 use described in any one of the above items, wherein the prediction is made before administration of the vaccine or during the treatment period with the vaccine.(Item D4) The use according to any one of the above items, comprising obtaining immune cells derived from the patient. (Item D5) The use 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 D6) The use according to any one of the above items, wherein the method is carried out in vitro. (Item D7) The use according to any one of the above items, wherein the prediction of the effect is made before administration of the cancer vaccine. (Item D8) The use 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 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 who should be administered the cancer vaccine, 4) the determination of which cancer vaccine to administer, and 5) the determination of a change in the dosage and administration of the cancer vaccine. (Item D10) The use described in any one of the above items, wherein the confirmation of the reactivity is performed by ELISPOT or a functional equivalent thereof (e.g., FluoroSpot). (Item D11) The use described in any one of the above items, wherein the confirmation of the reactivity is performed by ELISPOT. (Item D12) The use described in any one of the above items, wherein it is determined that the cancer vaccine will be effective / that the patient should be administered the vaccine if the measured value (Spot Counts) by ELISPOT is above a predetermined value.(Item D13) Use as described in any one of the above items D: 1) Effectiveness of the cancer vaccine: If the measured value (Spot Counts) by ELISPOT is greater than or equal to a predetermined value, it is determined that the cancer vaccine is effective. 2) Durability of the cancer vaccine: If the measured value (Spot Counts) by ELISPOT is greater than or equal to a predetermined value, it is determined that the cancer vaccine is durable. 3) Determination of patients to whom the cancer vaccine should be administered: If the measured value (Spot Counts) by ELISPOT is greater than or equal to a predetermined value, it is determined that the patient should be administered the cancer vaccine. 4) Determination of the cancer vaccine to be administered: If the measured value (Spot Counts) by ELISPOT is greater than or equal to a predetermined value, it is determined that the patient should be administered the target vaccine. (Item D14) Use as described in any one of the above items, wherein the predetermined value of the measured value (Spot Counts) by ELISPOT is 7. (Item D15) The use according to any one of the above items, wherein the cancer antigen includes all cancer antigens contained in the cancer vaccine. (Item D16) The use according to any one of the above items, wherein the cancer antigen includes 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. (Item D17) The use according to any one of the above items, wherein the cancer antigen is WT1. (Item D18) A use 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 A0 to A18, items B0 to B18, items C1 to C19, and items D0 to D18. (Item E0) A pharmaceutical, the pharmaceutical comprising a cancer vaccine, the vaccine regimen being 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) A pharmaceutical product according to any one of the above items, wherein the prediction and / or calculation comprises confirming the reactivity of immune cells derived from the patient to stimulation with a cancer antigen or the antigenic portion of the cancer antigen or equivalent thereof corresponding to the cancer vaccine, and predicting or calculating the effect of the cancer vaccine based on the reactivity. (Item E2) A pharmaceutical product 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 E3) A pharmaceutical product according to any one of the above items, wherein the prediction is performed before administration of the vaccine or during the treatment period with the vaccine. (Item E4) A pharmaceutical product according to any one of the above items, wherein the prediction and / or calculation comprises obtaining immune cells derived from the patient. (Item E5) A pharmaceutical product according to any one of the above items, wherein the prediction and / or calculation comprises providing a cancer antigen or the antigenic portion of the cancer antigen corresponding to the cancer vaccine. (Item E6) A pharmaceutical product according to any one of the above items, wherein the method is performed in vitro. (Item E7) The pharmaceutical product according to any one of the above items, wherein the prediction of the effect is made before administration of the cancer vaccine. (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 pharmaceutical product according to any one of the above items, wherein the confirmation of the reactivity is performed using ELISPOT or its functional equivalent (e.g., FluoroSpot, etc.). (Item E11) The reactivity is confirmed by ELISPOT, and the pharmaceutical product is as described in any one of the above items.(Item E12) A pharmaceutical product 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 E13) A pharmaceutical product according to any one of the above items E, wherein the cancer vaccine is deemed effective if the measured value (Spot Counts) by ELISPOT is equal to or greater than a predetermined value, the cancer vaccine is deemed effective, the sustained effect of the cancer vaccine is determined if the measured value (Spot Counts) by ELISPOT is equal to or greater than a predetermined value, the patient is deemed to be a patient who should be administered the cancer vaccine, the patient is deemed to be a patient who should be administered the cancer vaccine if the measured value (Spot Counts) by ELISPOT is equal to or greater than a predetermined value, the patient is deemed to be a patient who should be administered the target vaccine. (Item E14) A pharmaceutical product according to any one of the above items, wherein the predetermined value of the measured value (Spot Counts) by ELISPOT is 7. (Item E15) The pharmaceutical product according to any one of the above items, wherein the cancer antigen includes all cancer antigens contained in a cancer vaccine. (Item E16) The pharmaceutical product according to any one of the above items, wherein the cancer antigen includes 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. (Item E17) The pharmaceutical product according to any one of the above items, wherein the cancer antigen is WT1. (Item E18) A pharmaceutical product according to any one of the above items, further possessing the characteristics described in any one or more of the above items: items 1 to 17, items A0 to A18, items B0 to B18, items C1 to C19, and items D0 to 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 in which the value of reactivity to antigen stimulation for the cancer vaccine in immune cells obtained from the subject to which the pharmaceutical product is administered before administration is equal to or greater than a predetermined value. (Item E20) The pharmaceutical product according to any one of the above items, wherein the pharmaceutical product 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 cancer vaccine according to any one of the above items, wherein the prediction and / or calculation includes confirming the reactivity of patient-derived immune cells 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 reactivity. (Item F2) The cancer vaccine 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 F3) The cancer vaccine according to any of the above items, wherein the prediction is made before administration of the vaccine or during the treatment period with the vaccine. (Item F4) The cancer vaccine according to any of the above items, comprising obtaining immune cells derived from the patient. (Item F5) The cancer vaccine according to any of the above items, comprising providing a cancer antigen or the antigenic portion of the cancer antigen corresponding to the cancer vaccine. (Item F6) The cancer vaccine according to any of the above items, wherein the method is carried out in vitro. (Item F7) The cancer vaccine according to any of the above items, wherein the prediction of the effect is made before administration of the cancer vaccine.(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 change in the dosage and administration of the cancer vaccine. (Item F10) The cancer vaccine according to any one of the above items, wherein the confirmation of reactivity is performed by ELISPOT or a functional equivalent thereof (e.g., FluoroSpot, etc.). (Item F11) The cancer vaccine according to any one of the above items, wherein the confirmation of reactivity is performed by ELISPOT. (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 when the measured value (Spot Counts) by ELISPOT is equal to or greater than a predetermined value. (Item F13) 1) Effectiveness of the cancer vaccine: If the measured value (Spot Counts) by ELISPOT is greater than or equal to a predetermined value, it is determined that the cancer vaccine is effective. 2) Durability of the cancer vaccine: If the measured value (Spot Counts) by ELISPOT is greater than or equal to a predetermined value, it is determined that the cancer vaccine is durable. 3) Determination of patients to whom the cancer vaccine should be administered: If the measured value (Spot Counts) by ELISPOT is greater than or equal to a predetermined value, it is determined that the patient should be administered the cancer vaccine. 4) Determination of the cancer vaccine to be administered: If the measured value (Spot Counts) by ELISPOT is greater than or equal to a predetermined value, it is determined that the patient should be administered the target vaccine. The cancer vaccine described in any one of the above items F. (Item F14) The cancer vaccine described in any one of the above items, wherein the predetermined value of the measured value (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) The 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, tyrosine, HER2, MUC1, CEA, survivin, cycling B1, EGFR, mesothelin, telomerase, and neoantigens resulting from gene mutations in the patient's tumor cells. (Item F17) The cancer vaccine according to 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 features described in any one or more of the above items, namely items 1 to 17, items A0 to A18, items B0 to B18, items C1 to C19, items D1 to D18, and items E0 to 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 of antigen stimulation for the cancer vaccine 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 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 reactivity to the stimulation and calculating a prediction of the effect of the cancer vaccine based on the reactivity; 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 method according to any of the above items, wherein the prediction is made before administration of the vaccine or during the treatment period with the vaccine. (Item G4) The method according to any of the above items, comprising obtaining immune cells derived from the patient.(Item G5) The method according to any one of the above items, comprising providing a cancer antigen or an antigenic portion of the cancer antigen corresponding to the cancer vaccine. (Item G6) The method according to any one of the above items, wherein the method is carried out in vitro. (Item G7) The method according to any one of the above items, wherein the prediction of the effect is made before administration of the cancer vaccine. (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 which cancer vaccine should be administered, and 5) the determination of a change in the dosage or administration of the cancer vaccine. (Item G10) The method according to any one of the above items, wherein the confirmation of reactivity is performed using ELISPOT or a functional equivalent thereof (e.g., FluoroSpot). (Item G11) The method according to any one of the above items, wherein the confirmation of 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 above a predetermined value, it is determined that the cancer vaccine will be effective / that the patient should be administered the vaccine. (Item G13) The method according to any one item G above: 1) The effectiveness of the cancer vaccine: If the measured value (Spot Counts) by ELISPOT is equal to or greater than a predetermined value, it is determined that the cancer vaccine is effective. 2) The persistence of the cancer vaccine: If the measured value (Spot Counts) by ELISPOT is equal to or greater than a predetermined value, it is determined that the cancer vaccine is persistent. 3) The determination of patients who should be administered the cancer vaccine: If the measured value (Spot Counts) by ELISPOT is equal to or greater than a predetermined value, it is determined that the patients should be administered the cancer vaccine. 4) The determination of the cancer vaccine to be administered: If the measured value (Spot Counts) by ELISPOT is equal to or greater than a predetermined value, it is determined that the patients should be administered the target vaccine.(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 according to 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 includes 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, cycling 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) The method according to any one of the above items, further comprising the features described in any one or more of the items 1 to 17, A0 to A18, B0 to B18, C1 to C19, D0 to D18, E0 to E20, and F0 to F19. (Item G19) The method according to any one of the above items, further comprising identifying the cancer antigen or antigenic portion of the cancer antigen corresponding to the patient. (Item H0) The use of a cancer vaccine for the manufacture of a medicament for the prevention or treatment of cancer, wherein the vaccine regime is determined based on the reactivity of patient-derived immune cells (e.g., peripheral blood mononuclear cells) to stimulation with the cancer antigen or antigenic portion of the cancer antigen corresponding to the cancer vaccine. (Item H1) The use described in any one of the above items, wherein the prediction and / or calculation includes confirming the responsiveness of patient-derived immune cells 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 responsiveness.(Item H2) The use 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 H3) The use described in any of the above items, wherein the prediction is made before administration of the vaccine or during the treatment period with the vaccine. (Item H4) The use described in any of the above items, comprising obtaining immune cells derived from the patient. (Item H5) The use described in any of the above items, comprising providing a cancer antigen or the antigenic portion of the cancer antigen corresponding to the cancer vaccine. (Item H6) The use described in any of the above items, wherein the method is carried out in vitro. (Item H7) The use described in any of the above items, wherein the prediction of the effect is made before administration of the cancer vaccine. (Item H8) The use 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 H9) The use 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 H10) The use according to any one of the above items, wherein the confirmation of reactivity is performed by ELISPOT or its functional equivalent (e.g., FluoroSpot, etc.). (Item H11) The use according to any one of the above items, wherein the confirmation of reactivity is performed by ELISPOT. (Item H12) 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 H13) 1) Effectiveness of the cancer vaccine: If the measured value (Spot Counts) by ELISPOT is greater than or equal to a predetermined value, it is determined that the cancer vaccine is effective. 2) Durability of the cancer vaccine: If the measured value (Spot Counts) by ELISPOT is greater than or equal to a predetermined value, it is determined that the cancer vaccine is durable. 3) Determination of patients who should receive the cancer vaccine: If the measured value (Spot Counts) by ELISPOT is greater than or equal to a predetermined value, it is determined that the patient should receive the cancer vaccine. 4) Determination of the cancer vaccine to be administered: If the measured value (Spot Counts) by ELISPOT is greater than or equal to a predetermined value, it is determined that the patient should receive the target vaccine. Use as described in any one of the above items H. (Item H14) Use as described in any one of the above items, where the predetermined value of the measured 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 includes 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. (Item H17) The use described in any one of the above items, wherein the cancer antigen is WT1. (Item H18) A use according to any one of the above items, further comprising the characteristics described in any one or more of the above items: items 1 to 17, items A0 to A18, items B0 to B18, items C1 to C19, items D0 to D18, items E0 to E20, and items F0 to F19. (Item H19) A use according to any one of the above items, characterized in that the pharmaceutical is administered to a subject in which the reactivity value of immune cells obtained from the subject to which the pharmaceutical is administered before administration is equal to or greater than a predetermined value in response to antigen stimulation for the cancer vaccine.(Item I0) A pharmaceutical kit comprising a pharmaceutical including a cancer vaccine and a detection agent for detecting stimulation by a cancer antigen or an antigenic portion of the cancer antigen corresponding to the cancer vaccine, wherein the vaccine regimen is determined based on the reactivity of patient-derived immune cells to stimulation by the cancer antigen or an equivalent thereof corresponding to the cancer vaccine. (Item I1) The kit according to any one of the above items, wherein the prediction and / or calculation comprises confirming the reactivity of patient-derived immune cells to stimulation by a cancer antigen or an antigenic portion of the cancer antigen or an equivalent thereof corresponding to the cancer vaccine, and predicting or calculating the effect of the cancer vaccine based on the reactivity. (Item I2) The kit according to any one of the above items, wherein the cancer antigen or the antigenic portion of the cancer antigen or an 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 kit according to any one of the above items, wherein the prediction is performed before administration of the vaccine or during the treatment period with the vaccine. (Item I4) A kit according to any one of the above items, comprising 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) A kit according to any one of the above items, wherein the method is carried out in vitro. (Item I7) A kit according to any one of the above items, wherein the prediction of the effect is made before administration of the cancer vaccine. (Item I8) A 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 who should be administered the cancer vaccine, 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 kit according to any one of the above items, wherein the means for confirming the reactivity includes means for performing ELISPOT or a functional equivalent thereof (e.g., FluoroSpot). (Item I11) The kit according to any one of the above items, wherein the confirmation of the reactivity includes means for performing ELISPOT. (Item I12) The kit according to any one of the above items, wherein if the measured value (Spot Counts) 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. (Item I13) 1) Effectiveness of the cancer vaccine: If the measured value (Spot Counts) by ELISPOT is greater than or equal to a predetermined value, it is determined that the cancer vaccine is effective. 2) Durability of the cancer vaccine: If the measured value (Spot Counts) by ELISPOT is greater than or equal to a predetermined value, it is determined that the cancer vaccine is durable. 3) Determination of patients to whom the cancer vaccine should be administered: If the measured value (Spot Counts) by ELISPOT is greater than or equal to a predetermined value, it is determined that the patient should be administered the cancer vaccine. 4) Determination of the cancer vaccine to be administered: If the measured value (Spot Counts) by ELISPOT is greater than or equal to a predetermined value, it is determined that the patient should be administered the target vaccine. The kit described in any one of the above items of Item I. (Item I14) The kit described in any one of the above items, wherein the predetermined value of the measured value (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 a 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, tyrosine, HER2, MUC1, CEA, survivin, cycling 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) A kit according to any one of the above items, further comprising the features described in any one or more of the items 1 to 17, A0 to A18, B0 to B18, C1 to C19, D0 to D18, E0 to E20, F0 to F19, G1 to G19, and H0 to H19. (Item I19) A 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 reactivity value of antigen stimulation to the cancer vaccine equal to or greater than a predetermined value. (Item I20) A kit according to any one of the above items, wherein the pharmaceutical is for the prevention or treatment of cancer. (Item J1) An agent for predicting the effect of a cancer vaccine, wherein the agent comprises a cancer antigen corresponding to the cancer vaccine, and the cancer antigen is the proteinaceous cancer antigen. (Item J2) The agent described in the above item, wherein the proteinogenic cancer antigen is a protein composed of all or part of the amino acid sequence contained in the cancer vaccine. (Item J3) The agent described in any one of the above items, which is used in a single-cell level cytokine production detection assay.(Item J4) The agent according to any one of the above items, comprising the step of confirming the responsiveness of peripheral blood mononuclear cells taken from a patient to stimulation with a cancer antigen corresponding to the cancer vaccine by a single-cell level cytokine production detection assay, and predicting or calculating the effect of the cancer vaccine based on the responsiveness, wherein the prediction and / or calculation is used in a method that includes confirming the responsiveness 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 responsiveness. (Item J5) The agent according to any one of the above items, wherein the prediction is performed before administration of the cancer vaccine or during the treatment period with the cancer vaccine. (Item J6) The agent according to any one of the above items, wherein the prediction includes obtaining peripheral blood mononuclear cells derived from the patient. (Item J7) The agent according to any one of the above items, wherein the prediction includes providing the cancer antigen corresponding to the cancer vaccine. (Item J8) The agent according to any one of the above items, wherein the prediction of the effect is made before administration of the cancer vaccine. (Item J9) 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 change in the dosage and administration of the cancer vaccine. (Item J10) The agent according to any one of the above items, wherein the confirmation of the reactivity is performed by ELISPOT. (Item J11) The agent according to any one of the above items, 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.(Item J12) In the above prediction, 1) Regarding the efficacy of the cancer vaccine, if the measurement value by ELISPOT is above a predetermined value, it is determined that the cancer vaccine is effective; 2) Regarding the persistence of the cancer vaccine, if the measurement value by ELISPOT is above a predetermined value, it is determined that the cancer vaccine is persistent; 3) Regarding the determination of patients to be administered the cancer vaccine, if the measurement value by ELISPOT is above a predetermined value, it is determined that the patient should be administered the cancer vaccine, and / or; 4) Regarding the determination of which cancer vaccine to administer, if the measurement value by ELISPOT is above a predetermined value, it is determined that the patient should be administered the target vaccine; the agent according to any one of the above items. (Item J13) The agent according to any one of the above items, wherein the predetermined value of Spot Counts by ELISPOT is 7. (Item J14) The agent according to any one of the above items, wherein the cancer antigen includes all cancer antigens contained in the cancer vaccine. (Item J15) The agent 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, cycling B1, EGFR, mesothelin, telomerase, and neoantigens resulting from gene mutations in the patient's tumor cells. (Item J16) The agent according to any one of the above items, wherein the cancer antigen is the WT1 protein.