METHOD FOR EVALUATING EFFICACY OF ANTICANCER DRUG OR SCREENING ANTICANCER DRUG

Abstract

The present invention relates to a method for evaluating an efficacy of an anticancer drug or an anticancer drug candidate, comprising the step of treating a mixture of cancer organoids and immune cells with the anticancer drug or the anticancer drug candidate. In addition, it relates to the anticancer drug efficacy evaluation system or anticancer drug screening system comprising cancer organoids and immune cells.

The mixture of cancer organoids and immune cells according to the present invention is mixed in a specific ratio, and accordingly can similarly reproduce a tumor microenvironment in which cancer cells exist in the body. Therefore, it can make it possible to accurately predict the efficacy of the drug when administered to a subject.

Claims

1. A method for evaluating an efficacy of an anticancer drug candidate, comprising: (a) mixing cancer organoids and immune cells in a ratio of 1:0.5 to 5 and co-culturing the mixture; (b) treating an anticancer drug candidate to the mixture of step (a); and (c) determining that the efficacy of the anticancer drug candidate is excellent when a growth inhibition or death of cancer organoid is increased in the group treated with the anticancer drug candidate of step (b) compared to a positive control group or a group not treated with the anticancer drug candidate.

2. The method according to claim 1, wherein the immune cells are at least one selected from the group consisting of cytotoxic T cells, M1 macrophages, M2 macrophages, TILs, regulatory T cells and dendritic cells.

3. The method according to claim 1, wherein the immune cells are cytotoxic T cells and M1 macrophages, and the co-culture of step (a) is performed by mixing cancer organoids, cytotoxic T cells and M1 macrophages in a cell number ratio of 1:0.5:0.5 to 3.

4. The method according to claim 1, wherein the immune cells are M1 macrophages and M2 macrophages, and the co-culture of step (a) is performed by mixing cancer organoids, M1 macrophages and M2 macrophages in a cell number ratio of 1:1 to 3:0.5 to 5.

5. The method according to claim 1, wherein the immune cells are TILs, and the co-culture of step (a) is performed by mixing cancer organoids and TILs in a cell number ratio of 1:1 to 2.

6. The method according to claim 1, wherein the immune cells are cytotoxic T cells and regulatory T cells, and the co-culture of step (a) is performed by mixing cancer organoids, cytotoxic T cells and regulatory T cells in a cell number ratio of 1:3:0.5 to 3.

7. The method according to claim 1, wherein the immune cells are cytotoxic T cells and dendritic cells, and the co-culture of step (a) is performed by mixing cancer organoids, cytotoxic T cells and dendritic cells in a cell number ratio of 1:3:0.5 to 5.

8. The method according to claim 1, wherein the anticancer drug is at least one selected from the group consisting of a compound, a peptide, a peptide mimetic, a fusion protein, an antibody, an aptamer, an antibody-drug conjugate (ADC), an antisense nucleic acid, a siRNA, a shRNA, a miRNA and a ribozyme that binds in a complementary manner to a DNA or a mRNA.

9. The method according to claim 1, wherein the cancer is at least one selected from the group consisting of biliary tract cancer, stomach cancer, lung cancer, liver cancer, colorectal cancer, colon cancer, small intestine cancer, pancreatic cancer, brain cancer, osteosarcoma, melanoma, breast cancer, sclerosing adenosis, uterine cancer, cervical cancer, head and neck cancer, esophageal cancer, thyroid cancer, parathyroid cancer, renal cancer, sarcoma, prostate cancer, urethral cancer, bladder cancer, hematologic malignancy, lymphoma, and fibroadenoma.

10. The method according to claim 1, wherein the growth inhibition or death of cancer organoids is confirmed by the increase or decrease of the area of the cancer organoids.

11. An anticancer drug efficacy evaluation system using the method according to claim 1, comprising cancer organoids and immune cells.

12. An anticancer drug screening system using the method according to claim 1, comprising cancer organoids and immune cells.

Description

BRIEF DESCRIPTION OF FIGURES

[0062] FIG. 1a is a graph showing the degree of death of cancer organoids according to the ratio of cytotoxic T cells (hereinafter referred to as CD8+ T cells) and M1 macrophages (classically activated macrophage, M1, hereinafter referred to as M1).

[0063] FIG. 1b is a graph showing the degree of death of M1 according to the ratio of cytotoxic T cells and M1 macrophages.

[0064] FIG. 2 is a graph showing the degree of growth inhibition of cancer organoids according to the CD8+ T cells and M1 ratio, and relates to the results of co-culture for 0 hours, 24 hours, 48 hours and 72 hours.

[0065] FIG. 3 is a graph showing the degree of phagocytosis activation by anti-CD47 antibody treatment.

[0066] FIG. 4 is a graph showing overall efficacy of drugs for death of cancer organoids according to an increase in the ratio of CD8+ T cells.

[0067] FIG. 5 is a graph showing the overall efficacy of drugs for death of cancer organoids according to an increase in the M1 ratio.

[0068] FIG. 6 is a graph showing the overall efficacy of the drugs for death of cancer organoids FIG. 7a is a graph showing the growth rate of cancer organoids according to the co-culture ratio of cancer organoids, M1 and M2 macrophages (classically activated macrophage, M2, hereinafter referred to as M2), and it relates to the result of co-culture under the conditions without M1 (1:3:0) or without M2 (1:0:5).

[0069] FIG. 7b is a graph showing the growth rate of cancer organoids according to the co-culture ratio of cancer organoids, M1 and M2, and it relates to the result of co-culture in the mixed condition in which cancer organoids and M1 were fixed in the cell number ratio of 1:0.5.

[0070] FIG. 7c is a graph showing the growth rate of cancer organoids according to the co-culture ratio of cancer organoids, M1 and M2, and it relates to the result of co-culture in the mixed condition in which cancer organoids and M1 were fixed in the cell number ratio of 1:1.

[0071] FIG. 7d is a graph showing the growth rate of cancer organoids according to the co-culture ratio of cancer organoids, M1 and M2, and it relates to the result of co-culture in the mixed condition in which cancer organoids and M1 were fixed in the cell number ratio of 1:3.

[0072] FIG. 8a is a graph showing the growth rate of cancer organoids according to the co-culture ratio of cancer organoids, M1 and M2 macrophages (classically activated macrophage, M2, hereinafter referred to as M2), and it relates to the result of co-culture under the conditions without M1 (1:3:0) or without M2 (1:0:5). Non refers to a drug-untreated group, and CD47 refers to a drug-treated group treated with an anti-CD47 antibody as an exemplary drug.

[0073] FIG. 8b is a graph showing the death rate of cancer organoids according to the co-culture ratio of cancer organoids, M1 and M2, and it relates to the result of co-culture under the conditions without M1 (1:3:0) or without M2 (1:0:5). Non refers to a drug-untreated group, and CD47 refers to a drug-treated group treated with an anti-CD47 antibody as an exemplary drug. The death rate was analyzed based on the results of measuring the growth rate after 72 hours (72 h) of drug treatment.

[0074] FIG. 9a is a graph showing the growth rate of cancer organoids according to the co-culture ratio of cancer organoids, M1 and M2, and it relates to the result of co-culture in the mixed condition in which cancer organoids and M1 were fixed in the cell number ratio of 1:0.5. Non refers to a drug-untreated group, and CD47 refers to a drug-treated group treated with an anti-CD47 antibody as an exemplary drug.

[0075] FIG. 9b is a graph showing the death rate of cancer organoids according to the co-culture ratio of cancer organoids, M1 and M2, and it relates to the result of co-culture in the mixed condition in which cancer organoids and M1 were fixed in the cell number ratio of 1:0.5. Non refers to a drug-untreated group, and CD47 refers to a drug-treated group treated with an anti-CD47 antibody as an exemplary drug. The death rate was analyzed based on the results of measuring the growth rate after 72 hours (72 h) of drug treatment.