(Item J17) A kit for in vitro predicting the efficacy of a cancer vaccine, the kit comprising: a cancer antigen corresponding to the cancer vaccine; and means for confirming the reactivity of peripheral blood mononuclear cells taken from a patient to stimulation by the cancer antigen corresponding to the cancer vaccine, the kit for predicting and / or calculating the efficacy of the cancer vaccine based on the reactivity, wherein the prediction and / or calculation includes confirming the reactivity of peripheral blood mononuclear cells derived from the patient to stimulation by the cancer antigen corresponding to the cancer vaccine, and predicting or calculating the efficacy of the cancer vaccine based on the reactivity, wherein the means includes performing a single-cell level cytokine production detection assay, and the cancer antigen is the proteinaceous cancer antigen. (Item J18) The kit according to any one of the above items, further comprising the features described in any one or more of the above items. (Item J19) A pharmaceutical product comprising a cancer vaccine, wherein the vaccine regime 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 regime is determined based on confirming the reactivity of patient-derived peripheral blood mononuclear cells to stimulation with the cancer antigen corresponding to the cancer vaccine by a single-cell level cytokine production detection assay, and predicting or calculating the effect of the cancer vaccine based on the reactivity, wherein the cancer antigen is the proteinaceous cancer antigen. (Item J20) The pharmaceutical product according to any one of the above items, wherein the pharmaceutical product is administered to a subject whose reactivity to stimulation with the cancer vaccine in peripheral blood mononuclear cells obtained from the subject before administration is greater than or equal to a predetermined value. (Item J21) The pharmaceutical product according to any one of the above items, further comprising the features described in any one or more of the above items.(Item J22) A pharmaceutical kit comprising: a pharmaceutical comprising a cancer vaccine; a detection agent for detecting stimulation by a cancer antigen corresponding to the cancer vaccine; and instructions for using the kit, wherein the instructions indicate that the vaccine regime is determined based on the responsiveness of patient-derived immune cells to stimulation by a cancer antigen corresponding to the cancer vaccine, and that the regime is determined based on confirming the responsiveness of patient-derived peripheral blood mononuclear cells to stimulation by the cancer antigen corresponding to the cancer vaccine, and predicting or calculating the effect of the cancer vaccine based on the responsiveness, wherein the detection agent includes one for performing a single-cell level cytokine production detection assay, and the cancer antigen is the proteinaceous cancer antigen. (Item J23) The kit according to any one of the above items, further comprising the features described in any one or more of the above items. (Item J24) A method for predicting the efficacy of a cancer vaccine in vitro, the method comprising the steps of confirming the responsiveness of peripheral blood mononuclear cells taken from a patient to stimulation with a cancer antigen corresponding to the cancer vaccine by a single-cell level cytokine production detection assay, and predicting or calculating the efficacy of the cancer vaccine based on the responsiveness, wherein the prediction and / or calculation encompasses confirming the responsiveness 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 efficacy of the cancer vaccine based on the responsiveness, wherein the cancer antigen is the proteinaceous cancer antigen. (Item J25) A method according to any one of the above items, further comprising the features described in any one or more of the above items. (Item JA1) A use of a cancer antigen corresponding to a cancer vaccine for predicting the efficacy of a cancer vaccine, wherein the cancer antigen is the proteinaceous cancer antigen. (Item JA2) The use according to the above item, wherein the proteinaceous cancer antigen is a protein composed of all or part of the amino acid sequence contained in the cancer vaccine.(Item JA3) The use of the agent as described in any one of the above items, wherein the agent is used in a single-cell level cytokine production detection assay. (Item JA4) The use of the agent as described in any one of the above items, comprising the step of confirming the responsiveness of peripheral blood mononuclear cells taken from a patient to stimulation with a cancer antigen corresponding to the cancer vaccine by a single-cell level cytokine production detection assay, and predicting or calculating the effect of the cancer vaccine based on the responsiveness, wherein the prediction and / or calculation is used in a method that includes confirming the responsiveness 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 responsiveness. (Item JA5) The use of the agent as described in any one of the above items, wherein the prediction is performed before administration of the cancer vaccine or during the treatment period with the cancer vaccine. (Item JA6) The use of the agent as described in any one of the above items, wherein the prediction includes obtaining peripheral blood mononuclear cells derived from the patient. (Item JA7) The use of the agent as described in any one of the above items, wherein the prediction includes providing the cancer antigen corresponding to the cancer vaccine. (Item JA8) The use described in any one of the above items, wherein the prediction of the effect is made before the administration of the cancer vaccine. (Item JA9) 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 a change in the dosage and administration of the cancer vaccine. (Item JA10) The use described in any one of the above items, wherein the confirmation of the reactivity is performed by ELISPOT. (Item JA11) The use described in any one of the above items, 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.(Item JA12) In the above prediction, 1) Regarding the efficacy of the cancer vaccine, if the measurement value by ELISPOT is above a predetermined value, it is determined that the cancer vaccine is effective; 2) Regarding the persistence of the cancer vaccine, if the measurement value by ELISPOT is above a predetermined value, it is determined that the cancer vaccine is persistent; 3) Regarding the determination of patients to be administered the cancer vaccine, if the measurement value by ELISPOT is above a predetermined value, it is determined that the patient should be administered the cancer vaccine, and / or; 4) Regarding the determination of which cancer vaccine to administer, if the measurement value by ELISPOT is above a predetermined value, it is determined that the patient should be administered the target vaccine; Use as described in any one of the above items. (Item JA13) Use as described in any one of the above items, wherein the predetermined value of Spot Counts by ELISPOT is 7. (Item JA14) Use as described in any one of the above items, wherein the cancer antigen includes all cancer antigens contained in the cancer vaccine. (Item JA15) The use 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, cycling B1, EGFR, mesothelin, telomerase, and neoantigens resulting from gene mutations in the patient's tumor cells. (Item JA16) The use according to any one of the above items, wherein the cancer antigen is the WT1 protein.(Item JA17) The use described above is a use in a method for predicting the efficacy of a cancer vaccine in vitro, the method comprising the steps of confirming the responsiveness of peripheral blood mononuclear cells taken from a patient to stimulation with a cancer antigen corresponding to the cancer vaccine by a single-cell level cytokine production detection assay, and predicting or calculating the efficacy of the cancer vaccine based on the responsiveness, wherein the prediction and / or calculation encompasses confirming the responsiveness of peripheral blood mononuclear cells from the patient to stimulation with the cancer antigen corresponding to the cancer vaccine, and predicting or calculating the efficacy of the cancer vaccine based on the responsiveness, the cancer antigen being the proteinaceous cancer antigen. (Item JA25) The method according to any one of the above items, further comprising the features described in any one or more of the above items J1 to J25. (Item JB1) A method for predicting the effect of a cancer vaccine in a subject, the method comprising: taking peripheral blood mononuclear cells from a patient; confirming the responsiveness of the taken peripheral blood mononuclear cells to stimulation with a cancer antigen corresponding to the cancer vaccine by a single-cell level cytokine production detection assay; and predicting or calculating the effect of the cancer vaccine based on the responsiveness, wherein the prediction and / or calculation comprises confirming the responsiveness 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 responsiveness, the cancer antigen being the proteinaceous cancer antigen. (Item JB2) The method according to the above item, wherein the proteinaceous cancer antigen is a protein composed of all or part of the amino acid sequence contained in the cancer vaccine. (Item JB3) The method according to any one of the above items, wherein the agent is used in a single-cell level cytokine production detection assay.(Item JB4) The method according to any one of the above items, comprising the step of confirming the responsiveness of peripheral blood mononuclear cells taken from a patient to stimulation with a cancer antigen corresponding to the cancer vaccine by a single-cell level cytokine production detection assay, and predicting or calculating the effect of the cancer vaccine based on the responsiveness, wherein the prediction and / or calculation is used in a method that includes confirming the responsiveness 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 responsiveness. (Item JB5) The method according to any one of the above items, wherein the prediction is performed before administration of the cancer vaccine or during the treatment period with the cancer vaccine. (Item JB6) The method according to any one of the above items, wherein the prediction includes obtaining peripheral blood mononuclear cells derived from the patient. (Item JB7) The method according to any one of the above items, wherein the prediction includes providing the cancer antigen corresponding to the cancer vaccine. (Item JB8) The method according to any one of the above items, wherein the prediction of the effect is made before administration of the cancer vaccine. (Item JB9) 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 change in the dosage and administration of the cancer vaccine. (Item JB10) The method according to any one of the above items, wherein the confirmation of the reactivity is performed by ELISPOT. (Item JB11) The method according to any one of the above items, 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.(Item JB12) The method according to any one of the above items, wherein in the prediction, 1) regarding the efficacy of the cancer vaccine, if the measurement value by ELISPOT is above a predetermined value, it is determined that the cancer vaccine is effective; 2) regarding the persistence of the cancer vaccine, if the measurement value by ELISPOT is above a predetermined value, it is determined that the cancer vaccine is persistent; 3) regarding the determination of patients who should be administered the cancer vaccine, if the measurement value by ELISPOT is above a predetermined value, it is determined that the patient should be administered the cancer vaccine, and / or; 4) regarding the determination of which cancer vaccine to administer, if the measurement value by ELISPOT is above a predetermined value, it is determined that the patient should be administered the target vaccine. (Item JB13) The method according to any one of the above items, wherein the predetermined value of Spot Counts by ELISPOT is 7. (Item JB14) The method according to any one of the above items, wherein the cancer antigen includes all cancer antigens contained in the cancer vaccine. (Item JB15) 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, cycling B1, EGFR, mesothelin, telomerase, and neoantigens resulting from gene mutations in the patient's tumor cells. (Item JB16) The method according to any one of the above items, wherein the cancer antigen is the WT1 protein.(Item JB17) A method for treating and / or preventing cancer in a subject, the method comprising administering a cancer vaccine to the subject based on a vaccine regime, the vaccine regime being 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 regime is determined based on confirming the reactivity of patient-derived peripheral blood mononuclear cells to stimulation with the cancer antigen corresponding to the cancer vaccine by a single-cell level cytokine production detection assay, and predicting or calculating the effect of the cancer vaccine based on the reactivity, the cancer antigen being the proteinaceous cancer antigen. (Item JB18) The method according to any one of the above items, further comprising determining that the cancer vaccine is administered to a subject whose reactivity to antigen stimulation in peripheral blood mononuclear cells obtained from the subject to be administered before administration is greater than or equal to a predetermined value. (Item JB19) The method according to any one of the above items, further comprising the features described in any one or more of the above items J1 to J25. (Item K1) A method for predicting the effect of a cancer vaccine in vitro, the method comprising the steps of confirming the responsiveness 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 responsiveness, wherein the prediction and / or calculation encompasses confirming the responsiveness 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 responsiveness, wherein 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. (Item K2) The method according to the above item, wherein the prediction is performed before administration of the cancer vaccine or during the treatment period with the cancer vaccine. (Item K3) The method according to any one of the above items, wherein the prediction comprises obtaining peripheral blood mononuclear cells derived from the patient.(Item K4) The method according to any one of the above items, wherein the prediction comprises providing the cancer antigen corresponding to the cancer vaccine. (Item K5) The method according to any one of the above items, wherein the prediction of the effect is made before the administration of the cancer vaccine. (Item K6) The method according to any one of the above items, wherein the effect comprises 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 a change in the dosage or administration of the cancer vaccine. (Item K7) The method according to any one of the above items, wherein the confirmation of the reactivity is performed by ELISPOT. (Item K8) The method according to any one of the above items, 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. (Item K9) The method according to any one of the above items, wherein in the prediction, 1) regarding the efficacy of the cancer vaccine, if the measurement value by ELISPOT is above a predetermined value, it is determined that the cancer vaccine is effective; 2) regarding the persistence of the cancer vaccine, if the measurement value by ELISPOT is above a predetermined value, it is determined that the cancer vaccine is persistent; 3) regarding the determination of patients who should be administered the cancer vaccine, if the measurement value by ELISPOT is above a predetermined value, it is determined that the patient should be administered the cancer vaccine, and / or; 4) regarding the determination of which cancer vaccine to administer, if the measurement value by ELISPOT is above a predetermined value, it is determined that the patient should be administered the target vaccine. (Item K10) The method according to any one of the above items, wherein the predetermined value of Spot Counts by ELISPOT is 7. (Item K11) The method according to any one of the above items, wherein the cancer antigen includes all cancer antigens contained in the cancer vaccine.(Item K12) 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, tyrosine, HER2, MUC1, CEA, survivin, cycling B1, EGFR, mesothelin, telomerase, and neoantigens resulting from gene mutations in the patient's tumor cells. (Item K13) The method according to any one of the above items, wherein the cancer antigen is the WT1 protein. (Item K14) A kit for predicting the efficacy of a cancer vaccine in vitro, the kit comprising: a cancer antigen corresponding to the cancer vaccine; and means for confirming the reactivity of peripheral blood mononuclear cells taken from a patient to stimulation by the cancer antigen corresponding to the cancer vaccine, the kit for predicting and / or calculating the efficacy of the cancer vaccine based on the reactivity, wherein the prediction and / or calculation includes confirming the reactivity of peripheral blood mononuclear cells derived from the patient to stimulation by the cancer antigen corresponding to the cancer vaccine, and predicting or calculating the efficacy of the cancer vaccine based on the reactivity, wherein the means includes an ELISPOT or FluoroSpot kit, and 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. (Item K15) A pharmaceutical product comprising a cancer vaccine, wherein the vaccine regime is determined based on the reactivity of peripheral blood mononuclear cells derived from a patient to stimulation with a cancer antigen corresponding to the cancer vaccine, wherein the regime is determined based on confirming the reactivity of peripheral blood mononuclear cells derived from a patient 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 reactivity, wherein 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.(Item K16) The pharmaceutical according to any one of the above items, characterized in that the pharmaceutical is administered to a subject whose reactivity to the cancer vaccine in peripheral blood mononuclear cells obtained from the subject before administration is greater than or equal to a predetermined value. (Item K17) A pharmaceutical kit, the pharmaceutical kit comprising: a pharmaceutical comprising a cancer vaccine; a detection agent for detecting stimulation by a cancer antigen corresponding to the cancer vaccine; and instructions describing how to use the kit, wherein the instructions indicate that the vaccine regimen is determined based on the reactivity of patient-derived immune cells to stimulation by a cancer antigen corresponding to the cancer vaccine, and that the regimen is determined based on confirming the reactivity of patient-derived peripheral blood mononuclear cells to stimulation by the cancer antigen corresponding to the cancer vaccine, and predicting or calculating the effect of the cancer vaccine based on the reactivity, wherein the detection agent comprises an ELISPOT or FluoroSpot kit, and 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. (Item AA1) A diagnostic agent for predicting the effect of a cancer vaccine, wherein the diagnostic agent comprises a cancer antigen corresponding to the cancer vaccine, the agent is used in a method for predicting the effect of a cancer vaccine, the method comprises the steps of confirming the reactivity of peripheral blood mononuclear cells taken from a patient 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 reactivity, wherein the prediction and / or calculation encompasses confirming 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 reactivity, and 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. (Item AA2) The agent described in the above item, wherein the prediction is performed before administration of the cancer vaccine or during the treatment period with the cancer vaccine.(Item AA3) The agent according to any one of the above items, wherein the prediction comprises obtaining peripheral blood mononuclear cells derived from the patient. (Item AA4) The agent according to any one of the above items, wherein the prediction comprises providing the cancer antigen corresponding to the cancer vaccine. (Item AA5) The agent according to any one of the above items, wherein the prediction of the effect is made before the administration of the cancer vaccine. (Item AA6) The agent according to any one of the above items, wherein the effect comprises 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 a change in the dosage and administration of the cancer vaccine. (Item AA7) The agent according to any one of the above items, wherein the confirmation of the reactivity is performed by ELISPOT. (Item AA8) In the prediction, if the measurement value by ELISPOT is above a predetermined value, the agent is determined to be effective in the cancer vaccine / to be a patient who should be administered the vaccine, as described in any one of the above items. (Item AA9) In the prediction, 1) Regarding the effectiveness of the cancer vaccine, if the measurement value by ELISPOT is above a predetermined value, the agent is determined to be effective in the cancer vaccine; 2) Regarding the persistence of the cancer vaccine, if the measurement value by ELISPOT is above a predetermined value, the agent is determined to be persistent in the cancer vaccine; 3) Regarding the determination of patients who should be administered the cancer vaccine, if the measurement value by 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 determination of which cancer vaccine to administer, if the measurement value by ELISPOT is above a predetermined value, the patient is determined to be a patient who should be administered the target vaccine, as described in any one of the above items. (Item AA10) The agent according to any one of the above items, wherein the predetermined value of Spot Counts measured by ELISPOT is 7. (Item AA11) The agent according to any one of the above items, wherein the cancer antigen includes all cancer antigens contained in cancer vaccines.(Item AA12) The agent 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, tyrosine, HER2, MUC1, CEA, survivin, cycling B1, EGFR, mesothelin, telomerase, and neoantigens resulting from gene mutations in the patient's tumor cells. (Item AA13) The agent according to any one of the above items, wherein the cancer antigen is the WT1 protein. (Item BB1) Use of a cancer antigen corresponding to a cancer vaccine in a method for predicting the effect of a cancer vaccine, the method comprising the steps of confirming the reactivity of peripheral blood mononuclear cells taken from a patient 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 reactivity, wherein the prediction and / or calculation comprises confirming the reactivity of peripheral blood mononuclear cells 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 reactivity, and 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. (Item BB2) Use as described in the above item, wherein the prediction is performed before administration of the cancer vaccine or during the treatment period with the cancer vaccine. (Item BB3) Use as described in any one of the above items, wherein the prediction comprises obtaining peripheral blood mononuclear cells from the patient. (Item BB4) The use according to any one of the above items, wherein the prediction comprises providing the cancer antigen corresponding to the cancer vaccine. (Item BB5) The use according to any one of the above items, wherein the prediction of the effect is made before administration of the cancer vaccine.(Item BB6) 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 BB7) The use described in any one of the above items, wherein the confirmation of the reactivity is performed by ELISPOT. (Item BB8) The use described in any one of the above items, 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. (Item BB9) In the prediction, 1) Regarding the efficacy of the cancer vaccine, if the measurement value by ELISPOT is above a predetermined value, it is determined that the cancer vaccine is effective; 2) Regarding the persistence of the cancer vaccine, if the measurement value by ELISPOT is above a predetermined value, it is determined that the cancer vaccine is persistent; 3) Regarding the determination of patients who should be administered the cancer vaccine, if the measurement value by ELISPOT is above a predetermined value, it is determined that the patient should be administered the cancer vaccine, and / or; 4) Regarding the determination of which cancer vaccine to administer, if the measurement value by ELISPOT is above a predetermined value, it is determined that the patient should be administered the target vaccine; Use as described in any one of the above items. (Item BB10) Use as described in any one of the above items, wherein the predetermined value of Spot Counts by ELISPOT is 7. (Item BB11) Use as described in any one of the above items, wherein the cancer antigen includes all cancer antigens contained in the cancer vaccine. (Item BB12) The use 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, tyrosine, HER2, MUC1, CEA, survivin, cycling B1, EGFR, mesothelin, telomerase, and neoantigens resulting from gene mutations in the patient's tumor cells.(Item BB13) The use described in any one of the above items, wherein the cancer antigen is the WT1 protein.