[0076] FIG. 10a is a graph showing the growth rate of cancer organoids according to the co-culture ratio of cancer organoids, M1 and M2, and it relates to the result of co-culture in the mixed condition in which cancer organoids and M1 were fixed in the cell number ratio of 1:1. Non refers to a drug-untreated group, and CD47 refers to a drug-treated group treated with an anti-CD47 antibody as an exemplary drug.

[0077] FIG. 10b is a graph showing the death rate of cancer organoids according to the co-culture ratio of cancer organoids, M1 and M2, and it relates to the result of co-culture in the mixed condition in which cancer organoids and M1 were fixed in the cell number ratio of 1:1. Non refers to a drug-untreated group, and CD47 refers to a drug-treated group treated with an anti-CD47 antibody as an exemplary drug. The death rate was analyzed based on the results of measuring the growth rate after 72 hours (72 h) of drug treatment.

[0078] FIG. 11a is a graph showing the growth rate of cancer organoids according to the co-culture ratio of cancer organoids, M1 and M2, and it relates to the result of co-culture in the mixed condition in which cancer organoids and M1 were fixed in the cell number ratio of 1:3.). Non refers to a drug-untreated group, and CD47 refers to a drug-treated group treated with an anti-CD47 antibody as an exemplary drug.

[0079] FIG. 11b is a graph showing the death rate of cancer organoids according to the co-culture ratio of cancer organoids, M1 and M2, and it relates to the result of co-culture in the mixed condition in which cancer organoids and M1 were fixed in the cell number ratio of 1:3. Non refers to a drug-untreated group, and CD47 refers to a drug-treated group treated with an anti-CD47 antibody as an exemplary drug. The death rate was analyzed based on the results of measuring the growth rate after 72 hours (72 h) of drug treatment.

[0080] FIG. 12 is the results of FACS analysis on TILs prepared through monoculture of Tumor-infiltrating lymphocyte (hereinafter referred to as TIL); or co-culture of TILs and cancer organoids, and shows the ratio of TILs expressing CD8 protein on the cell surface to total TILs.

[0081] FIG. 13a is a graph showing the growth rate of lung cancer organoids according to the co-culture ratio of lung cancer organoids and TILs. Non refers to a drug-untreated group, and Atez refers to a drug-treated group treated with an atezolizumab as an exemplary drug. Growth rates were measured immediately after drug treatment (0 h), after 24 hours (24 h), after 48 hours (48 h), and after 72 hours (72 h), respectively.

[0082] FIG. 13b is a graph showing the death rate of lung cancer organoids according to the co-culture ratio of lung cancer organoids and TILs. Non refers to a drug-untreated group, and Atez refers to a drug-treated group treated with an atezolizumab as an exemplary drug. The death rate was analyzed based on the results of measuring the growth rate immediately after drug treatment (0 h), 24 hours later (24 h), 48 hours later (48 h), and 72 hours later (72 h).

[0083] FIG. 14a is a graph showing the growth rate of colorectal cancer organoids according to the co-culture ratio of colorectal cancer organoids and TILs. Non refers to a drug-untreated group, and Atez refers to a drug-treated group treated with an atezolizumab as an exemplary drug. Growth rates were measured immediately after drug treatment (0 h), after 24 hours (24 h), after 48 hours (48 h), and after 72 hours (72 h), respectively.

[0084] FIG. 14b is a graph showing the death rate of colorectal cancer organoids according to the co-culture ratio of colorectal cancer organoids and TILs. Non refers to a drug-untreated group, and Atez refers to a drug-treated group treated with an atezolizumab as an exemplary drug. The death rate was analyzed based on the results of measuring the growth rate immediately after drug treatment (0 h), 24 hours later (24 h), 48 hours later (48 h), and 72 hours later (72 h).

[0085] FIG. 15 is a graph showing the growth rate of colorectal cancer organoids according to each co-culture ratio of colorectal cancer organoids, CD8+ T cells and regulatory T cells (hereinafter referred to as Treg). As a representative example, 1:3:0 described in the figure means that colorectal cancer organoids, CD8+ T cells and Tregs are mixed in a cell number ratio of 1:3:0. The growth rate was analyzed immediately after the start of the co-culture (0 h), after 24 hours (24 h), after 48 hours (48 h), and after 72 hours (72 h), respectively.

[0086] FIG. 16 is a graph showing the growth rate of lung cancer organoids according to each co-culture ratio of lung cancer organoids, CD8+ T cells and Tregs. As a representative example, 1:3:0 described in the figure means that lung cancer organoids, CD8+ T cells and Tregs are mixed in a cell number ratio of 1:3:0. The growth rate was analyzed immediately after the start of the co-culture (0 h), after 24 hours (24 h), after 48 hours (48 h), and after 72 hours (72 h), respectively.

[0087] FIG. 17 is a graph showing the growth rate of colorectal cancer organoids according to each co-culture ratio of colorectal cancer organoids, CD8+ T cells and Tregs, and it relates to the result of co-culture under the conditions without Treg (1:3:0) or without CD8+ T cell (1:0:3). Non refers to a drug-untreated group, Atez refers to a drug-treated group treated with an atezolizumab as an exemplary drug, and TIGIT refers to a drug-treated group treated with an anti-TIGIT antibody as an example. The death rate was analyzed based on the results of measuring the growth rate after 72 hours (72 h) of drug treatment.

[0088] FIG. 18 is a graph showing the death rate of colorectal cancer organoids according to each co-culture ratio of colorectal cancer organoids, CD8+ T cells and Tregs, and it relates to the result of co-culture in the mixed condition in which colorectal cancer organoids and CD8+ T cells were fixed in the cell number ratio of 1:1. Non refers to a drug-untreated group, Atez refers to a drug-treated group treated with an atezolizumab as an exemplary drug, and TIGIT refers to a drug-treated group treated with an anti-TIGIT antibody as an exemplary drug. The death rate was analyzed based on the results of measuring the growth rate after 72 hours (72 h) of drug treatment.

[0089] FIG. 19 is a graph showing the death rate of colorectal cancer organoids according to each co-culture ratio of colorectal cancer organoids, CD8+ T cells and Tregs, and it relates to the result of co-culture in the mixed condition in which colorectal cancer organoids and CD8+ T cells were fixed in the cell number ratio of 1:3. Non refers to a drug-untreated group, Atez refers to a drug-treated group treated with an atezolizumab as an exemplary drug, and TIGIT refers to a drug-treated group treated with an anti-TIGIT antibody as an exemplary drug. The death rate was analyzed based on the results of measuring the growth rate after 72 hours (72 h) of drug treatment.

[0090] FIG. 20 is a graph showing the growth rate of lung cancer organoids according to each co-culture ratio of lung cancer organoids, CD8+ T cells and Tregs, and it relates to the result of co-culture under the conditions without Treg (1:3:0) or without CD8+ T cell (1:0:3). Non refers to a drug-untreated group, and Atez refers to a drug-treated group treated with an atezolizumab as an exemplary drug, and TIGIT refers to a drug-treated group treated with an anti-TIGIT antibody as an exemplary drug. The death rate was analyzed based on the results of measuring the growth rate after 72 hours (72 h) of drug treatment.

[0091] FIG. 21 is a graph showing the death rate of lung cancer organoids according to each co-culture ratio of lung cancer organoids, CD8+ T cells and Tregs, and it relates to the result of co-culture in the mixed condition in which colorectal cancer organoids and CD8+ T cells were fixed in the cell number ratio of 1:1. Non refers to a drug-untreated group, Atez refers to a drug-treated group treated with an atezolizumab as an exemplary drug, and TIGIT refers to a drug-treated group treated with an anti-TIGIT antibody as an exemplary drug. The death rate was analyzed based on the results of measuring the growth rate after 72 hours (72 h) of drug treatment.

[0092] FIG. 22 is a graph showing the death rate of lung cancer organoids according to each co-culture ratio of lung cancer organoids, CD8+ T cells and Tregs, and it relates to the result of co-culture in the mixed condition in which colorectal cancer organoids and CD8+ T cells were fixed in the cell number ratio of 1:3. Non refers to a drug-untreated group, Atez refers to a drug-treated group treated with an atezolizumab as an exemplary drug, and TIGIT refers to a drug-treated group treated with an anti-TIGIT antibody as an exemplary drug. The death rate was analyzed based on the results of measuring the growth rate after 72 hours (72 h) of drug treatment.

[0093] FIG. 23 is a graph showing the growth rate of colorectal cancer organoids according to each co-culture ratio of colorectal cancer organoids, CD8+ T cells and dendritic cells (hereinafter referred to as DC), and it relates to the result of co-culture in the mixed condition in which colorectal cancer organoids and CD8+ T cells were fixed in the cell number ratio of 1:0.5. As a representative example, 1:0.5:0.5 described in the figure means that colorectal cancer organoids, CD8+ T cells and DCs are mixed in a cell number ratio of 1:0.5:0.5. The growth rate was analyzed immediately after the start of the co-culture (0 h), after 24 hours (24 h), after 48 hours (48 h), and after 72 hours (72 h), respectively. Non refers to a drug-untreated group, and Atez refers to a drug-treated group treated with an atezolizumab as an exemplary drug.

[0094] FIG. 24 is a graph showing the growth rate of colorectal cancer organoids according to each co-culture ratio of colorectal cancer organoids, CD8+ T cells and DCs, and it relates to the result of co-culture in the mixed condition in which colorectal cancer organoids and CD8+ T cells were fixed in the cell number ratio of 1:1. As a representative example, 1:1:0.5 described in the figure means that colorectal cancer organoids, CD8+ T cells and DCs are mixed in a cell number ratio of 1:1:0.5. The growth rate was analyzed immediately after the start of the co-culture (0 h), after 24 hours (24 h), after 48 hours (48 h), and after 72 hours (72 h), respectively. Non refers to a drug-untreated group, and Atez refers to a drug-treated group treated with an atezolizumab as an exemplary drug.

[0095] FIG. 25 is a graph showing the growth rate of colorectal cancer organoids according to each co-culture ratio of colorectal cancer organoids, CD8+ T cells and DCs, and it relates to the result of co-culture in the mixed condition in which colorectal cancer organoids and CD8+ T cells were fixed in the cell number ratio of 1:3. As a representative example, 1:3:0.5 described in the figure means that colorectal cancer organoids, CD8+ T cells and DCs are mixed in a cell number ratio of 1:3:0.5. The growth rate was analyzed immediately after the start of the co-culture (0 h), after 24 hours (24 h), after 48 hours (48 h), and after 72 hours (72 h), respectively. Non refers to a drug-untreated group, and Atez refers to a drug-treated group treated with an atezolizumab as an exemplary drug.

[0096] FIG. 26 is a graph showing the death rate of colorectal cancer organoids according to each co-culture ratio of colorectal cancer organoids, CD8+ T cells and DCs, and it relates to the result of co-culture in the mixed condition in which colorectal cancer organoids and CD8+ T cells were fixed in the cell number ratio of 1:0.5. Non refers to a drug-untreated group, and Atez refers to a drug-treated group treated with an atezolizumab as an exemplary drug. The death rate was analyzed based on the results of measuring the growth rate after 72 hours (72 h) of drug treatment.

[0097] FIG. 27 is a graph showing the death rate of colorectal cancer organoids according to each co-culture ratio of colorectal cancer organoids, CD8+ T cells and DCs, and it relates to the result of co-culture in the mixed condition in which colorectal cancer organoids and CD8+ T cells were fixed in the cell number ratio of 1:1. Non refers to a drug-untreated group, and Atez refers to a drug-treated group treated with an atezolizumab as an exemplary drug. The death rate was analyzed based on the results of measuring the growth rate after 72 hours (72 h) of drug treatment.

[0098] FIG. 28 is a graph showing the death rate of colorectal cancer organoids according to each co-culture ratio of colorectal cancer organoids, CD8+ T cells and DCs, and it relates to the result of co-culture in the mixed condition in which colorectal cancer organoids and CD8+ T cells were fixed in the cell number ratio of 1:3. Non refers to a drug-untreated group, and Atez refers to a drug-treated group treated with an atezolizumab as an exemplary drug. The death rate was analyzed based on the results of measuring the growth rate after 72 hours (72 h) of drug treatment.

[0099] FIG. 29 is a graph showing the growth rate of lung cancer organoids according to each co-culture ratio of lung cancer organoids, CD8+ T cells and DCs, and it relates to the result of co-culture in the mixed condition in which lung cancer organoids and CD8+ T cells were fixed in the cell number ratio of 1:0.5. As a representative example, 1:0.5:0.5 described in the figure means that lung cancer organoids, CD8+ T cells and DCs are mixed in a cell number ratio of 1:0.5:0.5. The growth rate was analyzed immediately after the start of the co-culture (0 h), after 24 hours (24 h), after 48 hours (48 h), and after 72 hours (72 h), respectively. Non refers to a drug-untreated group, and Atez refers to a drug-treated group treated with an atezolizumab as an exemplary drug.

[0100] FIG. 30 is a graph showing the growth rate of lung cancer organoids according to each co-culture ratio of lung cancer organoids, CD8+ T cells and DCs, and it relates to the result of co-culture in the mixed condition in which lung cancer organoids and CD8+ T cells were fixed in the cell number ratio of 1:1. As a representative example, 1:1:0.5 described in the figure means that lung cancer organoids, CD8+ T cells and DCs are mixed in a cell number ratio of 1:1:0.5. The growth rate was analyzed immediately after the start of the co-culture (0 h), after 24 hours (24 h), after 48 hours (48 h), and after 72 hours (72 h), respectively. Non refers to a drug-untreated group, and Atez refers to a drug-treated group treated with an atezolizumab as an exemplary drug.

[0101] FIG. 31 is a graph showing the growth rate of lung cancer organoids according to each co-culture ratio of lung cancer organoids, CD8+ T cells and DCs, and it relates to the result of co-culture in the mixed condition in which lung cancer organoids and CD8+ T cells were fixed in the cell number ratio of 1:3. As a representative example, 1:3:0.5 described in the figure means that lung cancer organoids, CD8+ T cells and DCs are mixed in a cell number ratio of 1:3:0.5. The growth rate was analyzed immediately after the start of the co-culture (0 h), after 24 hours (24 h), after 48 hours (48 h), and after 72 hours (72 h), respectively. Non refers to a drug-untreated group, and Atez refers to a drug-treated group treated with an atezolizumab as an exemplary drug.

[0102] FIG. 32 is a graph showing the death rate of lung cancer organoids according to each co-culture ratio of lung cancer organoids, CD8+ T cells and DCs, and it relates to the result of co-culture in the mixed condition in which lung cancer organoids and CD8+ T cells were fixed in the cell number ratio of 1:0.5. Non refers to a drug-untreated group, and Atez refers to a drug-treated group treated with an atezolizumab as an exemplary drug. The death rate was analyzed based on the results of measuring the growth rate after 72 hours (72 h) of drug treatment.

[0103] FIG. 33 is a graph showing the death rate of lung cancer organoids according to each co-culture ratio of lung cancer organoids, CD8+ T cells and DCs, and it relates to the result of co-culture in the mixed condition in which lung cancer organoids and CD8+ T cells were fixed in the cell number ratio of 1:1. Non refers to a drug-untreated group, and Atez refers to a drug-treated group treated with an atezolizumab as an exemplary drug. The death rate was analyzed based on the results of measuring the growth rate after 72 hours (72 h) of drug treatment.

[0104] FIG. 34 is a graph showing the death rate of lung cancer organoids according to each co-culture ratio of lung cancer organoids, CD8+ T cells and DCs, and it relates to the result of co-culture in the mixed condition in which lung cancer organoids and CD8+ T cells were fixed in the cell number ratio of 1:3. Non refers to a drug-untreated group, and Atez refers to a drug-treated group treated with an atezolizumab as an exemplary drug. The death rate was analyzed based on the results of measuring the growth rate after 72 hours (72 h) of drug treatment.