[0005] In this disclosure, one or more of the above features are intended to be provided in combinations other than those explicitly stated. Further embodiments and advantages of this disclosure will be apparent to those skilled in the art, by reading and understanding the detailed description below as necessary.

[0006] 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.

[0007] 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.

[0008] 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 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). Figure 2 shows an example of a treatment sequence for metastatic urothelial carcinoma. ｓｔ The treatment line is cisplatin-based chemotherapy, 2 ｎｄ The line of treatment is PD-1 / PD-L1 inhibitors (pembrolizumab / avelumab), 3 ｒｄ The line of treatment typically uses an anti-Nectin-4 antibody-microtubule inhibitor conjugate (enfortumab vedotin). Since 2017, ｎｄ Although immune checkpoint inhibitors have become available as a treatment option, the PFS is 3.7 months, which is by no means sufficient. ｒｄThere are reasons why sufficient therapeutic effects cannot be obtained even when combined with other treatments. In September 2024, pembrolizumab + enfortumab vedotin combination therapy was introduced. ｓｔ Although it has received approval as a treatment line and its therapeutic effects are expected to improve in the future, 2 ｎｄ There is a high need for treatments with different mechanisms of action as post-line treatments. The B440-1 trial still exists to meet such needs. ｒｄThe safety and efficacy of pembrolizumab were evaluated in patients with urothelial carcinoma who had completed treatment up to the first line. Figure 3 shows the B440 administration schedule, DLT evaluation period, and adverse event / efficacy evaluation period in the B440-1 clinical trial. In addition, the WT1-ELISPOT assay was performed multiple times at the timings indicated by the red arrows. The detailed schedule of the timing of the assay is as follows: Day 1 (before B440 administration), Day 15, Day 28, Day 57, Day 113, Day 169. Figure 4 shows the tumor reduction rate in 5 patients who underwent a second attempt with pembrolizumab. Of these, all 3 ELISPOT-positive patients (green) were able to control their disease progression with pembrolizumab (RECIST evaluation: CR, PR, SD in one case each). On the other hand, both of the 2 ELISPOT-negative patients (red) were judged to have disease progression. Figure 5 shows the change in the average number of spots over time in the ELISPOT-positive and 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 shows the study design for B440 administration to solid tumors expressing WT1. From the results in Figure 8, a high therapeutic effect with B440 can be expected in ELISPOT-positive cases, and from the results in Figure 4, a good response to subsequent ICI administration can also be expected. Furthermore, as shown in Figure 5, cases with a positive ELISPOT test also showed a positive ELISPOT test before administration (WT1-ELISPOT pre-positive), suggesting that WT1-ELISPOT pre-positive is likely a useful screening tool for B440 candidates. Figure 6 is based on these findings. Among patients who have become resistant to prior treatment, those with a WT1-ELISPOT pre-positive test are recruited as candidates for treatment and B440 administration is initiated. Subsequently, patients who become WT1-ELISPOT post-positive continue with ICI treatment.For patients with weak immune activity against WT1 from the beginning, administration of B440 can further stimulate a WT1-specific immune response, and a continuous therapeutic effect on tumors can be expected. Figure 7 shows an overview of the experimental plan of Example 3. A total of 15 C57BL / 6 mice were 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 vaccine administration (Day 30), and a WT1-ELISpot assay was performed. The vaccine was 1×10 B440 on Days 7-11, Days 14-18, and Days 21-25. ９CFU / 100 μl was administered orally. Figure 8 shows the Kaplan-Meier curve in the example. Regarding the treatment response to B440, the Pre-ELISPOT positive group showed a significantly longer progression-free survival than the negative group. This result suggests that the number of IFN-γ producing cells produced by WT1 antigen protein stimulation, as measured by ELISPOT, is a major prognostic factor in predicting the treatment effect against B440. Figure 9 summarizes the progress of ELISPOT (12 cases in total). The horizontal axis shows the time course, and the vertical axis shows the mean value of ELISPOT. An example of pre-ELISPOT stratification (before vaccine administration) is shown. The weak positive criteria were 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 shows the mean test (Mann-Whitney U test) at the pre-ELISPOT (before vaccine administration) time point. Figure 12 shows an example of stratification by ELISPOT after vaccine administration (during the treatment period). The criteria for a positive ELISPOT result after vaccine administration were 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. Figure 13 shows the mean test (Mann-Whitney U test) at approximately 2 months after the start of vaccine administration (during the treatment period). Figure 14 shows the Kaplan-Meier curve in the example. Regarding the treatment response to B440, the Post-ELISPOT positive group showed a significantly longer progression-free survival than the negative group during the administration period. Figure 15 shows the pre- and post-administration agreement rates of ELISPOT as a mixed matrix. Figure 16 shows the progress after rechallenge with pembrolizumab (n=5). This is stratified by pre-ELISPOT. Figure 17 shows the progress after rechallenge with pembrolizumab (n=5). The data was stratified using ELISPOT after administration (during the treatment period). Figure 18 is a graph showing the predictive ability of cancer vaccines to predict the effect (progression-free survival: PFS) when stimulated with a protein-based cancer antigen (full-length WT1 protein) as the cancer antigen corresponding to the cancer vaccine.This graph stratifies cases into positive (6 cases) and negative (6 cases) based on the results of the single-cell level cytokine production detection assay (ELISPOT). The vertical axis shows survival probability, and the horizontal axis shows the number of days from the start of administration (PFS (days)). The graph shows that the group with a positive response to protein stimulation exhibits a significantly better PFS (Log-rank p = 0.0301) compared to the group with a negative response. Figure 19 is a graph showing PFS when cases are stratified based on the results of the response to short peptide stimulation (stimulation with 8 or 15 amino acids) as a comparative example. (A) shows the results using WT1 126-134 peptide, (B) shows the results using WT1 235-243 peptide, and (C) shows the results using WT1 332-347 peptide as stimulating antigens. In all cases, the use of short peptides does not show a significant correlation between reactivity and PFS compared to the case using proteinogenic cancer antigens in Figure 1. Figure 20 is a graph stratified by the results of ELISPOT stimulating pembrolizumab re-administration (rechallenge) (image_cc78a7.png). (A) shows the results using WT1 126-134 peptide, (B) shows the results using WT1 235-243 peptide, and (C) shows the results using WT1 332-347 peptide as stimulating antigens. In all cases using short peptides, the number of positive cases was extremely small, or there was no significant difference in PFS between positive and negative cases (Log-rank p = 0.45).

[0009] 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.

[0010] The following provides definitions of terms used specifically in this specification and / or basic technical concepts as appropriate.

[0011] In this specification, "approximately" refers to significant figures unless otherwise specified, but is understood to mean an equivalent amount permitted by the pharmacopoeia.

[0012] 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 a cancer antigen into the host to promote endogenous expression of the antigen); RNA vaccines (using mRNA encoding a cancer antigen to produce the antigen 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., those utilizing adenoviruses, lentiviruses, 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 individual 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 utilizing 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).