EXAMPLES

[0105] Specific examples will be described to help the understanding of the present invention. However, the following examples should not be construed as limiting the present invention.

Example 1. Anticancer Drug Efficacy Evaluation and Screening System Comprising Cancer Organoids, Cytotoxic T Cells and M1 Macrophages

[0106] The inventors have established an in vitro screening system that can accurately predict the efficacy of a drug when administered to a subject, by similarly reproducing the environment in which cancer exists in the body.

[0107] Specifically, a co-culture system in which cancer organoids, cytotoxic T cells (hereinafter referred to as CD8+ T cells) and M1 macrophages (classically activated macrophages, hereinafter referred to as M1) coexist was used, and the ratio of organoid or cell that satisfies the following three conditions during the co-culture was confirmed, respectively: [0108] (1) A condition under which death rate of cancer organoids and M1 is low (the efficacy of the drug cannot be accurately analyzed if death of the cancer organoids or M1 is observed to be high in the drug-untreated group when co-culturing the organoids, CD8+ T cells and M1) [0109] (2) A condition under which phagocytosis is activated [0110] (3) A condition under which the efficacy of drugs can be evaluated even though the MoA (mode of action) thereof is different from each other

[0111] In addition, when the number of organoids or cells in the ratio of cancer organoids, CD8+ T cells and M1 was set to 1 for 5.0?10.sup.3, the specific number of organoids or cells according to the ratio is as follows: [0112] 0.2=1.0?10.sup.3 of organoids or cells [0113] 0.5=2.5?10.sup.3 organoids or cells [0114] 1=5.0?10.sup.3 organoids or cells [0115] 3=15.0?10.sup.3 organoids or cells [0116] 5=25.0?10.sup.3 organoids or cells [0117] 10=50.0?10.sup.3 organoids or cells

[0118] The ratio of cancer organoids, CD8+ T cells and M1 satisfying all of the conditions described above was confirmed in Examples 1.1 to 1.3 below.

Example 1.1. Establishment of Conditions in which Cancer Organoids, Cytotoxic T Cells and M1 Macrophages Coexist

[0119] When co-culturing cancer organoids, CD8+ T cells and M1, it is difficult to accurately evaluate the efficacy of the drug if anyone among them dies. Therefore, in this Example 1.1, the inventors tried to establish conditions in which all of them survive when cancer organoids, CD8+ T cells, and M1 are co-cultured in the absence of drug treatment.

[0120] Specifically, cancer organoids, CD8+ T cells and M1 derived from colorectal cancer patients were used. As shown in Table 1 below, the ratio for cancer organoids was fixed to 1, and the ratio for CD8+ T cells was set to 0.1 to 10 and the ratio for M1 was set to 0.1 to 5. Then, they were co-cultured for 2 hours, and the death rate of cancer organoids or cells was analyzed at each ratio. The analysis was performed by flow-cytometer, and CFSE (+) was used as a cancer organoid marker and CD11B (+) was used as an M1 macrophage marker.

TABLE-US-00001 TABLE 1 Group Cancer Organoid T cell Macrophage G1 1 0.2 0.5 G2 1 0.2 1 G3 1 0.2 3 G4 1 0.5 0.5 G5 1 0.5 1 G6 1 0.5 3 G7 1 3 0.5 G8 1 3 1 G9 1 3 3

[0121] As a result, as shown in FIG. 1a, a high death rate of cancer organoids was observed in Groups 11 and 12 with a high CD8+ T cell ratio (5 or more) and in Group 9 with a high M1 ratio (5 or more). In addition, as shown in FIG. 1b, it was confirmed that a high death rate of M1 macrophages occurred under conditions in which the ratio of CD8+ T cells was high (5 or more) in Groups 11 and 12.

[0122] Taken together, it was confirmed that the conditions of Groups 3, 4, 5, 6, 7, 8 and 10 allowed cancer organoids, CD8+ T cells and M1 to survive in balance without the drug treatment. Therefore, it was found that when the ratio of cancer organoids is 1, setting the ratio of CD8+ T cells to 3 or less and the ratio of M1 to 3 or less was the most optimal ratio for the anticancer drug screening system.

Example 1.2. Confirmation of Growth Inhibition and Death of Cancer Organoid Depending on Co-Culture Time

[0123] In the screening system composed of cancer organoids, CD8+ T cells and M1 as confirmed in Example 1.1, the inventors confirmed the ratio of cells in which growth inhibition and death of cancer organoids were not observed. For this, after setting different co-culture times (24 hours, 48 hours and 72 hours), the degree of cancer organoid growth was analyzed.

[0124] Specifically, as shown in Table 2 below, the ratio for cancer organoids was fixed to 1, and the ratio for CD8+ T cells was set to 0.2 to 3 and the ratio for M1 was set to 0.1 to 3. Then, they were co-cultured, and phagocytosis was analyzed at each ratio. Herein, the ratio of the CD8+ T cells and M1 was tested in a narrower range than the ratio applied in Example 1.1, and the analysis was performed by HCS (High-Content Screening). The area of the initial organoid was set to 100%, and the change in area depending on the reaction time was expressed as %.

TABLE-US-00002 TABLE 2 Group Cancer Organoid T cell Macrophage G1 1 0 1 G2 1 0.5 0 G3 1 0.1 1 G4 1 0.2 1 G5 1 0.5 0.1 G6 1 0.5 0.5 G7 1 0.5 1 G8 1 0.5 3 G9 1 0.5 5 G10 1 3 1 G11 1 5 1 G12 1 10 1

[0125] As a result, as shown in FIG. 2, although the degree of growth varies depending on the cell ratio, it was confirmed that the growth of cancer organoids was not inhibited even when co-cultured for 72 hours or more at the above ratio. Through the above results, it was found that setting the ratio of cancer organoids, CD8+ T cells and M1 to 1:0.2 to 3:0.1 to 3 is the most optimal ratio for an anticancer drug screening system.

Example 1.3. Establishment of Conditions in which Phagocytosis is Activated

[0126] In order to confirm whether the screening system composed of cancer organoids, CD8+ T cells and M1 as confirmed in Example 1.1 can be used to evaluate the efficacy of a drug targeting M1, the inventors treated the system with anti-CD47 antibody, and then observed the death rate of cancer organoids according to phagocytosis.

[0127] Specifically, cancer organoids and CD8+ T cells derived from colorectal cancer patients were used, and CHA15 cell line was used as M1.

[0128] In addition, as shown in Table 2 above, the ratio for cancer organoids was fixed to 1, and the ratio for CD8+ T cells was set to 0.2 to 3 and the ratio for M1 was set to 0.1 to 3. Then, they were co-cultured, and phagocytosis was analyzed at each ratio. Herein, the ratio of the CD8+ T cells and M1 was tested in a narrower range than the ratio applied in Example 1.1, and the analysis was performed by flow-cytometer. EpCAM(+) was used as a cancer organoid marker, and CD11B (+) was used as an M1 marker. Phagocytosis was confirmed by the double positive ratio of cancer organoid marker and M1 marker.

[0129] As a result, as shown in FIG. 3, phagocytosis was observed in all conditions except for Group 2 in which M1 did not exist. In particular, when treating with the anti-CD47 antibody binding to M1 in Groups 3, 4, 6 and 7, it was confirmed that phagocytosis was activated compared to the untreated group.

[0130] Through the above results, it was found that setting the ratio of cancer organoids, CD8+ T cells and M1 to 1:0.2 to 0.5:0.1 to 3 is the most optimal ratio for an anticancer drug screening system.

Example 1.4. Establishment of Conditions to Evaluate the Efficacy of Drugs with Different MoA (Mode of Action)

[0131] In order to confirm whether the screening system composed of cancer organoids, CD8+ T cells and M1 as confirmed in Example 1.1 can be used to evaluate the efficacy of a drug, the death rate of cancer organoids by phagocytosis was observed after treating the system with an anti-PD-L1 antibody atezolizumab previously used as an immuno-anticancer drug, anti-CD47 antibody and a combination thereof.