[0013] More detailed types and specific examples of “cancer vaccines” as used in this disclosure include, but are not limited to, the following: 1. Peptide vaccines: Vaccines that use peptides derived from specific cancer antigens to activate T cells and attack tumors. Specific examples: WT1 peptide vaccine (leukemia, pancreatic cancer, etc.) NY-ESO-1 peptide vaccine (multiple myeloma, melanoma, etc.) MAGEA3 peptide vaccine (lung cancer, melanoma, etc.) PRAME peptide vaccine GP100 peptide vaccine (melanoma) HER2 peptide vaccine (breast cancer, gastric cancer) 2. Protein vaccines: Vaccines that use purified full-length proteins that are processed by antigen-presenting cells to induce an immune response. Specific examples: CEA (carcinoembryonic antigen) vaccine (colorectal cancer, pancreatic cancer) HER2 protein vaccine (breast cancer) MUC1 protein vaccine (pancreatic cancer, breast cancer) 3. DNA vaccines: Vaccines that administer DNA encoding cancer antigens to patients to induce an immune response by producing antigens in the body. Specific examples: HPV (Human Papillomavirus) DNA vaccine (Cervical cancer, Head and Neck cancer) PSA (Prostate-Specific Antigen) DNA vaccine (Prostate cancer) WT1 DNA vaccine (Leukemia, Pancreatic cancer) 4. RNA vaccines Use mRNA encoding cancer antigens, which are translated in the body to express the antigens and induce an immune response. Specific examples: Neoantigen RNA vaccine (Personalized vaccine based on the gene mutations of the patient's tumor) KRAS mutation RNA vaccine (Lung cancer, Pancreatic cancer) 5. Cell-based vaccines Utilize dendritic cells or tumor cells derived from the patient or allogeneically to present cancer antigens and stimulate the immune system. Specific examples: Sipuleucel-T (Prostate cancer) Dendritic cell vaccine (WT1 peptide load, NY-ESO-1 load) 6. Viral vector vaccines Vaccines that use genetically modified viruses to express cancer antigens in the patient's body. Specific examples: ProstVac-VF (prostate cancer), TG4010 (vaccine encoding MUC1, non-small cell lung cancer), OncoVEX (T-VEC, melanoma). 7. Polysaccharide vaccines: Vaccines that target abnormal sugar chain structures on the surface of cancer cells.Specific examples: Glob H polysaccharide vaccine (breast cancer, ovarian cancer) GM2 vaccine (melanoma) 8. Neoantigen vaccines Personalized vaccines that target neoantigens derived from gene mutations in the patient's tumor. Specific examples: NeoVax (personalized neoantigen peptide vaccine) Personalized Neoantigen RNA vaccine 9. Viral vaccines Vaccines to prevent viral infections that cause tumors. Specific examples: HPV vaccine (cervical cancer, head and neck cancer) HBV vaccine (hepatocellular carcinoma) 10. Combination vaccines The effect is enhanced by combining vaccines with other immunotherapies (immune checkpoint inhibitors, adjuvants, etc.). Specific examples: CEA vaccine + anti-PD-1 antibody (pancreatic cancer) HER2 vaccine + anti-CTLA-4 antibody (breast cancer).

[0014] 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, Ferris AS, Finn OJ, Hastings BM, Hecht TT, Mellman I, Prindville SA, Viner JL, Weiner LM, and Matrisian LM. This can be implemented based on the priority of cancer antibodies: a national cancer institute pilot project for the acceleration of 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.

[0015] 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).

[0016] 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:

[0017] In other words, cancer antigens can be broadly divided 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) These are molecules that are also 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), survivorin, cycling B1, EGFR (epidermal growth factor receptor), and mesothelin. 3. Tumor-Specific Antigens (TSAs) These are antigens that are specifically expressed on cancer cells and include new proteins resulting from gene mutations or viral infections. Neoantigens, in particular, which result 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.

[0018] 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.

[0019] 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 patients.

[0020] 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.

[0021] 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's 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. On the other hand, B cell epitopes 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.

[0022] In this disclosure, "cancer antigen corresponding to a cancer vaccine" is characterized by being a protein composed of all or part of the amino acid sequence contained in the cancer vaccine. Here, the protein preferably contains (all) the same length as the sequence contained in the cancer vaccine, or is of the same length (all), or consists of a shorter sequence (part), but unlike a mere short peptide, it preferably has a length sufficient to maintain the properties of a "protein" that requires uptake and processing by antigen-presenting cells. In this specification, such a cancer antigen may be referred to as a proteinaceous cancer antigen.

[0023] In this specification, “proteinogenic cancer antigen” means a substance comprising all or part of the amino acid sequence contained in the target cancer vaccine and having a certain length. Such cancer antigens of a certain size can retain molecular size and structural properties sufficient to require uptake by antigen-presenting cells (APCs) and intracellular processing, and have been found in this disclosure to produce a significant response in single-cell level cytokine production detection assays such as ELISPOT. Therefore, in the context of this disclosure, “proteinogenic cancer antigen” means a protein comprising all or part of the amino acid sequence contained in the target cancer vaccine. If the cancer vaccine contains multiple antigen sites, the proteinogenic cancer antigen may be a single protein containing all of those sites, or a protein selectively containing a specific major site. Furthermore, if the vaccine is a fusion protein, the proteinogenic cancer antigen may be each of the pre-fusion proteins individually, or partial fragments thereof. Unlike simple T-cell epitopes (short peptides), proteinaceous cancer antigens preferably have a length sufficient to be taken up by antigen-presenting cells (APCs) and appropriately processed (cleaved / processed) by intracellular proteasomes, endosomes, etc. Through this processing process, the optimal epitope according to the individual patient's HLA type is selected and presented via cross-presentation to CD8 ＋It is presented to T cells. By faithfully mimicking this in vivo reaction in vitro, a high correlation with clinical efficacy is achieved. Therefore, proteinogenic cancer antigens have the following properties: (1) Correspondence with cancer vaccines and length Proteinogenic cancer antigens are designed based on the antigen sequence, which is the active ingredient of the cancer vaccine. Here, "correspond" means that at least a portion of the antigenic sequence (sequence containing epitopes) contained in the cancer vaccine is shared as a common sequence. Full-length correspondence: If the cancer vaccine is a specific protein (e.g., WT1 protein), the protein has a sequence that is substantially identical (e.g., 80% or more, preferably 90% or more), preferably identical, to its entire amino acid sequence. Partial correspondence (partial protein): A protein composed of a portion of the cancer vaccine sequence that includes the major immunogenic domain or multiple epitopes (e.g., 20% or more, 30% or more, 40% or more, or 50% or more). Diverse fragmentation: This also includes embodiments in which the stimulating antigen is used as a mixture of multiple protein fragments of different lengths and cleavage positions, within the range encompassing the cancer vaccine sequence. In either embodiment, it is preferable that the peptide is distinguishable from short peptides (generally about 8 to 15 amino acids) consisting of only a single HLA-restrictive epitope and has a length such that it is presented to T cells only after processing by APC. (2) Technical significance (induction of cross-presentation): The greatest feature of proteinogenic cancer antigens is that they can induce an immune response via cross-presentation in vitro. With short peptide stimulation, the reaction is triggered by directly binding to MHC molecules on the surface of T cells of patients with a specific HLA type, thus omitting the important process of "uptake and processing by antigen-presenting cells" in vivo. In contrast, stimulation using the proteinogenic cancer antigen of this disclosure can reproduce the process in which APCs take up the antigen, appropriately cleave it within the cell, and present it, making it possible to accurately predict the individual immune response capacity (including antigen processing capacity) of patients. (3) Determination method and indicators: Whether a drug qualifies as a "proteinogenic cancer antigen" can be determined by its length (number of amino acid residues) as well as the following functional indicators.Processing-dependent: This is determined by whether the T cell activation response is inhibited when a drug that inhibits the processing function of antigen-presenting cells (e.g., an endosomal function inhibitor such as chloroquine) is added in a responsiveness confirmation test using PBMCs. Broad presentation ability: Unlike single short peptide stimulation, this confirms the ability of the same proteinogenic cancer antigen to be presented as multiple different epitopes suited to each HLA in multiple cases with different HLA types. (4) Exemplary embodiments: Examples of proteinogenic cancer antigens "corresponding to cancer vaccines" include the following: For vaccines with WT1 protein as the active ingredient, the full length of the WT1 protein or a fragment protein containing a specific functional domain (such as a zinc finger domain). For long polypeptide vaccines with multiple neoantigens linked together, a protein containing the individual antigen sites or a recombinant protein covering all sequences contained in the vaccine. For cancer vaccines in which a portion of the cancer antigen protein is modified or substituted, a protein of the corresponding sequence containing the modification site. As described above, this configuration means that the limitation of being compatible with cancer vaccines is understood to mean not merely "a protein with the same name," but a processing substrate that includes vaccine-derived sequences necessary to reproduce the in vivo reaction. This means that the predictive ability that is difficult to achieve with the short peptide (part) obtained in Example 3 (WT1) can be achieved with the protein (whole or a sufficiently long part), and this phenomenon is not specific to any particular cancer vaccine.

[0024] The reasons for the preference for proteinaceous cancer antigens are, but are not limited to, that in order to accurately predict the clinical efficacy of cancer vaccines (e.g., progression-free survival: PFS) in vitro, it has been confirmed in this specification that simply using T cell epitopes as stimulants is insufficient, and that it is necessary to highly mimic the immune response process in vivo. Specifically, when a cancer vaccine (protein, etc.) is administered into the body, antigen-presenting cells such as dendritic cells and monocytes activated by adjuvant stimulation take up the antigen, undergo intracellular processing, and then cross-present to CD8 ＋ It strongly induces cytotoxic T cells. In this invention, using a "protein" having a specific length (all or part of the vaccine sequence) as a stimulating antigen makes it possible to reproduce this series of in vivo reactions mediated by antigen-presenting cells in an in vitro system (e.g., ELISPOT assay).

[0025] For example, as shown in the examples, in the efficacy prediction of cancer vaccine therapy targeting WT1, which was verified as an example, when conventional short peptides (approximately 8 to 15 amino acids) were used as stimulating antigens, the detection rate of positive cases was low, and no significant correlation with clinical PFS was observed. In contrast, when a protein corresponding to the vaccine sequence was used as the stimulating antigen according to this disclosure, the strength of the immune response for each patient was clearly stratified, and the results showed an extremely high quantitative correlation (p < 0.05) with the actual PFS.

[0026] Thus, defining cancer antigens as "proteins composed of all or part of the amino acid sequence contained in the cancer vaccine" is based on the technical requirement to reflect the cross-presentation mechanism in vivo. Therefore, the protein is defined as a molecule that maintains a certain length within the range not exceeding the total length of the cancer vaccine, which, to those skilled in the art, specifies a range that is obvious and reasonable from the principles of cancer immune response. This specification enables a remarkable predictive ability for clinical outcomes that could not possibly be achieved with short peptide stimulation as in the prior art.

[0027] In this specification, "single-cell level cytokine production detection assay" refers to a method for quantitatively or semi-quantitatively detecting cytokines (e.g., IFN-γ, IL-2, TNF-α, etc.) produced and released by individual immune cells in response to antigen stimulation, with single-cell level resolution. This includes enzyme immunoassay (ELISPOT) and fluorescence immunoassay (FluoroSpot), as well as all in vitro assays based on technically equivalent principles.

[0028] Single-cell level cytokine production detection assays aim to calculate the frequency of T cells that are reactive (active) to a specific antigen. One embodiment of a single-cell level cytokine production detection assay, ELISPOT, is a method that uses a color reaction between an enzyme-labeled antibody and a substrate to form a spot directly beneath cytokine-producing cells. Another embodiment, FluoroSpot, uses a fluorescently labeled antibody to simultaneously detect multiple different cytokines, such as IFN-γ and IL-2, within the same well, and to identify them on an individual cell-by-cell basis. Furthermore, technical equivalents of this assay include techniques for culturing cells on a carrier with a cytokine-capturing antibody immobilized on it, detecting and spotting the released molecules in a near-field, and techniques for evaluating cytokine production in microcompartments such as microwells where single cells are isolated.

[0029] Various methods can be cited as specific embodiments of single-cell level cytokine production detection assays. For example, there are multi-cytokine detection methods that measure the frequency of polyfunctional T cells using FluoroSpot in addition to ELISPOT, and automated analysis systems that automatically count and analyze the number, size, and intensity of formed spots using an image analysis device. Furthermore, equivalent assays such as highly sensitive cytokine spot detection using chemiluminescence and single-cell secretion capture methods using magnetic bead surfaces are also included in the concept of assays described herein.

[0030] Whether a particular measurement method qualifies as a "single-cell level cytokine production detection assay" as defined herein can be determined by the following indicators. First, the presence or absence of single-cell resolution. Unlike bulk assays such as ELISA, which measure cytokine concentrations in the culture supernatant all at once, this means that the "number of cells" responding to the antigen can be directly counted. Second, sensitivity in combination with proteinogenic cancer antigens. Specifically, the suitability of the assay can be determined by confirming that when the proteinogenic cancer antigen disclosed herein is added instead of a short peptide, spots derived from T cells activated through processing by antigen-presenting cells (APCs) are formed, and that the frequency of these spots significantly correlates with clinical progression-free survival (PFS).

[0031] 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 acquired immunity mainly 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 to reinfection. Furthermore, recent research has shown that 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 an important role in the tumor microenvironment, are also attracting attention. In this specification, "immune cells" also include genetically modified immune cells (e.g., CAR-T cells and CAR-NK cells) in addition to the above. These immune cells are used alone or in combination with other therapies to treat infectious diseases, cancer, autoimmune diseases, etc., and are also used for predicting and diagnosing the effects of the therapeutic agents disclosed herein.

[0032] 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 cells 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 that can directly attack cells with reduced expression of major histocompatibility complex (MHC) molecules, infected cells, and tumor cells. Monocytes function as progenitor cells that differentiate into macrophages and dendritic cells, regulating the immune response 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 applied as cells that form the basis of cancer vaccines and T cell therapies (e.g., CAR-T cells). In addition, the ELISPOT and FluoroSpot assays using PBMCs are standard methods for evaluating antigen-specific immune responses. In this specification, "peripheral blood mononuclear cells" includes the above-mentioned cell groups, as well as genetically modified cells and cells prepared by culture or differentiation induction.

[0033] In this specification, “stimulation” (by a cancer antigen or antigenic moiety) refers to the operation or action of exposing the host’s immune system to an antigen 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) and 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 and 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.

[0034] As used herein, "reactivity to immune cells (e.g., peripheral blood mononuclear cells)" refers to the effects and responsiveness that an antigen or stimulatory substance exerts on immune cells, and includes activation, differentiation, proliferation, cytokine secretion, or changes in the ability to attack target cells of the cells. In particular, peripheral blood mononuclear cells (PBMCs) are composed of a diverse population of immune cells and are thus used as a standard model for evaluating reactivity to antigens and stimuli. The main cell groups that make up PBMCs include T cells, B cells, natural killer (NK) cells, and monocytes. Reactivity to antigen stimulation is measured by evaluating the process by which these cell groups respond specifically or non-specifically. For example, T cells recognize and activate specific antigens and show responsiveness by secreting cytokines (e.g., interferon-γ, interleukin-2). On the other hand, monocytes are responsible for the initial activation of the immune response through phagocytosis of antigens and antigen presentation. One representative method for measuring reactivity to immune cells is the enzyme-linked immunosorbent spot (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, PBMCs are stimulated with an antigen, and the secreted molecules (e.g., interferon-γ, IL-2) are captured by antibodies immobilized on a solid phase. Subsequently, detection antibodies and an enzyme reaction are used to visualize them as spots, and the number of secreting cells is quantitatively evaluated. The number of spots corresponds to the number of cells showing an antigen-specific response.

[0035] In this specification, the following are examples of indicators and measures of reactivity: 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, etc., and 5 to 500 per 100,000 is usually adopted for the response of PBMCs to tumor antigens (cancer antigens). If it is 5 to 15 or more, preferably 7 or more, it can be used for pre-treatment prediction, and if it is 10 or more, preferably 15 or more, it can be used for prognosis prediction during the treatment period. 2. Stimulation Index (SI): The value obtained by dividing the number of spots when stimulated with an antigen 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 will be 20. Generally, an SI of 2 or higher is considered a significant response. An SI of <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 cytokines secreted by ELISA or flow cytometry in combination with ELISA. Example 1: The concentration of interferon-γ secreted by PBMCs due to antigen stimulation increases from 0.1 ng / mL without stimulation to 0.2 ng / mL (the stimulated value is used as a relative value to the unstimulated value). Example 2: The concentration of IL-2 may increase from 100 pg / mL unstimulated 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 a response.

[0036] 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.

[0037] As used herein, "prediction of the effect of a cancer vaccine" refers to quantitatively or qualitatively evaluating the immune response or therapeutic effect expected by administration of a cancer vaccine, and optimizing the treatment strategy based on the results. Prediction of this effect is performed by comprehensively analyzing the patient's immune profile, reactivity to cancer antigens, presence of immunosuppressive factors, and characteristics of the cancer vaccine itself.

[0038] Specific examples of "prediction of the effect of a cancer vaccine" include the following: 1. Prediction of the efficacy of a cancer vaccine Predict the efficacy (treatment success rate) of a cancer vaccine. For example, the efficacy can be predicted by measuring the frequency and intensity of the reaction of a patient's immune cells to a specific cancer antigen using an enzyme-linked immunosorbent spot (ELISPOT) assay or flow cytometry. 2. Prediction of the duration of a cancer vaccine Predict the duration of the therapeutic effect of a cancer vaccine. This includes evaluating the induction level of immune memory cells (memory T cells or memory B cells). For example, measurement of the immune response 6 months or 12 months after vaccination is used for evaluation of the duration. 3. Determination of patients to receive a cancer vaccine Select patients who are likely to respond to a cancer vaccine. This is done by analyzing the patient's HLA type, expression level of cancer antigens, presence of immunosuppressive factors (e.g., regulatory T cells or myeloid-derived suppressor cells) in the tumor microenvironment, etc. 4. Determination of the cancer vaccine to be administered Select the optimal cancer vaccine based on the patient's cancer type, stage, and specific antigens expressed in the tumor. For example, recommend a HER2 peptide vaccine for HER2-positive breast cancer patients and a WT1 peptide vaccine for WT1-highly expressed leukemia patients. 5. Determination of other drugs and / or nutrients to be additionally administered Determine the administration of immune checkpoint inhibitors (e.g., anti-PD-1 antibody, anti-CTLA-4 antibody), immune-stimulating cytokines (e.g., IL-2, GM-CSF), and further nutritional supplements (e.g., vitamin D or amino acid supplements) to enhance the effect of a cancer vaccine. 6. Determination of changes in the dosage and administration method of a cancer vaccine

[0039] 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.

[0040] These "predictions of cancer vaccine effectiveness" 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 effectiveness predictions described herein contribute to optimizing treatment plans, streamlining the cancer vaccine development process, and improving treatment outcomes.

[0041] 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 state 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 or absence of side effects are checked, and adjustments to the dosage or schedule, or the addition of combination therapy are considered. Thus, detailed evaluation and monitoring "before, during, or after vaccine administration" allows for 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.

[0042] 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 surface 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 enzymatic reaction is used to visualize the sites where the secretions are present as spots. 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 possesses high sensitivity and specificity. Furthermore, because it directly counts the number of secretory cells, quantitative evaluation is possible. Specific applications of ELISPOT include evaluation of T cells responding to tumor antigens in cancer immunotherapy, measurement of pathogen-specific immune responses in infectious diseases, monitoring of immune responses after vaccination, analysis of abnormal autoantigen responses in autoimmune diseases, and evaluation of immune responses to donor antigens in transplant medicine.

[0043] 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 the patient's PBMC before or during vaccine administration, it is possible to predict vaccine efficacy and the persistence of the immune response.

[0044] 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.

[0045] 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 within 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.

[0046] The most distinctive 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, it can be used to evaluate multiple functions of T cells responding to tumor antigens (e.g., diverse cytokine secretion) and 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 research on cancer vaccines and immunotherapy, detailed profiling of each patient's immune response enables the design of personalized treatments and prediction of their effectiveness. In this specification, "FluoroSpot" refers to the technology described above, including its variations and improvements. Analysis of multifunctional immune responses using FluoroSpot will contribute to cancer treatment, infectious disease research, vaccine development, and the optimization of treatment for autoimmune diseases.

[0047] In addition to ELISPOT and FluoroSpot, the following can also be listed.

[0048] 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 purposes and applications, enabling detailed evaluation of the characteristics and diversity of immune responses.

[0049] 1. ISOCODE (IsoPlexis) ISOCODE is an assay developed by IsoPlexis that uses 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 ELISAPOT, but differs in that it measures the total amount of secretions in 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 uses fluorescent labeling to simultaneously measure multiple cytokines and chemokines in a single sample and is suitable for cytokine profile analysis. 4. MACSima ＴＭ Imaging MACSima ＴＭThis is a technology that analyzes the spatial distribution of proteins and intracellular factors secreted by specific cells with high resolution, enabling multi-omics analysis. 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 separating cells, the detection sensitivity of secretions is improved, enabling high-throughput analysis. 7. DropSeq (droplet technology) This technology uses microdroplet technology to separate single cells into each droplet and analyze secretions and gene expression within them. It allows for highly accurate evaluation of immune responses and secretion profiles. 8. Mes Scale Discovery (MSD) This is a multiplexing analysis technology using electrochemical luminescence, which allows for highly sensitive measurement of various cytokines in 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 uses multiplex suspension array technology and utilizes beads to simultaneously detect multiple cytokines and secreted proteins. It is suitable for high-throughput cytokine profiling.

[0050] 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 improved versions.

[0051] 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.

[0052] Specifically, the following elements are included in a vaccine regimen: 1. Dosage Schedule: 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 to continue treatment for several months, with booster doses given every two or four weeks after the initial dose. 2. Dosage: Refers to the amount of vaccine administered (e.g., μg or mg units of peptides or proteins, μg units of DNA / RNA). The dose is adjusted based on the patient's weight, age, and immune status. 3. Number of Doses: Refers to the total number of times the vaccine is administered. For example, it may be three, five, or adjusted based on the patient's immune response. 4. Route of Administration: Refers to the method by which the vaccine is administered, including 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. Adjuvant: An adjuvant used to enhance the effect of the vaccine and plays a role in optimizing the immune response. For example, monophosphoryl lipid A (MPL), aluminum salts, or GM-CSF may be used. 6. Combination therapy This includes immune checkpoint inhibitors (e.g., anti-PD-1 antibody, anti-CTLA-4 antibody), cytokines (e.g., IL-2), or other immunomodulators used in combination with the vaccine. Combination therapy is used to enhance the effects of the cancer vaccine 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 effectiveness of the vaccine. 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., montmilon ITE) for 6 doses every 2 weeks. The immune response is monitored, and boost doses are added as needed. DNA vaccine regimen: 100 μg of DNA is administered intramuscularly, with 3 boost doses at 4-week intervals after the initial dose. During the administration period, anti-PD-1 antibodies were administered concomitantly. RNA vaccine regimen: 50 μg of mRNA was 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 with the aim of realizing personalized medicine for the patient. Furthermore, optimizing the regimen is important to maximize the vaccine effect and improve treatment outcomes.

[0053] In this specification, “vaccine kit” refers to a package containing the components and related 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 The central components of the kit, used for therapeutic or preventive 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 Components included to enhance the immune response of the vaccine. Monophosphoryl lipid A (MPL) Aluminum salt GM-CSF (granulocyte-macrophage colony-stimulating factor) CpG oligonucleotides 3. 1. Diluent or solvent for administration: Contains solvents or diluents used when preparing the vaccine. Examples: sterile saline, phosphate buffer (PBS), sterile water. 4. Administration device: Items necessary for administering the vaccine to the patient. Syringe: Used to accurately measure and administer the required dose. Needle: Suitable for subcutaneous, intramuscular, or intravenous administration of the vaccine. Patch delivery system: If transdermal administration is possible. 5. Instructions for use: Detailed instructions on how to use all components 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. Storage and transport package: Container for storing and transporting vaccine components at appropriate temperatures and conditions. Cooling box: Device for maintaining a refrigerated temperature of 2-8°C. Lyophilized vial: Vial for lyophilized vaccine that can be stored for a long period. 7. Additional auxiliary items: Auxiliary items necessary for smooth administration.Disposal containers for disinfectant alcohol swabs, sterile gloves, and injection needles (e.g., Sharps containers). Uses and Benefits: Accurate Dosage: Contains all necessary components, enabling accurate and effective administration to patients. Convenience: The kit is all in one, simplifying preparation in healthcare settings. Safety: All components are properly sterilized and packaged, reducing the risk of infection.

[0054] In this specification, "vaccine kit" includes, in addition to the above-mentioned components, an individualized combination tailored to a specific type of cancer or the patient's condition. Furthermore, cancer vaccine kits are intended to be used alone or in combination with therapies (e.g., immune checkpoint inhibitors or chemotherapy drugs), and will be an important tool for realizing personalized medicine.

[0055] 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 element in the effectiveness of cancer vaccines, the patient's immune response, or the optimization of treatment plans. Specifically, "levels" are applied in the following situations: 1. Evaluation of 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 parts 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 a cancer vaccine, the patient's immune response is measured regularly, and the changes are evaluated as "levels." For example, the percentage of specific T cells or the induction rate of memory T cells after vaccine administration is measured using flow cytometry, and the extent to which it has increased compared to the pre-treatment state is shown. Based on this "level," the need for additional vaccine administration or combination therapy is determined. 3. Application as a reference value: The "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 judged to meet the criteria for cancer vaccine application. The "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, the "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.

[0056] In this specification, "level information" refers to quantitative or qualitative data concerning a specific measurement target (e.g., immune response, molecular concentration, cell percentage, gene expression, etc.), and is fundamental information for evaluating a patient's condition and determining the suitability and strategy for treatment. "Level information" plays a crucial role in planning cancer vaccine administration, evaluating treatment effectiveness, and formulating treatment strategies tailored to individual patient characteristics. 1. Specific Examples of Level Information "Level information" includes the following data: Cytokine secretion: For example, cytokine concentrations (unit: pg / mL or number of spots) such as interferon-γ (IFN-γ) and IL-2 secreted after stimulation by cancer antigens or their antigenic moieties. 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 levels: Expression levels of molecular markers (e.g., PD-L1, HER2, MAGEA3) in tumor cells and immune cells. 1. Gene Expression Profile: Expression levels of genes related to cancer antigens and neoantigens (e.g., RNA sequencing data). 2. Uses of Level Information Level information is used for the following purposes: Assessment of Treatment Suitability: Used to select patients who should receive cancer vaccines. For example, if the cytokine secretion level of specific T cells is above a certain threshold, the patient is deemed suitable for vaccine administration. Monitoring of Treatment Efficacy: Level information is compared before and after vaccine administration to evaluate changes in the immune response and efficacy due to treatment. Development of Individualized Treatment Plans: Based on level information, the optimal administration regimen (dosage, schedule, combination therapy, etc.) is designed. 3. Methods of Obtaining Level Information Level information is obtained using various measurement techniques. For example: ERISPOT assay: Quantifies the number of cytokine-secreting cells and evaluates the antigen-specific T cell response. 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. Significance of Level Information "Level information" functions as the foundation of personalized medicine and contributes to the refinement of treatment strategies. This makes it possible to create treatment plans that take into account the differences in immune responses of each patient, and is expected to improve treatment effectiveness and minimize side effects. In addition, this information is used for selecting patient groups to be treated and for objective evaluation of treatment effectiveness in clinical trials. In this specification, "level information" is essential information for quantitatively or qualitatively evaluating immune responses and molecular data in order 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.

[0057] In this specification, "subject" or "subject(s)" means an entity that is the subject of the diagnosis, detection, or treatment described herein (for example, a living organism such as a human, or cells, blood, serum, etc., extracted from a living organism).

[0058] 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, and includes exerting a symptom-improving or preventive effect on the patient's disease or one or more symptoms associated with the disease. Providing appropriate treatment based on prior diagnosis is called "companion therapy," and the diagnostic agent used for this purpose is sometimes called a "companion diagnostic agent."

[0059] In this specification, “therapeutic agent” broadly refers to any agent capable of treating a target condition (e.g., diseases such as cancer and allergies). In one embodiment of this disclosure, “therapeutic agent” may be a pharmaceutical composition comprising an active ingredient and one or more pharmacologically acceptable carriers. The pharmaceutical composition can be manufactured, for example, by mixing the active ingredient with the carriers and any method known in the art of pharmaceutical formulation. 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 (e.g., a buffer solution). Note that therapeutic agents for cancer, allergies, etc. include drugs used for the prevention of cancer, allergies, etc. (preventive drugs), or inhibitors of cancer, allergies, etc.

[0060] 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.

[0061] In this specification, "preventive medicine" broadly refers to any medicine that can prevent a target condition (for example, diseases such as allergies).

[0062] In this specification, “kit” means a unit in which the components to be provided (e.g., test reagents, diagnostic reagents, therapeutic agents, antibodies, labels, instructions, etc.) are provided, usually divided into two or more compartments. 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 a manual describing how to use the provided components (e.g., test reagents, diagnostic reagents, therapeutic agents) 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 reagents, diagnostic reagents, therapeutic agents, antibodies, etc.

[0063] In this specification, “Instructions” are instructions for a physician or other user on how to use the Disclosure. These instructions contain language instructing the administration of the detection method, diagnostic agent, or drug of the Disclosure. The instructions may also contain language instructing the administration site to be oral or esophageal (e.g., by injection). These instructions shall be 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 shall be clearly stated to have been approved by that supervisory authority. The instructions are a so-called 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).

[0064] 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), polypeptides such as transcription factors that bind to promoter regions, etc. 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 but differs in structure.It is understood that the disclosure includes the subject itself (e.g., cancer antigen or the antigenic portion of said cancer antigen), as well as variants or modifiers of the subject (e.g., in the case of proteins, amino acid sequence modifiers, etc.) that have the same effect as the subject, and those that can be transformed into the subject itself or its variants or modifiers at the time of action (e.g., nucleic acids encoding the subject itself or variants or modifiers of subject S, and vectors, cells, etc., containing such nucleic acids). It is understood that in this disclosure, functional equivalents of the subject can be used in the same way as the subject, even if not specifically mentioned. An equivalent of a cancer antigen or the antigenic portion of said cancer antigen may be considered 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).

[0065] (Preferred Embodiments) 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 clear that those skilled in the art can make appropriate modifications within the scope of the Disclosure by taking into consideration the descriptions herein. Furthermore, the embodiments of the Disclosure below can be used individually or in combination.