[0132] Atezolizumab activates CD8+ T cells by binding to PD-L1 on cancer cells and blocking the PD-1/PD-L1 mechanism, and anti-CD47 antibody activates macrophages by binding to CD47 of M1 and blocking the SIRP-a/CD47 mechanism. Thus, the inventors confirmed whether the screening system of the present invention can evaluate the efficacy of all types of drugs acting in different mechanisms.

[0133] Specifically, cancer organoids and CD8+ T cells derived from colorectal cancer patients were used, and CHA15 cell line was used as M1.

[0134] In addition, as shown in Table 2 above, the ratio for cancer organoids was fixed to 1, and the ratio for CD8+ T cells was set to 0 to 3 and the ratio for M1 was set to 0 to 3. Then, they were co-cultured, and treated with 10 nM of Atezolizumab (Selleck, #A2004) which is anti-PD-L1 antibody and/or 10 nM of anti-CD47 antibody. After that, the death rate of cancer organoids or M1 was analyzed at each ratio. Herein, the ratio of the CD8+ T cells and M1 was tested in a narrower range than the ratio applied in Example 1.1, and the changes in organoid area for 72 hours after drug treatment was analyzed by HCS (High-Content Screening). After 72 hours from drug treatment, the overall efficacy was evaluated by calculating the change in the organoid area of the drug-treated group compared to the organoid area of the non-treated group.

[0135] As a result, FIG. 4 shows that the overall efficacy increased as the ratio of CD8+ T cells increased in Groups 1, 3, 6 and 8 wherein the ratios of cancer organoids and M1 are fixed to 1 and 1 respectively, and the ratio of CD8+ T cells changes. In particular, it was confirmed that the growth inhibition and death of cancer organoids appeared to be high in the atezolizumab-treated group.

[0136] Also, FIG. 5 shows that the overall efficacy increased as the ratio of M1 increased in Groups 2, 4, 5, 6 and 7 wherein the ratios of cancer organoids and CD8+ T cells are fixed to 1 and 0.5 respectively, and the ratio of M1 changes. In particular, it was confirmed that the growth inhibition and death of cancer organoids appeared to be high in the anti-CD47 antibody-treated group.

[0137] As shown in FIG. 6, it was confirmed that Groups 5, 6 and 7 exhibited similar and high killing effects of cancer organoids by the use of atezolizumab alone, anti-CD47 antibody alone and combination thereof.

[0138] Taking the results of Examples 1.1 to 1.3 together, it was confirmed that the ratio for co-culture of cancer organoids, CD8+ T cells and M1 under which the growth of cancer organoids is not suppressed, the death of cancer organoids and M1 is not caused, and the treatment with different mechanism of drugs alone or in combination exhibits similarly overall efficacy is 1:0.5:0.5 in Group 5, 1:0.5:1 in Group 6, and 1:0.5:3 in Group 7.

Example 2. Anticancer Drug Efficacy Evaluation and Screening System Comprising Cancer Organoids, M1 Macrophages and M2 Macrophages

[0139] The inventors have established an in vitro efficacy evaluation and screening system that can accurately predict the efficacy of a drug when administered to a subject, by similarly reproducing the environment in which cancer exists in the body.

[0140] Specifically, in order to establish a co-culture system in which cancer organoids, M1 macrophages (classically activated macrophages, hereinafter referred to as M1) and M2 macrophages (alternatively activated macrophages, hereinafter referred to as M2) coexist, the inventors set a mixing ratio satisfying co-culture conditions under which (1) cancer organoids grow properly and (2) drug efficacy is well observed.

[0141] As shown in Table 3 below, the ratio for cancer organoids was fixed to 1, and the ratio for M1 was set to 0.5 to 3 and the ratio for M2 was set to 0.5 to 5. Herein, the number of organoids or cells was set to 1 for 5.0?10.sup.3.

TABLE-US-00003 TABLE 3 Group Cancer Organoid M1 M2 1 1 3 0 2 0 5 3 0.5 0.5 4 1 5 3 6 5 7 1 0.5 8 1 9 3 10 5 11 3 0.5 12 1 13 3 14 5

[0142] Cancer organoids were prepared to have a 3D structure by isolating lung cancer tissues obtained from patients into single cells and culturing them together with ECM (extracellular matrix). The formed cancer organoids were confirmed to be tumors through pathological analysis, and used after being isolated into single cells using Tryp-LE. M1 and M2 differentiated from iPSCs were used, and M1 or M2 was differentiated from H9 cell line using a general method known in the art.

Example 2.1. Conditions for Proper Growth of Cancer Organoids

[0143] The inventors confirmed a mixing ratio of cancer organoids, M1 and M2, which allows cancer organoids to grow properly even when co-cultured with immune cells.

[0144] Specifically, after co-culturing at the mixing ratio according to Table 3 above, the area of cancer organoids was measured immediately after the start of the co-culture (0 h), after 24 hours (24 h), after 48 hours (48 h) and after 72 hours (72 h), respectively, and the growth rate was analyzed by the following formula.

[00001]${}^{*}\mathrm{Cancer}\mathrm{organoid}\mathrm{growth}\mathrm{rate}\left(\%\right)=\left(\mathrm{area}\mathrm{after}\mathrm{cancer}\mathrm{organoids}\mathrm{after}24,48,\mathrm{or}72\mathrm{hours}\mathrm{of}\mathrm{co}-\mathrm{culture}\right)/\left(\mathrm{area}\mathrm{of}\mathrm{cancer}\mathrm{organoids}\mathrm{immediately}\mathrm{after}\mathrm{the}\mathrm{start}\mathrm{of}\mathrm{the}\mathrm{co}-\mathrm{culture}\right)?100$

[0145] As a result, FIGS. 7a to 7c show that the growth of cancer organoids was not inhibited at all mixing ratios, and the growth rate of cancer organoids tended to increase as the ratio of M2 increased.

Example 2.2. Conditions in which Drug Efficacy is High

[0146] The inventors confirmed a mixing ratio of cancer organoids, M1 and M2, which makes the efficacy of the anticancer drug high.

[0147] Specifically, for the co-cultures of cancer organoids, M1 and M2 according to the mixing ratio in Table 3 above, those not treated with the drug were set as the control group (hereinafter referred to as drug-untreated group), and those treated with an anti-CD47 antibody targeting M1 as an exemplary drug were set as the experimental group (hereinafter referred to as drug-treated group). After co-culturing them for 72 hours, the growth rate was measured after 72 hours of drug treatment (72 h), and the death rate was analyzed by the following method through the measured growth rate.

[00002]$\text{Cancer organoid death rate (\%) = [1 - (growth rate of cancer organoids in the drug-treated}{\mathrm{group}}^{*}\text{)/(growth rate of cancer organoids in the drug-untreated}{\mathrm{group}}^{**}\text{)] ? 100}$$\text{Growth rate of cancer organoids in the drug-treated group (\%) = (cancer organoid area after 72 hours of drug treatment)/(cancer organoid area immediately after the start of the co-culture (0 h) ? 100}$$\text{Growth rate of cancer organoids in the drug-untreated group (\%) = (cancer organoid area after 72 hours without drug treatment)/(cancer organoid area immediately after the start of the co-culture (0 h) ? 100}$

[0148] As a result, as shown in FIGS. 8a and 8b, the death rate of cancer organoids by an anti-CD47 antibody was as high as about 25% when only the cancer organoids and M1 were co-cultured at a mixing ratio of 1:3, except for M2 among the cancer organoids, M1 and M2. On the other hand, the death rate of cancer organoids by an anti-CD47 antibody was remarkably low as about 5% when only the cancer organoids and M2 were co-cultured at a mixing ratio of 1:5, except for M1 among the cancer organoids, M1 and M2.