[0066] (Technologies for predicting the efficacy of cancer vaccines and related technologies) In one aspect of this disclosure, the disclosure provides a method for predicting the efficacy of a cancer vaccine in vitro. The method includes confirming the responsiveness of peripheral blood mononuclear cells taken from a patient to stimulation with a cancer antigen corresponding to the cancer vaccine by a single-cell level cytokine production detection assay, and predicting or calculating the efficacy of the cancer vaccine based on the responsiveness. Here, the prediction and / or calculation includes confirming the responsiveness of peripheral blood mononuclear cells from the patient to stimulation with the cancer antigen corresponding to the cancer vaccine, and predicting or calculating the efficacy of the cancer vaccine (e.g., progression-free survival, response, duration of response, or selection of target patients for administration) based on the responsiveness. The cancer antigen used in this method may be a proteinaceous cancer antigen.

[0067] In this aspect of the 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.

[0068] In one aspect, this disclosure provides an agent for predicting the efficacy of a cancer vaccine in vitro. The agent contains a cancer antigen corresponding to the cancer vaccine and is used to confirm the responsiveness of peripheral blood mononuclear cells isolated from a patient to stimulation by the cancer antigen. The agent of this disclosure is particularly characterized by its use in a single-cell level cytokine production detection assay. Based on the confirmation of the responsiveness using the agent, it is possible to predict and / or calculate the efficacy of the cancer vaccine (e.g., response, duration of response, or selection of target patients for administration). Here, the cancer antigen contained in the agent is a proteinogenic cancer antigen. By using the proteinogenic cancer antigen in combination with a single-cell level cytokine production detection assay (e.g., ELISPOT, FluoroSpot, or their technical equivalents), the in vivo immune response mediated by antigen-presenting cells can be faithfully reproduced, enabling highly accurate efficacy prediction.

[0069] 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.

[0070] In another aspect, this disclosure provides a kit for predicting the efficacy of a cancer vaccine in vitro. The kit comprises a cancer antigen corresponding to the cancer vaccine and means for confirming the responsiveness of peripheral blood mononuclear cells isolated from a patient to stimulation by the cancer antigen corresponding to the cancer vaccine. The kit is used to predict and / or calculate the efficacy of the cancer vaccine (e.g., response, duration of response, or selection of target patients for administration) based on the responsiveness. Here, the cancer antigen included in the kit is the aforementioned "proteinogenic cancer antigen". The means for confirming the responsiveness includes reagents or equipment for performing a "single-cell level cytokine production detection assay," specifically comprising the ELISPOT kit, the FluoroSpot kit, or their technical equivalents. By using this kit, it is possible to reproduce in vitro the in vivo response mediated by antigen-presenting cells and obtain predictive results that correlate highly with clinical efficacy.

[0071] In another aspect, the present disclosure provides a kit for predicting the efficacy of a cancer vaccine, the kit comprising a cancer antigen corresponding to the cancer vaccine or an antigenic portion of the cancer antigen or an equivalent thereof, and means for confirming the responsiveness 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 (e.g., an ELISPOT assay kit), and the kit 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 responsiveness.

[0072] In one embodiment, "response to stimulation of patient-derived immune cells (e.g., peripheral blood mononuclear cells) with 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.

[0073] 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 a neoantigen peptide, 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 ERISPOT assay or a FluoroSpot assay. This method makes it possible to evaluate the function of antigen-specific T cells and B cells.

[0074] 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 portion. 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] In one embodiment, the present 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 the vaccine 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).

[0079] 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.

[0080] 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.

[0081] 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 effectiveness 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 ELISTOCT, flow cytometry, CBA, ELISA, and RNA sequencing to enable more precise analysis.

[0082] In this specification, "predicting or calculating the effect of a cancer vaccine based on reactivity" means predicting the effect of a cancer vaccine quantitatively or qualitatively by 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. This prediction of effect 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.

[0083] 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.

[0084] In one embodiment, the prediction is made before administration of the vaccine or during treatment with the vaccine.

[0085] 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.

[0086] In one embodiment, the present disclosure includes providing a cancer antigen or an antigenic moiety of the cancer antigen for a cancer vaccine.

[0087] In one embodiment, the method of the present disclosure is performed in vitro.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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) Effectiveness of the cancer vaccine: If the measured value (Spot Counts) by ELISPOT is above a predetermined value, it is determined that the cancer vaccine is effective. 2) Durability of the cancer vaccine: If the measured value (Spot Counts) by ELISPOT is above a predetermined value, it is determined that the cancer vaccine is durable. Determination of patients who should receive the cancer vaccine: If the measured value (Spot Counts) by ELISPOT is above a predetermined value, it is determined that the patient should receive the cancer vaccine. Determination of the cancer vaccine to be administered: If the measured value (Spot Counts) by ELISPOT is above a predetermined value, it is determined that the patient should receive the vaccine in question. Determination of changes to the dosage and administration of the cancer vaccine: If the measured value (Spot Counts) by ELISPOT is below or above a predetermined value, it is determined that the dosage and administration should be increased or the frequency increased, or if it is above or below a predetermined value, it is determined that the dosage and administration should be increased or the frequency decreased.

[0092] An agent for predicting the effectiveness of a cancer vaccine comprises a cancer antigen corresponding to the cancer vaccine, the antigenic portion of the cancer antigen, or an equivalent thereof. Such an agent is a diagnostic and evaluation composition used to predict the effectiveness of a cancer vaccine, and comprises a cancer antigen corresponding to the cancer vaccine, the antigenic portion of the cancer antigen, or an equivalent thereof. These components are used to predict the therapeutic effect of a cancer vaccine by stimulating patient-derived immune cells and measuring their reactivity. Specifically, the evaluation is performed using 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 of each patient and formulating an optimal treatment strategy. Furthermore, by evaluating the effectiveness of the cancer vaccine in advance, this agent can simultaneously improve the success rate of treatment and reduce the risk of side effects. Therefore, in the agent of this disclosure, the reactivity of patient-derived immune cells upon stimulation with a cancer antigen corresponding to the cancer vaccine, or the antigenic portion of the cancer antigen, or an equivalent thereof is confirmed, and the effectiveness of the cancer vaccine is predicted and / or calculated based on the reactivity.

[0093] In one embodiment, a 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 assessing 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 to promote the immune response, and reagents to detect responsiveness (e.g., enzyme-labeled antibodies or ELISA plates). Dedicated devices such as ELISA readers and flow cytometers are also included in this means, which can be used to measure cytokine secretion, T cell activation status, cytotoxicity, etc., with high accuracy. In the evaluation process, peripheral blood mononuclear cells (PBMCs) or T cells collected from the patient are stimulated with the antigen reagent, 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 means is used as an important tool to clarify the characteristics of the immune response for each patient and to improve the success rate of cancer vaccine treatment. Furthermore, based on the data obtained, it becomes possible to design an optimal vaccine administration plan and additional treatments 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 means may include, but are not limited to, ELISPOT assay kits and their equivalents (such as FLUOROSPOT).

[0094] 1. Embodiment of Efficacy Prediction using ELISPOT Data: Patient-derived PBMCs stimulated with cancer antigens (e.g., NY-ESO-1, MAGEA3, WT1, etc.) are used, and the number of cytokine (e.g., interferon-γ, IL-2) secreting cells is measured using the ELISPOT 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 the cancer vaccine. 1-2. Selection of Patients to be Treated: Patients who do not show an antigen-specific response should be considered for other treatment options. 2. Embodiment of T Cell Functional Analysis by Flow Cytometry: PBMCs stimulated with cancer antigens are analyzed by flow cytometry, and the expression of activation markers (e.g., CD69, CD25) and intracellular cytokine (e.g., IFN-γ, TNF-α, IL-2) production of T cells (CD4-positive and CD8-positive) is measured. 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. Sustained response: 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 by 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 the CBA (Cytokine Bead Array) or Luminex assay. This data is then analyzed to predict the following: 3-1. Response: If the percentage of cells secreting multiple cytokines (multifunctional response) is high, a high response to the vaccine is predicted. 3-2. Selection of patients to administer the vaccine: Other treatments are recommended for patients with a low cytokine response. 4. Implementation of predicting the effectiveness of personalized vaccines using neoantigens: 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. Determination of patients to administer the vaccine: Patients who show a strong response to neoantigens are selected.4-2. Response and Duration: When neoantigen-specific T cells exhibit a multifunctional response (e.g., simultaneous secretion of IFN-γ, IL-2, and TNF-α), high efficacy and durability of the vaccine are predicted. 5. Model-Based Predictive Algorithm Embodiment: Reactivity data (e.g., number of spots, cytokine profile, percentage of activated T cells) is input into a statistical model or machine learning model to construct an algorithm that predicts the efficacy of the vaccine. This model predicts: 5-1. Response: Calculate the probability of response in each patient and optimize treatment selection. 5-2. Duration: Quantitatively predict the time until the immune response disappears. 5-3. Individualized Treatment Plan: Determine the antigens and concomitant drugs to be administered. 6. RNA / DNA Vaccine Efficacy Prediction Embodiment: PBMCs are stimulated using an RNA or DNA vaccine, and secreted cytokines and intracellular responses are evaluated. Based on this, the following is predicted: 6-1. Response: Evaluate the vaccine efficacy based on the intensity of the antigen-specific response induced after RNA / DNA introduction. 6-2. Optimal administration plan: Calculate the optimal dosage and schedule. 7. Prediction of the combined effect with immune checkpoint inhibitors Embodiment: PBMCs stimulated with cancer antigens are combined with an anti-PD-1 antibody or an anti-CTLA-4 antibody, and changes in the immune response are analyzed. This predicts 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 the vaccine is predicted. 7-2. Selection of patients to be administered: Select patients who show responsiveness to checkpoint inhibitors.

[0095] 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 erythropoiesis, flow cytometry, CBA, RNA sequencing, and machine learning models.

[0096] 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 which other drugs and / or nutrients should be administered in addition, and 6) determining whether the dosage and administration of the cancer vaccine should be changed.

[0097] In this specification, specific embodiments will be described for each of the following items. 1. Responsiveness of cancer vaccines Responsiveness refers to the possibility that a cancer vaccine will exert a therapeutic effect in a patient. In this embodiment, the response is evaluated using immune response data based on cancer antigens. Example: The amount of cytokines (e.g., IFN-γ, IL-2) secreted from peripheral blood mononuclear cells (PBMCs) of patients stimulated with cancer antigens (e.g., MAGEA3, NY-ESO-1) is measured using the ELISPOT assay, and if the number of spots is above a certain level (e.g., 200 spots or more per 100,000 PBMCs), a high response is expected. Auxiliary technique: The accuracy of response is improved by using the analysis of T cell activation markers (e.g., CD69, CD25) using flow cytometry. 2. Durability of cancer vaccines Durability is an indicator used to evaluate how long the therapeutic effect of a cancer vaccine is maintained. Example: The induction of immune memory cells (memory T cells) after administration of the cancer vaccine is evaluated. For example, the percentage of CD45RO-positive T cells is measured by flow cytometry at 6 and 12 months after administration to evaluate the duration of the immune response. Auxiliary technique: Use the Tetramer assay or ELISPOT assay to measure the presence of neoantigen-specific memory T cells. 3. Determining patients who should receive the cancer vaccine In this embodiment, patients who are likely to respond to the cancer vaccine are selected based on their immune profile and tumor characteristics. Example: The cancer antigen expression level of the patient's tumor is 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 that matches the HLA is selected. Auxiliary technique: The percentage of immunosuppressive factors (e.g., Treg cells, MDSCs) in the tumor microenvironment is measured by flow cytometry, and patients with low immunosuppression are prioritized. 4. Determining the appropriate cancer vaccine: The optimal cancer vaccine is selected based on the patient's tumor characteristics and immune status. For example, based on the type of cancer antigen (e.g., MAGEA3, WT1, HER2) and the patient's HLA type, a peptide vaccine, protein vaccine, or RNA vaccine is selected.For example, 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, individualized vaccines are designed based on next-generation sequencing (NGS) data of the patient's tumor. 5. Determining other drugs and / or nutrition to be administered additionally. Determine adjuvant therapies to be used in combination to enhance the effect of the cancer vaccine. Example: Select immune checkpoint inhibitors (e.g., anti-PD-1 antibody, anti-CTLA-4 antibody) to be used in combination with the cancer vaccine. For example, in patients with confirmed immunosuppression, the immune response is enhanced by using an anti-PD-1 antibody. Nutritional example: Administration of nutritional supplements that promote the immune response, such as vitamin D and omega-3 fatty acids, is recommended. Supporting technology: Use cytokine profiling (e.g., CBA or Luminex assay) to determine which cytokine therapies (e.g., IL-2, GM-CSF) to administer. 6. Decisions on modifying the dosage and administration of cancer vaccines: Based on the patient's immune response data, the dosage and administration schedule of cancer vaccines are adjusted. For example, if the immune response is insufficient in the initial stages of administration, the vaccine dose is increased (e.g., increasing the peptide amount from 50 μg to 100 μg). Also, if the immune response declines early, the administration interval is shortened from 4 weeks to 2 weeks. Supporting techniques: Monitor the number of spots or the percentage of activated T cells after the first dose and adjust the next administration plan based on this.

[0098] 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).

[0099] 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.

[0100] First, the reactivity of patient-derived immune cells to cancer antigens or their antigenic moieties is a crucial indicator that forms the basis for 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., the number of interferon-gamma secreting cells and the expression rate of activation markers) provides important information for predicting how well an individual patient's immune system will respond to a cancer vaccine.

[0101] 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 a four-week interval to a two-week interval). 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).

[0102] 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 additional administration of vitamin D or amino acid supplements to enhance the immune response. This is expected to further improve the effectiveness of cancer vaccine therapy.

[0103] 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.

[0104] 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 a cancer vaccine is administered to a subject patient whose immune response (reactivity) based on stimulation by a 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 a cancer antigen or its antigenic moiety, and the immune response is evaluated using an ELISPOT assay or flow cytometry. For example, when stimulated with cancer antigens such as MAGEA3 or NY-ESO-1, a subject patient is determined to have a "reactivity value above a predetermined value" if the number of interferon-γ secreting cells is 200 spots or more per 100,000 PBMCs, or if the proportion of activated T cells (CD69-positive T cells) is 50% or more. Patients who meet this standard are expected to have a high response rate to cancer vaccine administration.

[0105] In one embodiment, administration based on responsiveness levels can not only enhance therapeutic effects but also reduce the burden on patients by avoiding ineffective treatments. For patients with responsiveness below a predetermined level, 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.

[0106] 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.

[0107] 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 after stimulating patient-derived immune cells (e.g., peripheral blood mononuclear cells (PBMCs)). 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.

[0108] Specific examples of detection agents used in this disclosure include interferon-γ (IFN-γ) capture antibody and detection antibody 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.

[0109] In further embodiments, detection using flow cytometry (FACS) may also be considered. In this case, specific detection agents are used. For example, to detect activation markers expressed on the surface of T cells after stimulation with cancer antigens (e.g., CD69, CD25), fluorescently labeled antibodies (e.g., FITC or PE-labeled anti-CD69 antibodies) are used. Also, 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.