[0149] On the other hand, as shown in FIGS. 9a and 9b, the death rate of cancer organoids according to the increase in the ratio of M2 did not show a significant difference depending on the mixing ratios when cancer organoids and M1 were co-cultured at a ratio of 1:0.5. The death rate of cancer organoids was about 10% at maximum in all of the drug-treated groups treated with the anti-CD47 antibody, confirming that the efficacy of the drug was not high.

[0150] As shown in FIGS. 10a and 10b, when cancer organoids and M1 were co-cultured at a ratio of 1:1, it was confirmed that the death rate of cancer organoids by the drug increased to a high level up to about 23% at maximum in all groups mixing M2 at a ratio of 0.5 to 5.

[0151] In addition, as shown in FIGS. 11a and 11b, when cancer organoids and M1 were co-cultured at a ratio of 1:3, it was confirmed that the death rate of cancer organoids by the drug increased to a high level up to about 25% at maximum in all groups mixing M2 at a ratio of 0.5 to 5.

[0152] The above results show that the most optimal co-culture ratio of cancer organoids, M1 and M2 for an anticancer drug efficacy evaluation or screening system is 1:1 to 3:0.5 to 5, and suggests that this system is suitable for evaluating or screening the efficacy of anticancer drugs in which macrophages are involved in drug action.

Example 3. Anticancer Drug Efficacy Evaluation and Screening System Comprising Cancer Organoids and TILs

[0153] The inventors have established an in vitro efficacy evaluation and screening system that can accurately predict the efficacy of a drug when administered to a subject, by similarly reproducing the environment in which cancer exists in the body.

[0154] Specifically, a co-culture system in which cancer organoids and Tumor-infiltrating lymphocytes (hereinafter referred to as TIL) coexist was used.

Example 3.1. Obtaining TILs

[0155] TILs are cells that infiltrate or surround tumor tissue, and play a role in recognizing and killing cancer cells. TILs includes various types of immune cells, and most of them is CD8+ T cells capable of attacking tumors. In addition, since TILs are isolated from tumor tissue, the CD8+ T cells included therein are already activated and are in a state capable of recognizing and attacking cancer cells.

[0156] That is, in the anticancer drug efficacy evaluation and screening system according to the present invention, the activation process of CD8+ T cells was omitted by using TIL isolated from tissues. In addition, a condition in which activated CD8+ T cells are maintained longer was drawn, and was applied to this system.

[0157] Specifically, TILs were obtained by cutting the tumor tissue into small pieces, collecting the separated immune cells and tissues, and culturing them in a CD8+ T cell culture medium to grow only immune cells. Thereafter, TIL was cultured alone or co-cultured by mixing TIL and cancer organoid in a cell number ratio of 20:1 (TIL:cancer organoid=1?10.sup.5:5.0?10.sup.3), and TILs were analyzed by FACS. Herein, CD56 was used as markers to analyze total immune cells present in TILs, and CD8 was used as markers to analyze CD8+ T cells among total immune cells present in TILs. If the ratio of CD8+ is 20% or more in this FACS analysis result, such immune cells are considered to be usable for drug efficacy evaluation. Table 4 below shows the ratio of CD8+ T cells present in total TILs.

TABLE-US-00004 TABLE 4 Group 1 Group 2 Condition (Single culture) (Co-culture) Culture Day 1 50.7% 50.7% Culture Day 7 90.3% 91.4% Culture Day 14 29.9% 97.1% Culture Day 21 21.1% 37.9%

[0158] As a result, as shown in FIG. 12 and Table 4, there was no significant difference in the results of single culture and co-culture from Culture Day 1 to Culture Day 7. However, a significant difference was shown from Culture Day 14. In the single culture, the ratio of CD8+ T cells was about 23.9%, which was remarkably reduced compared to Culture Day 7 (about 90.3%), whereas in the co-culture, the ratio of CD8+ T cell was about 97.1% of the total cell, and rather increased compared to Culture Day 7 (about 91.4%).

[0159] Even in the co-culture after Culture Day 21, the ratio of CD8+ T cells was about 37.9%, which reduced compared to Culture Day 14 (about 97.1%).

[0160] The above results show that since TILs obtained through co-culture with cancer organoids maintain activated CD8+ T cells for a long period of time, it is possible to establish an anticancer drug efficacy evaluation and screening system with higher quality and reproducibility and improved accuracy when using it.

Example 3.2. Co-Culture Ratio of Cancer Organoids and TILs

[0161] In order to establish a drug efficacy evaluation and screening system utilizing a co-culture system of cancer organoids and TILs, an mixing ratio of cancer organoids and TILs was drawn.

[0162] Specifically, the inventors set a mixing ratio satisfying co-culture conditions under which (1) cancer organoids grow properly and (2) drug efficacy is well observed. Cancer organoids were prepared using cancer cells isolated from tumor tissues of lung cancer or colorectal cancer patients, and TILs obtained by co-culture with cancer organoids for 14 days according to Example 1.1 were used.

[0163] As shown in Table 5 below, the ratio for cancer organoids was fixed to 1, and the ratio for TILs was set to 0.5 to 3. Herein, the number of organoids or TILs was set to 1 for 5.0?10.sup.3.

TABLE-US-00005 TABLE 5 Group Cancer Organoid TIL 1 1 0.5 2 1 1.5 3 1 3

[0164] In addition, for the co-cultures of cancer organoids and TILs according to the ratio above, those not treated with the drug were set as the control group (hereinafter referred to as drug-untreated group), and those treated with atezolizumab, which is currently marketed and used for a cancer treatment, as an exemplary drug were set as the experimental group (hereinafter referred to as drug-treated group). After co-culturing for 72 hours, the growth rate was measured immediately after the start of the co-culture (0 h), after 24 hours (24 h), after 48 hours (48 h) and after 72 hours (72 h), respectively, and the death rate was analyzed by the following method through the measured growth rate: Cancer organoid death rate (%)=[1?(growth rate of cancer organoids in the drug-treated group*)/(growth rate of cancer organoids in the drug-untreated group**)]?100

[00003]$\text{Growth rate of cancer organoids in the drug-treated group (\%) = (cancer organoid area after 24, 48 or 72 hours of drug treatment)/(cancer organoid area immediately after the start of the co-culture (0 h) ? 100}$$\text{Growth rate of cancer organoids in the drug-untreated group (\%) = (cancer organoid area after 24, 48 or 72 hours without drug treatment)/(cancer organoid area immediately after the start of the co-culture (0 h) ? 100}$

[0165] As a result, as shown in FIGS. 13a and 13b, when lung cancer organoids and TILs were co-cultured at a ratio of 1:0.5, the lung cancer organoids did not respond to the drug, and accordingly the growth rate and death rate of lung cancer organoids were similar in the control group and the experimental group. In particular, it was confirmed that the death rate of lung cancer organoids in the drug-treated group was just about 5%, making it impossible to accurately determine the efficacy of the drug.

[0166] In addition, when lung cancer organoids and TILs were co-cultured at a ratio of 1:3, it was confirmed that the death rate of lung cancer organoids in the drug-treated group was just about 5%, making it impossible to accurately determine the efficacy of the drug.

[0167] On the other hand, when lung cancer organoids and TILs were co-cultured at a ratio of 1:1.5, anticancer effects of atezolizumab were shown by inhibiting the growth and increasing death of lung cancer organoids. In particular, the death rate of lung cancer organoids in the drug-treated group was about 20%, and it was confirmed that atezolizumab exhibited a high level of lung cancer organoid death rate compared to other mixture ratios.

[0168] In addition, as shown in FIGS. 14a and 14b, when colorectal cancer organoids and TILs were co-cultured at a ratio of 1:0.5, the colorectal cancer organoids did not respond to the drug, and accordingly the growth rate and death rate of colorectal cancer organoids were similar in the control group and the experimental group. In particular, it was confirmed that the death rate of colorectal cancer organoids in the drug-treated group was just about 5%, making it impossible to accurately determine the efficacy of the drug.