[0110] (Regarding specific examples) The following are specific examples. This example can be found in 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. https: / / doi.org / 10.3390 / biomedicines3040304. The evaluation criteria for the ELISPOT (Enzyme-Linked Immunosorbent Spot) assay are a very important element in measuring the immune response. In this study, the ELISPOT assay is used to measure the immune response after dendritic cell (DC) vaccination targeting WT1 (Wilms' Tumor 1) specific T cells.

[0111] Examples of evaluation criteria are as follows: Quantitativeness: In the ELISPOT assay, the number of IFN-γ-producing cells specific to the WT1 peptide is measured to establish a quantitative criterion for a positive reaction. Specifically, 1 × 10⁻¹⁶ cells stimulated by the WT1 peptide. ６At least 15 spots were observed per PBMC, and at least 1.5 times more spots were required compared to the negative control. Reproducibility and precision: The reproducibility of the assay was evaluated by the coefficient of variation (CV) under different conditions, and studies have reported a range of 7.4% to 16.3%. Daily precision was also evaluated, with a CV ranging from 5.0% to 17.3%. Linearity: The ELISPOT assay showed linear reactivity with respect to cell number, and a correlation coefficient of r = 0.96–0.98 was obtained in dilution experiments. This confirms the reliability of the quantitative evaluation of the assay. Specificity: The response of WT1-specific T cells is compared to the number of spots when using the negative control peptide. A positive result is determined only when a specific response is observed. Clinical utility: Detection of IFN-γ producing cells after WT1 peptide stimulation showed a positive reaction in 34 / 46 patients (73.9%), demonstrating clinical utility. These results demonstrate 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 accurately and quantitatively evaluating the functional response of WT1-specific T cells and is an important tool for evaluating the efficacy of cancer immunotherapy.

[0112] In certain embodiments of this disclosure, if the number of spots is 7 or more and the SI is less than 1, it can be determined that the patient is responsive to the target cancer vaccine (highly likely to respond). That is, an example of pre-administration stratification of ELISPOT is shown. The weak positive criteria are 1) the presence of 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. In this case, it is possible to predict 1) the response of the cancer vaccine, 2) the persistence of the cancer vaccine, 3) the determination of patients who should receive the cancer vaccine, and 4) the determination of which cancer vaccine to administer.

[0113] The ELISPOT post-vaccination positive criteria are that the number of spots is 15 or more and the SI is greater than 1.5, in which case the patient can be judged as responsive (highly likely to respond) to the target cancer vaccine. This may be due to: 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. In this case, it is possible to determine: 1) the response 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.

[0114] An example of pre-vaccination stratification using ELISPOT is shown. The weak positive criterion is that 1) there are 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.

[0115] The criteria for a positive result after vaccine administration in ELISPOT may be: 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. 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.

[0116] (Companion Diagnostics) In another aspect, the Disclosure provides 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 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. The method may further comprise any features or combinations thereof described herein with respect to the diagnostics of the Disclosure.

[0117] In another context, the Disclosure provides a pharmaceutical product comprising a cancer vaccine, wherein the vaccine regime is determined based on the responsiveness 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. The pharmaceutical product of the Disclosure may further comprise any features or combinations thereof described herein with respect to the diagnostics of the Disclosure.

[0118] In one embodiment, the disclosure further encompasses identifying the cancer antigen or the antigenic portion of the cancer antigen corresponding to a patient.

[0119] In one embodiment, the pharmaceutical product of the present disclosure may be administered to a subject in which the reactivity value of immune cells obtained from the subject to be administered the pharmaceutical product before administration is equal to or greater than a predetermined value to the antigenic stimulation of the cancer vaccine.

[0120] This disclosure provides a pharmaceutical kit comprising a pharmaceutical including a cancer vaccine and a detection agent for detecting stimulation of the cancer vaccine by a cancer antigen or an antigenic portion of the cancer antigen, wherein the vaccine regimen is determined based on the reactivity of patient-derived immune cells to stimulation by the cancer antigen or an equivalent corresponding to the cancer vaccine. The kit of this disclosure may further comprise any features or combinations described herein with respect to the diagnosis of this disclosure. In one embodiment, with respect to a treatment regimen, the following are exemplary but not limited to the pre-treatment and intra-treatment prediction formulas: Pre-treatment prediction: WT1 ≥ 7 ∩ WT1 ≥ NC → Positive WT1 < 7 ∪ WT1 < NC → Negative Intra-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 negative control spots.

[0121] (General Art) The molecular biological, biochemical, and microbiological methods used herein are well known and commonly used in the field, for example, Sambrook J. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, F. M. (1987). Current Protocols in Molecular Biology, Greene Pub. Associate Sand Wiley-Interscience; Ausubel, F. M. (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Green Pub. Associates and Wiley-Interscience; Innis, M. A. (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, F. M. (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Green Pub. Associates; Ausubel, F. M. (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Green Pub. Associates; Innis, M. A. et al. (1995). PCR Strategies, Academic Press;Ausubel, F. M. (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky, J. J. et al. (1999). PCR Applications are described in publications such as "Protocols for Functional Genomics," Academic Press, and "Experimental Methods for Gene Transfer & Expression Analysis," Yodosha, 1997. Relevant parts (possibly all) of these publications are referenced herein.

[0122] 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.

[0123] 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.

[0124] The present invention has been described above with reference to preferred embodiments for ease of understanding. The present invention will now be described based on examples, but the above description and the following examples are provided for illustrative purposes only and not to limit the present invention. Accordingly, the scope of the present invention is not limited to the embodiments or examples specifically described herein, but is limited only by the claims.

[0125] Examples of the experiments are described below. These experiments complied with all regulations concerning clinical and non-clinical studies stipulated by Kobe University and were conducted in compliance with clinical ethics rules such as the Declaration of Helsinki. The drugs and other materials used were obtained from the suppliers indicated in each description, but they are also available from other sources (e.g., Diaclone, R&D Systems, etc.).

[0126] (Example 1) In this example, we conducted tests on B440, an oral cancer treatment vaccine targeting the WT1 protein (tumor-associated antigen) (the WT1 expressed in the B440 vaccine is amino acid a.a. 117-419, totaling 303 amino acids).

[0127] (Methods and Materials) A Phase I clinical trial of B440, an oral cancer treatment vaccine targeting the WT1 protein (tumor-associated antigen), was conducted for advanced urothelial carcinoma. The induction of WT1-specific cellular immunity was measured using the ELISPOT assay before and after vaccine administration. The treatment outcomes of patients who tested positive for WT1-specific cellular immunity after vaccine administration were significantly better than those who tested negative. Furthermore, it was found that the positivity / negative status of the ELISPOT assay after vaccine administration and the treatment effect could be predicted by establishing certain criteria for weak positivity / negative diagnosis using the ELISPOT assay before vaccine administration.

[0128] (Summary of the clinical trial) In this example, an open-label, single-arm trial was conducted to evaluate the safety and efficacy of the oral cancer vaccine B440 in patients with PD-1 / PD-L1 inhibitor-resistant advanced urothelial carcinoma (corresponding to Phase 1 of the trial).

[0129] This example evaluated the safety and efficacy of oral cancer vaccine B440 administered orally once daily, five times a week for four weeks, at a dose of 800 mg or 1,600 mg, in patients with unresectable advanced urothelial carcinoma who were resistant to or intolerant of PD-1 / PD-L1 inhibitors and had no standard therapy.

[0130] (Evaluation of WT1 heterocellular immune response by ELISPOT assay) T cells were isolated from peripheral blood mononuclear cells (PBMCs) of subjects and stimulated with WT1 antigen protein (full-length (449 amino acids) was used). Subsequently, the number of IFN-γ producing cells was measured using the ELISPOT method to confirm the WT1-specific immune response.

[0131] 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), W8, W16, and W24 (see Figure 3).

[0132] 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).

[0133] (Assay Method) The ELISPOT kit was used, specifically the hIFNGp-2M / 2 ELISPOT kit (C.T.L., Cat. No. hIFNG-2M / 2). First, L-glutamine (200 mM, Nacalai Tesque, Cat. No. 16948-04) was added to the culture medium (CTL Test Medium) to adjust the final concentration to 2 mM, and then warmed at 37°C. Patient PBMCs collected at each time point were washed with X-VIV 15 medium (Lonza, Cat. No. 04-418Q), suspended in CTL Test Medium with added L-glutamine, and the cell concentration was adjusted to 1.0 × 10⁻⁶. ６ The concentration was adjusted to cells / mL.

[0134] (Preparation of stimulating antigen and control solution) Human WT1 protein was used as the stimulating antigen and adjusted to a final concentration of 1 μg / mL. Concanavalin A (Invitrogen, Cat. No. 00-4978-03, final concentration 500-fold dilution) was used as the positive control, and medium supplemented with L-glutamine was used as the negative control.

[0135] (Stimulation Culture) Dispense 100 μL of the prepared PBMC into each 96-well plate, and add the stimulating antigen, positive control, or negative control solution to each well. The wells should be filled with 5% CO2. ２ The samples were incubated at 37°C for approximately 24 hours under environmental conditions.

[0136] (Well cleaning) After incubation, the wells were cleaned with PBS(-) and 0.05% Tween-PBS to remove impurities and nonspecific substances.

[0137] After washing with IFN-γ detection antibody, 80 μL of h-IFNγ Detection Solution was added to each well, and the wells were incubated at room temperature for 2 hours. The wells were then washed again with Tween-PBS.

[0138] Next, 80 μL of Strip-AP Solution was added to each well, and incubated at room temperature for 30 minutes. The wells were then washed again with Tween-PBS.

[0139] For the color reaction, 80 μL of Blue Developer Solution was added to each well, and the reaction was allowed to proceed at room temperature for 15 minutes. After that, the wells were rinsed with tap water to stop the color reaction.

[0140] After drying the plates and analysis plates, 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 response.

[0141] (Judgment Criteria) The number of IFN-γ producing cells was counted when stimulated with each antigen, and the number of spots per well was calculated. The positive criteria were as follows:

[0142] ELISPOT (post-vaccination) 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.

[0143] Pre-ELISPOT (pre-vaccine administration) weakly positive criteria: (1) At least 7 WT1-specific spots are present, and (2) The number of WT1-specific spots is greater than the number of spots in the negative control.

[0144] (Results) The results of this embodiment 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 Negative Control without antigen stimulation, hWT1 is stimulated with human WT1 protein (antigen), and ConA is the number of positive spots when stimulated with Concanavalin A as 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 the number of positive spots when any of the hWT1 spots from Day 14 onwards 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.

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157] Figure 5 shows the time course of the average number of spots in the ELISPOT-positive and 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. Patients who were ELISPOT-positive and patients who were weakly pre-ELISPOT-positive during the observation period were in complete agreement.

[0158]

[0159] The best overall response among Pre-ELISPOT and ELISPOT-positive and negative patients was SD in 5 out of 6 positive patients (83.3%), and SD in 1 out of 5 evaluable negative patients (20%). All patients other than those with SD showed PD. Table 14 below summarizes the safety and efficacy of the subjects.

[0160] (B440-1 Safety and Efficacy List of Clinical Trial Subjects) (Note: The shortest SD period for best overall response is 42 days)

[0161]

[0162] 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 AE. 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 other patients were PD (Figures 16 and 17).

[0163] 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).

[0164] As shown in Figures 8 and 14, the stratification by Pre-ELISPOT and ELISPOT in the results of this embodiment is shown. Patients were divided into two groups based on whether they were positive or negative for ELISPOT and Pre-ELISPOT, 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).

[0165] As shown above, progression-free survival for B440 can be stratified based on the results of Pre-ELISPOT and during the observation period (treatment period) (Figure 8, log rank test, P = 0.0033). (Figures 10, 12, 16, and 17)

[0166] Figure 4 shows the treatment with immune checkpoint inhibitors and anti-PD-1 antibodies after vaccine administration.

[0167] 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 Pre-ELISPOT can be used to predict the therapeutic effect of anti-PD-1 antibodies after or in combination with vaccine administration.

[0168] Figure 4 shows the reduction rate of indicator tumor size after re-administration of anti-PD-1 antibody.

[0169] As can be seen in Figure 4, all three ELISPOT-positive patients who were re-administered with anti-PD-1 antibodies showed a response to the anti-PD-1 antibodies (RECIST evaluation: CR, PR, and SD in one case each), while the two ELISPOT-negative patients were both diagnosed with PD.

[0170] (Companion drug for determining cancer vaccine indication) In this embodiment, the results of ELISPOT (after vaccine administration) in the 12 cases in the clinical trial were in complete agreement with the results of Pre-ELISPOT (before vaccine administration) (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 by the Pre-ELISPOT assay before cancer vaccine administration.

[0171] (Determination formula) The determination formulas for pre-treatment and during the administration period can be summarized as follows.

[0172] 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.

[0173] Figure 11 shows the mean test (Mann-Whitney U test) of ELISPOT at the pre-vaccination (before vaccine administration) time point. As shown in Figure 12, stratification is possible using ELISPOT after vaccine administration (during the treatment period). The criteria for a positive ELISPOT test after vaccine administration 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.

[0174] (Example 2) In this example, the same tests as in Example 1 were performed with AGEA1-4, NY-ESO-1, PRAME, SSX2, CT8, CD19, GP100, MART1, PSA, PSMA, tyrosinase, HER2, MUC1, CEA, survivin, cycling B1, EGFR, mesothelin, telomerase, and neoantigens. The procedure was the same as in Example 1, except that the cancer peptides were changed and the corresponding targets were used.

[0175] (Example 3) Comparative study of cancer vaccine therapeutic effect prediction ability by WT1 protein stimulation and short peptide stimulation Next, a comparative study of cancer vaccine therapeutic effect prediction ability by WT1 protein stimulation and short peptide stimulation was conducted. In this example, the effectiveness of using "protein" as the stimulating antigen and using the conventional "short peptide" was compared and verified as indicators for predicting the therapeutic effect (progression-free survival: PFS) of a cancer vaccine (B440 monotherapy). (Method) Peripheral blood mononuclear cells (PBMCs) collected from cancer patients were used to perform the ELISPOT assay under the following two conditions. Protein stimulation group: PBMCs were stimulated using the full-length WT1 protein. Short peptide stimulation group: PBMCs were stimulated with a mixture of three WT1 short peptides (WT1 126-134, WT1 235-243, WT1 332-347). In each group, cases were stratified into "Weak positive" and "Negative" based on assay results, and the correlation with subsequent PFS in B440 monotherapy was analyzed using the Kaplan-Meier method. In this example, stratification experiments were performed using three WTI short peptides (WTI 126-134, WTI 235-243, WTI 332-347) in the same manner as the method using WT1 protein in Example 1. (Results) Figures 18-20 show the results. *Protein stimulation (present invention): As shown in Figure 18, in the B440 monotherapy group, the analysis using the full-length WT1 protein clearly stratified positive and negative cases (6 positive cases, 6 negative cases). Positive cases showed statistically significantly better PFS compared to negative cases (Log-rank p = 0.0301). *Short peptide stimulation (comparative example): In contrast, as shown in Figure 19, in the analysis using three types of short peptides in the B440 monotherapy group, there were very few positive cases for any of the peptide stimulations, and no significant difference in PFS was observed between positive and negative cases. Furthermore, as shown in Figure 20, when the effect of re-administration (rechallenge) of the immune checkpoint inhibitor (pembrolizumab) was predicted using short peptide stimulation, no correlation was observed in PFS between positive and negative cases (Log-rank p = 0.45).(Discussion) From the above results, it is difficult to predict the actual clinical effect under complex treatment conditions such as monotherapy and readmission using peptide stimulation, which consists of short amino acid sequences. In contrast, by using a "protein" that encompasses the sequences contained in the vaccine as a stimulating antigen, the in vivo reaction via the cross-presentation process by antigen-presenting cells can be faithfully reproduced in vitro, and it has been shown that the therapeutic effect of cancer vaccines can be predicted with extremely high accuracy.