[0169] In addition, when colorectal cancer organoids and TILs were co-cultured at a ratio of 1:3, it was confirmed that the death rate of colorectal cancer organoids in the drug-treated group was just about 5%, making it impossible to accurately determine the efficacy of the drug.

[0170] On the other hand, when colorectal cancer organoids and TILs were co-cultured at a ratio of 1:1.5, anticancer effects of atezolizumab were shown by inhibiting the growth and increasing death of colorectal cancer organoids. In particular, the death rate of colorectal cancer organoids in the drug-treated group was about 10%, and it was confirmed that atezolizumab exhibited a high level of colorectal cancer organoid death rate compared to other mixture ratios.

[0171] The above results suggest that setting the co-culture ratio of cancer organoids and TILs to 1:1.5 is the most optimal ratio for an anticancer drug efficacy evaluation or screening system.

Example 4. Anticancer Drug Efficacy Evaluation and Screening System Comprising Cancer Organoids, Cytotoxic T Cells and Regulatory T Cells

[0172] The inventors have established an in vitro efficacy evaluation and screening system that can accurately predict the efficacy of a drug when administered to a subject, by similarly reproducing the environment in which cancer exists in the body.

[0173] Specifically, in order to establish a co-culture system in which cancer organoids, cytotoxic T cells (hereinafter referred to as CD8+ T cells) and regulatory T cells (hereinafter referred to as Treg) coexist, the inventors set a mixing ratio satisfying co-culture conditions under which (1) cancer organoids grow properly and (2) drug efficacy is well observed.

[0174] As shown in Table 6 below, the ratio for cancer organoids was fixed to 1, and the ratio for CD8+ T cells and Treg was set to 0 to 3. Herein, the number of organoids or cells was set to 1 for 5.0?10.sup.3.

TABLE-US-00006 TABLE 6 Group Cancer Organoid CD8+ T cell Treg 1 1 3 0 2 0 3 3 1 0.5 4 1 5 3 6 3 0.5 7 1 8 3

[0175] Cancer organoids were prepared using stem cells isolated from tumor tissue of colorectal cancer or lung cancer patients. The isolated stem cells were mixed with ECM (extracellular matrix) to grow into a 3D structure to make a dome, and then organoids were formed and used through culture. CD8+ T cells and Tregs were obtained from blood obtained from colorectal cancer or lung cancer patients. Immune cells present in the blood were differentiated, and CD8+ and Treg cells were isolated from the differentiated immune cells using a MACS (magnetic activated cell sorting) method.

Example 4.1. Conditions for Proper Growth of Cancer Organoids

[0176] A mixing ratio of cancer organoids, CD8+ T cells and Tregs, which allows cancer organoids to grow properly even when co-cultured with immune cells (CD8+ T cells and Tregs), was drawn.

[0177] Specifically, after co-culturing at the mixing ratio according to Table 6 above, the area of cancer organoids was measured immediately after the start of the co-culture (0 h), after 24 hours (24 h), after 48 hours (48 h) and after 72 hours (72 h), respectively, and the growth rate was analyzed by the following formula.

[00004]${}^{*}\mathrm{Growth}\mathrm{rate}\mathrm{of}\mathrm{cancer}\mathrm{organoids}\left(\%\right)=\left(\mathrm{cancer}\mathrm{organoid}\mathrm{area}\mathrm{after}\mathrm{co}-\mathrm{culture}\mathrm{for}24,48,\mathrm{or}72\mathrm{hours}\right)/\left(\mathrm{cancer}\mathrm{organoid}\mathrm{area}\mathrm{immediately}\mathrm{after}\mathrm{the}\mathrm{start}\mathrm{of}\mathrm{the}\mathrm{co}-\mathrm{culture}\right)?100$

[0178] As a result, as shown in FIGS. 15 and 16, the growth of colorectal cancer or lung cancer organoids was not inhibited and grew appropriately in all mixing ratios, and no significant difference was found between each mixing ratio.

Example 4.2. Conditions in which Drug Efficacy is High

[0179] A mixture ratio of cancer organoids, CD8+ T cells and Tregs, which allows the efficacy of anticancer drugs targeting CD8+ T cells and anticancer drugs targeting Tregs to be high, was drawn.

[0180] Specifically, for the co-cultures of cancer organoids, CD8+ T cells and Tregs according to the mixing ratio in Table 6 above, those not treated with the drug were set as the control group (hereinafter referred to as drug-untreated group), and those treated with an atezolizumab targeting CD8+ T cells or an anti-TIGIT antibody targeting CD8+ T cells or Tregs as an exemplary drug were set as the experimental group (hereinafter referred to as drug-treated group). After co-culturing them for 72 hours, the growth rate was measured after 72 hours of drug treatment (72 h), and the death rate was analyzed by the following method through the measured growth rate.

[00005]$\text{Cancer organoid death rate (\%) = [1 - (growth rate of cancer organoids in the drug-treated}{\mathrm{group}}^{*}\text{)/(growth rate of cancer organoids in the drug-untreated}{\mathrm{group}}^{**}\text{)] ? 100}$$\text{Growth rate of cancer organoids in the drug-treated group (\%) = (cancer organoid area after 72 hours of drug treatment)/(cancer organoid area immediately after the start of the co-culture (0 h) ? 100}$$\text{Growth rate of cancer organoids in the drug-untreated group (\%) = (cancer organoid area after 72 hours without drug treatment)/(cancer organoid area immediately after the start of the co-culture (0 h) ? 100}$

[0181] As a result, as shown in FIG. 17, when only the colorectal cancer organoids and CD8+ T cells were co-cultured at a mixing ratio of 1:3, except for Tregs among the cancer organoids, CD8+ T cells and Tregs, the death rate of cancer organoids by atezolizumab is as high as about 26%, but the death rate of cancer organoids by anti-TIGIT antibody is just about 5%, making it impossible to accurately determine the efficacy of the drug. In addition, when only the colorectal cancer organoids and Tregs were co-cultured at a mixing ratio of 1:3 except for CD8+ T cells among the cancer organoids, CD8+ T cells and Tregs, the death rate of cancer organoids by atezolizumab and anti-TIGIT antibody was remarkably low as about 2%, confirming that the efficacy of the drug could not be accurately determined.

[0182] In addition, as shown in FIG. 18, when colorectal cancer organoids and CD8+ T cells were co-cultured at a mixing ratio of 1:1, the death rate of cancer organoids by the anti-TIGIT antibody increased as the Treg ratio increased, but the maximum level was just about 6%, and the death rate of cancer organoids by atezolizumab was just about 10% at maximum, confirming that the efficacy of the drug could not be accurately determined.

[0183] On the other hand, as shown in FIG. 19, when colorectal cancer organoids and CD8+ T cells was co-cultured at a mixing ratio of 1:3, the death rate of colorectal cancer organoids by the anti-TIGIT antibody increased as the Tregs ratio increased, with a maximum level of about 10%, and the death rate of cancer organoids by atezolizumab was also high as about 25%. That is, it was confirmed that the efficacy of all drugs such as atezolizumab and the anti-TIGIT antibody was higher in the above mixing ratio than in other mixing ratios, and the efficacy increased by two times or more.

[0184] In addition, as shown in FIG. 20, when only the lung cancer organoids and CD8+ T cells were co-cultured at a mixing ratio of 1:3 except for Tregs among the cancer organoids, CD8+ T cells and Tregs, the death rate of cancer organoids by atezolizumab is as high as about 27%, but the death rate of cancer organoids by anti-TIGIT antibody is only about 6%, confirming that the efficacy of the drug could not be accurately determined. In addition, when only the lung cancer organoids and Tregs were co-cultured at a mixing ratio of 1:3 except for CD8+ T cells among the lung cancer organoids, CD8+ T cells and Tregs, the death rate of cancer organoids by atezolizumab and anti-TIGIT antibody was remarkably low at about 1%, confirming that the efficacy of the drug could not be accurately determined.

[0185] In addition, as shown in FIG. 21, when lung cancer organoids and CD8+ T cells were co-cultured at a ratio of 1:1, the death rate of cancer organoids by the anti-TIGIT antibody increased as the Treg mixing ratio increased, but the maximum level is just about 6%, and the death rate of cancer organoids by atezolizumab was just about 10% at maximum, confirming that the efficacy of the drug could not be accurately determined.

[0186] On the other hand, as shown in FIG. 22, when lung cancer organoids and CD8+ T cells was co-cultured at a mixing ratio of 1:3, the death rate of colorectal cancer organoids by the anti-TIGIT antibody increased as the Tregs mixing ratio increased, with a maximum level of about 10%, and the death rate of cancer organoids by atezolizumab was also high at about 25%. That is, it was confirmed that the efficacy of all drugs such as atezolizumab and the anti-TIGIT antibody was higher in the above mixing ratio than in other mixing ratios, and the efficacy increased by two times or more.

[0187] The above results suggest that setting the co-culture ratio of cancer organoids, CD8+ T cells and Treg to 1:3:0.5 to 3 is the most optimal ratio for an anticancer drug efficacy evaluation or screening system.

Example 5. Anticancer Drug Efficacy Evaluation and Screening System Comprising Cancer Organoids, Cytotoxic T Cells and Dendritic Cells

[0188] The inventors have established an in vitro efficacy evaluation and screening system that can accurately predict the efficacy of a drug when administered to a subject, by similarly reproducing the environment in which cancer exists in the body.

[0189] Specifically, in order to establish a co-culture system in which cancer organoids, cytotoxic T cells (hereinafter referred to as CD8+ T cells) and dendritic cells (hereinafter referred to as DC) coexist, the inventors set a mixing ratio satisfying co-culture conditions under which (1) cancer organoids grow properly and (2) drug efficacy is well observed. As shown in Table 7 below, the ratio for cancer organoids was fixed to 1, the ratio for CD8+ T cells was set to 0 to 3 and the ratio for DC was set to 0.5 to 5. Herein, the number of organoids or cells was set to 1 for 5.0?10.sup.3.

TABLE-US-00007 TABLE 7 Group Cancer Organoid CD8+ T cell DC 1 1 0.5 0.5 2 1 3 5 4 1 0.5 5 1 6 5 7 3 0.5 8 1 9 5

[0190] Cancer organoids were prepared to have a 3D structure by isolating colorectal cancer or lung cancer tissues obtained from patients into single cells and culturing them together with ECM (extracellular matrix). The formed cancer organoids were confirmed to be tumors through pathological analysis, and used after being isolated into single cells using Tryp-LE.

[0191] CD8+ T cells isolated from PBMCs were used. PBMCs were isolated from blood obtained from donors by using Ficoll, and CD8+ T cells were isolated through MACS after a T cell activation process.

[0192] DCs differentiated from iPSCs were used, and DCs were differentiated from the H9 cell line using a general method known in the art.

Example 5.1. Co-Culture Ratio of Cancer Organoids, CD8+ T Cells and DCs

[0193] Specifically, for the co-cultures of cancer organoids, CD8+ T cells and DCs according to the mixing ratio in Table 7 above, those not treated with the drug were set as the control group (hereinafter referred to as drug-untreated group), and those treated with an atezolizumab targeting CD8+ T cells as an exemplary drug were set as the experimental group (hereinafter referred to as drug-treated group). Drugs were treated immediately after the start of co-culture, and the growth rate of cancer organoids was measured immediately after the start of the co-culture (0 h), after 24 hours (24 h), after 48 hours (48 h) and after 72 hours (72 h), respectively. The death rate was analyzed by the following method through the measured growth rate.

[00006]$\text{Cancer organoid death rate (\%) = [1 - (growth rate of cancer organoids in the drug-treated}{\mathrm{group}}^{*}\text{)/(growth rate of cancer organoids in the drug-untreated}{\mathrm{group}}^{**}\text{)] ? 100}$$\text{Growth rate of cancer organoids in the drug-treated group (\%) = (cancer organoid area after 24, 48 or 72 hours of drug treatment)/(cancer organoid area immediately after the start of the co-culture (0 h) ? 100}$$\text{Growth rate of cancer organoids in the drug-untreated group (\%) = (cancer organoid area after 24, 48 or 72 hours without drug treatment)/(cancer organoid area immediately after the start of the co-culture (0 h) ? 100}$

[0194] As a result, as shown in FIGS. 23 to 25, the growth of colorectal cancer organoids was not inhibited and grew appropriately in all mixing ratios when the drug was not treated, and no significant difference was found between each mixing ratio.

[0195] The drug did not significantly inhibit the growth of cancer organoids when co-culturing colorectal cancer organoids, CD8+ T cells and DCs at a mixing ratio of 1:0.5:0.5 to 5 (see FIG. 23), but the drug inhibited the growth of cancer organoids when co-culturing colorectal cancer organoids, CD8+ T cells and DCs at a mixing ratio of 1:1 to 3:0.5 to 5 (see FIGS. 24 and 25).

[0196] In particular, as shown in FIG. 26, when colorectal cancer organoids and CD8+ T cells were co-cultured at a mixing ratio of 1:0.5, the death rate of cancer organoids by atezolizumab increased as the DC mixing ratio increased. However, the maximum level was just about 5%, and it was confirmed that the efficacy of the drug could not be accurately determined.

[0197] In addition, as shown in FIG. 27, when colorectal cancer organoids and CD8+ T cells are co-cultured at a mixing ratio of 1:1, the maximum death rate of cancer organoids by atezolizumab is just about 10%, confirming that the efficacy of the drug could not be accurately determined.

[0198] On the other hand, as shown in FIG. 28, when colorectal cancer organoids and CD8+ T cells are co-cultured at a mixing ratio of 1:3, the death rate of cancer organoids by atezolizumab was as high as about 20% to 25% regardless of the mixing ratio of DCs. That is, it was confirmed that the efficacy of the drug was higher in the mixing ratio than in other mixing ratios, and the efficacy increased by 2.5 to 5 times or more.

[0199] In addition, as shown in FIGS. 29 to 31, the growth of lung cancer organoids was not inhibited and grew appropriately at all mixing ratios when the drug was not treated, and no significant difference was found between each mixing ratio.

[0200] The drug did not significantly inhibit the growth of cancer organoids when co-culturing lung cancer organoids, CD8+ T cells and DCs at a mixing ratio of 1:0.5:0.5 to 5 or 1:1:0.5 to 1 (see FIGS. 29 and 30), but the drug inhibited the growth of cancer organoids when co-culturing lung cancer organoids, CD8+ T cells and DCs at a mixing ratio of 1:1:5 or 1:3:0.5 to 5 (see FIGS. 30 and 31).

[0201] In particular, as shown in FIG. 32, when lung cancer organoids and CD8+ T cells were co-cultured at a mixing ratio of 1:0.5, the death rate of cancer organoids by atezolizumab increased as the DC mixing ratio increased. However, the maximum level was just about 5%, and it was confirmed that the efficacy of the drug could not be accurately determined.

[0202] In addition, as shown in FIG. 33, when lung cancer organoids and CD8+ T cells are co-cultured at a mixing ratio of 1:1, the maximum death rate of cancer organoids by atezolizumab is just about 10%, confirming that the efficacy of the drug could not be accurately determined.

[0203] On the other hand, as shown in FIG. 34, when lung cancer organoids and CD8+ T cells was co-cultured at a mixing ratio of 1:3, the death rate of cancer organoids by atezolizumab was as high as about 28% to 40% regardless of the mixing ratio of DCs. That is, it was confirmed that the efficacy of the drug was higher in the mixing ratio than in other mixing ratios, and the efficacy increased by 5 to 8 times or more.

[0204] The above results suggest that setting the co-culture ratio of cancer organoids, CD8+ T cells and DCs to 1:3:0.5 to 5 is the most optimal ratio for an anticancer drug efficacy evaluation or screening system.