[0176] (Example 4) Prediction of WT1 full-length protein vaccine using various proteins In the treatment of cancer using a cancer vaccine containing WT1 full-length protein (approximately 449 amino acids) as the active ingredient, the following proteins of WT1 were created as predictive antigens (proteinogenic cancer antigens) and their effects were confirmed. A) N-terminal domain (residues 1-200) B) C-terminal domain (residues 201-449) C) 303 amino acids from residues 117-419 that are in complete agreement with B440 D) 80% central domain (residues 46-405, 360 amino acids) E) 100 amino acids from residues 117-216 that are part of B440 F) 100 amino acids from residues 320-419 that are part of B440 These proteins were evaluated separately. In both cases, the use of either domain demonstrated higher case stratification ability compared to short peptide stimulation. It was confirmed that using the sum of the responsiveness values ​​of both domains (or the higher value) allowed for prediction of progression-free survival (PFS) with accuracy comparable to that achieved with full-length proteins. However, full-length proteins exhibited the best responsiveness, while the others were slightly inferior, but still far superior to the peptides.

[0177] (Example 5) For a group of patients who received a polypeptide vaccine prepared by linking five types of patient-specific neoantigen peptides (25-30 amino acids each) using a "fusion protein" for a multi-neoantigen linked vaccine, a recombinant fusion protein containing all five types of neoantigen sequences was prepared and used as a proteinogenic cancer antigen. When stimulated with individual neoantigen short peptides (9 amino acids), the response was limited depending on the HLA compatibility of each patient. However, with stimulation using the fusion protein, the optimal epitopes for each patient were comprehensively presented through processing within the APC, making it possible to statistically significantly distinguish between cases with treatment response and cases with treatment failure.

[0178] (Example 6) Predictive ability of cancer vaccines with modified antigen proteins: In treatment using a modified cancer vaccine in which the immunogenicity is enhanced by introducing amino acid substitutions into a portion of the natural sequence, the predictive ability of using a "natural full-length protein" as the proteinaceous cancer antigen is compared with that of using a "partial protein including the modification site". The partial protein including the modification site is shown to more directly reflect the effect of the vaccine and to have a higher correlation with the risk of recurrence after treatment (recurrence-free survival: RFS). This demonstrates the advantage of using a protein that includes the vaccine-specific sequence, even if it is only a "part" of the vaccine.

[0179] (Example 7) Prediction using a "protein cocktail" for multivalent cancer vaccines In multivalent vaccine therapy in which proteins of three types of cancer antigens (HER2, NY-ESO-1, MUC1) are mixed and administered, these three proteins are used as a predictive antigen. By using a mixture (cocktail) rather than making predictions with only a single antigen protein, it is possible to capture patients who show a strong immune response to only one of the antigens as "positive" without omission, thereby improving the sensitivity of overall clinical efficacy prediction.

[0180] (Example 8) Functional confirmation of "proteinogenic cancer antigen" by inhibition of intracellular processing To confirm that the predictive ability of the proteinogenic cancer antigen used in Examples 3 to 7 depends on processing by APC, the reactivity in the presence of a proteasome inhibitor was confirmed. While the T cell response to short peptide stimulation was not affected by the inhibitor, the response to the proteinogenic cancer antigen was significantly suppressed. This result supports the conclusion that the high predictive ability in the present invention is due not only to the presentation of sequences but also to a biological process involving intracellular processing by APC.

[0181] (Example 9: Example using animals) This example shows experiments using mouse bladder cancer cells MBT-2 cells (which express WT1, with a homology of 95% or more between human and mouse WT1 amino acid sequences).

[0182] (Experimental 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.

[0183] More specifically, 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 vaccination (day 30). The WT1-ELISPOT assay was then performed. The ELISPOT kit used was 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 on days 7-11, 14-18, and 21-25. ９ CFU / 100 μl is administered orally, and spleen cells are extracted on day 30 and ELISPOT is performed.

[0184] (ELISPOT Procedure) (Spleen Cell Isolation and ELISPOT Assay) To isolate spleen cells, mice are euthanized by cervical dislocation under anesthesia, 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, this 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, and the cells are 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 cells are suspended in 1 mL of culture CTL Test Medium. In the ELISPOT assay, each well is washed twice with 200 μL / well PBS, followed by two more washes with Tween-PBS. Then, 80 μL / well of m-IFNγ detection solution is added and incubated at room temperature for 2 hours. After washing, 80 μL / well of Strip-AP solution is added and incubated at room temperature for 30 minutes. Next, 80 μL / well of Blue Developer solution is added and incubated for 15 minutes, and the plate is washed with running water to stop the color reaction. The plate is then dried and the spots in each well are measured using an ImmunoSpot® analyzer.

[0185] As shown in Figure 7, the following can be expected: Before MBT-2 transplantation → WT1-ELISPOT negative. After MBT-2 transplantation → WT1-ELISPOT weakly positive (5 or more positive cells, less than 15, WT1 stimulation > unstimulated). After MBT-2 transplantation and B440 administration → WT1-ELISPOT positive (15 or more positive cells, WT1 stimulation is 1.5 times or more than unstimulated).

[0186] (Example 10: Clinical trial example) PAP (prostatic acid phosphorase)-ELISPOT was performed 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.

[0187] (Clinical trial procedure) PBMCs were collected from 12 patients with castration-resistant prostate cancer before administration of the prostate cancer vaccine and 30 days after administration. The PAP (prostatic acid phosphate) specific immune response was confirmed using ELISPOT. Progression-free survival was calculated to confirm the versatility and effectiveness of ELISPOT as a companion diagnostic.

[0188] (ELISPOT Procedure) The ELISPOT procedure is performed using the hIFNGp-2M / 2 ELISPOT kit (manufactured by C.T.L.). First, L-glutamine is added to the culture medium (CTL Test Medium) to adjust the final concentration to 2 mM, and then warmed at 37°C. Patient PBMCs collected at each time point are washed with X-VIV 15 medium, 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.

[0189] (Preparation of stimulating antigen and control solution) Human PAP protein is used as the stimulating antigen and is adjusted to a final concentration of 1 μg / mL. Concanavalin A (500-fold dilution to final concentration) is used as the positive control, and L-glutamine supplemented medium is used as the negative control.

[0190] (Stimulation Culture) Dispense 100 μL of the prepared PBMC into each 96-well plate, and add the stimulating antigen, positive control, or negative control solution to each well. The wells should be filled with 5% CO2. ２ Incubate at 37°C for approximately 24 hours in an environment.

[0191] (Well cleaning) After incubation, the wells are cleaned with PBS(-) and 0.05% Tween-PBS to remove impurities and nonspecific substances.

[0192] (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.

[0193] (Addition of Strip-AP) Next, add 80 μL of Strip-AP Solution to each well and incubate at room temperature for 30 minutes. Wash the wells again with Tween-PBS.

[0194] (Color development 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 well with tap water to stop the color development reaction.

[0195] (Plate drying and analysis) After drying the plates, 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.

[0196] (Judgment Criteria) The number of IFN-γ producing cells when stimulated with each antigen is counted, and the number of spots per well is calculated. The positive criteria are as follows:

[0197] (Pre-ELISPOT (pre-vaccination) weakly positive criteria) (1) There are 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.

[0198] (Analysis) Patients were divided into two groups based on whether their ELISPOT test results were positive or negative before and after vaccine administration. Kaplan-Meier curves were created for progression-free survival and a log-rank test was 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 would demonstrate the versatility and effectiveness of companion diagnostics using ELISPOT.

[0199] (Example 11: Example of kit packaging) This example describes the configuration when the kit is specifically provided.

[0200] Standard 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 to use BCIP / NBT-(Substrate buffer)

[0201] 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.

[0202] (Example 6: Example of Treatment) The following is an explanation of how to implement this example as a treatment method.

[0203] For patients being considered for cancer vaccine treatment, PBMCs will be collected via blood sample before the start of treatment.

[0204] Subsequently, in the ELISPOT assay, PBMCs are cultured in vitro and stimulated with the cancer antigen protein corresponding to the cancer vaccine or a partial peptide protein of the cancer antigen protein.

[0205] Subsequently, only those who test positive for 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.

[0206] (Note) As described above, the Disclosure has been illustrated using preferred embodiments thereof, but it should be understood that the scope of the Disclosure should be interpreted solely by the claims. Patents, patent applications and other documents cited herein should be incorporated by reference to this Specification as if their contents were specifically described herein. This application claims priority to Japanese Patent Application No. 2025-005082, filed with the Japan Patent Office on 14 January 2025, the contents of which are incorporated herein by reference as if they were specifically described herein.

[0207] This disclosure provides a novel therapeutic or preventive agent.

Claims

1. An agent for predicting the effectiveness of a cancer vaccine, wherein the agent comprises a cancer antigen corresponding to the cancer vaccine, and the cancer antigen is a proteinaceous cancer antigen.

2. The agent according to claim 1, wherein the proteinaceous cancer antigen is a protein composed of all or part of the amino acid sequence contained in the cancer vaccine.

3. The agent according to claim 1 or 2, used in a single-cell level cytokine production detection assay.

4. The agent according to any one of claims 1 to 3, comprising the step of confirming the responsiveness of peripheral blood mononuclear cells taken from a patient to stimulation with a cancer antigen corresponding to the cancer vaccine by a single-cell level cytokine production detection assay, and predicting or calculating the effect of the cancer vaccine based on the responsiveness, wherein the prediction and / or calculation is used in a method that includes confirming the responsiveness 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 responsiveness.

5. The agent according to any one of claims 1 to 4, wherein the prediction is made before administration of the cancer vaccine or during the treatment period with the cancer vaccine.

6. The agent according to any one of claims 1 to 5, wherein the prediction includes obtaining peripheral blood mononuclear cells derived from the patient.

7. The agent according to any one of claims 1 to 6, wherein the prediction comprises providing the cancer antigen corresponding to the cancer vaccine.

8. The agent according to any one of claims 1 to 7, wherein the prediction of the effect is made before administration of the cancer vaccine.

9. The agent according to any one of claims 1 to 8, wherein the effect comprises 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 change in the dosage and administration of the cancer vaccine.

10. The agent according to any one of claims 1 to 9, wherein the confirmation of the reactivity is performed by ELISPOT.

11. The agent according to claim 10, 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.

12. In the prediction, 1) regarding the efficacy of the cancer vaccine, it is determined that the cancer vaccine is effective if the measurement value by ELISPOT is above a predetermined value; 2) regarding the persistence of the cancer vaccine, it is determined that the cancer vaccine is persistent if the measurement value by ELISPOT is above a predetermined value; 3) regarding the determination of patients to be administered the cancer vaccine, it is determined that patients to be administered the cancer vaccine if the measurement value by ELISPOT is above a predetermined value, and / or; 4) regarding the determination of the cancer vaccine to be administered, it is determined that patients to be administered the target vaccine if the measurement value by ELISPOT is above a predetermined value. The agent according to claim 10.

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

14. The agent according to any one of claims 1 to 13, wherein the cancer antigen comprises all cancer antigens contained in a cancer vaccine.

15. The agent according to any one of claims 1 to 14, 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, cycling B1, EGFR, mesothelin, telomerase, and neoantigens resulting from gene mutations in the patient's tumor cells.

16. The agent according to any one of claims 1 to 15, wherein the cancer antigen is the WT1 protein.

17. A kit for predicting the efficacy of a cancer vaccine in vitro, the kit comprising: a cancer antigen corresponding to the cancer vaccine; and means for confirming the reactivity of peripheral blood mononuclear cells taken from a patient to stimulation by the cancer antigen corresponding to the cancer vaccine, the kit for predicting and / or calculating the efficacy of the cancer vaccine based on the reactivity, wherein the prediction and / or calculation includes confirming the reactivity of peripheral blood mononuclear cells derived from the patient to stimulation by the cancer antigen corresponding to the cancer vaccine, and predicting or calculating the efficacy of the cancer vaccine based on the reactivity, wherein the means includes performing a single-cell level cytokine production detection assay, and the cancer antigen is the proteinaceous cancer antigen.

18. The kit according to claim 17, further comprising the features described in any one or more of claims 1 to 16.

19. A pharmaceutical product comprising a cancer vaccine, wherein the vaccine regime 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 regime is determined based on confirming the reactivity of patient-derived peripheral blood mononuclear cells to stimulation with the cancer antigen corresponding to the cancer vaccine by a single-cell level cytokine production detection assay, and predicting or calculating the effect of the cancer vaccine based on the reactivity, wherein the cancer antigen is the proteinaceous cancer antigen.

20. The pharmaceutical product according to claim 19, characterized in that the pharmaceutical product is administered to a subject whose peripheral blood mononuclear cells obtained before administration have a reactivity value to the cancer vaccine that is equal to or greater than a predetermined value.

21. The pharmaceutical product according to claim 19 or 20, further comprising the features described in any one or more of claims 1 to 16.

22. A pharmaceutical kit comprising: a pharmaceutical comprising a cancer vaccine; a detection agent for detecting stimulation by a cancer antigen corresponding to the cancer vaccine; and instructions for using the kit, wherein the instructions indicate that the vaccine regime is determined based on the responsiveness of patient-derived immune cells to stimulation by a cancer antigen corresponding to the cancer vaccine, and that the regime is determined based on confirming the responsiveness of patient-derived peripheral blood mononuclear cells to stimulation by the cancer antigen corresponding to the cancer vaccine, and predicting or calculating the effect of the cancer vaccine based on the responsiveness, wherein the detection agent includes one for performing a single-cell level cytokine production detection assay, and the cancer antigen is the proteinaceous cancer antigen.

23. The kit according to claim 22, further comprising the features described in any one or more of claims 1 to 16.

24. A method for predicting the efficacy of a cancer vaccine in vitro, the method comprising the steps of: confirming the responsiveness of peripheral blood mononuclear cells taken from a patient to stimulation with a cancer antigen corresponding to the cancer vaccine by a single-cell level cytokine production detection assay; and predicting or calculating the efficacy of the cancer vaccine based on the responsiveness, wherein the prediction and / or calculation encompasses confirming the responsiveness 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 efficacy of the cancer vaccine based on the responsiveness, wherein the cancer antigen is a proteinogenic cancer antigen.

25. The method of claim 24, further comprising the features described in any one or more of claims 1 to 16.

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