New application of PBMC (peripheral blood mononuclear cell)-derived cytotoxic T cell

Abstract

The present invention relates to: a method for evaluating the efficacy of an anti-cancer drug, a method for screening an anti-cancer drug, a system for evaluating the efficacy of an anti-cancer drug, or a system for screening an anti-cancer drug, using a mixture of peripheral blood mononuclear cells (PBMC) and a cancer organoid. Specifically, the present invention relates to: a method for evaluating the efficacy of an anti-cancer drug, a method for screening an anti-cancer drug, a system for evaluating the efficacy of an anti-cancer drug, or a system for screening an anti-cancer drug, using a mixture of cytotoxic T cells differentiated from PBMC and cancer organoids. According to the method for evaluating the efficacy of an anti-cancer drug, the screening method, the efficacy evaluation system, or the screening system of the present invention, a tumor microenvironment, i.e. The form in which cancer cells are present in a living body, can be very closely reproduced. Furthermore, since PBMCs derived from normal individuals are used and differentiated into cancer patient-specific cytotoxic T cells, the burden of collecting biological samples such as blood and tissue samples from cancer patients can be reduced, and the efficacy of an anti-cancer drug can be evaluated with high accuracy.

Description

Technical Field

[0001] The present invention relates to a method for evaluating the efficacy of an anticancer drug using a mixture of peripheral blood mononuclear cells (PBMC) and cancer organoids, a method for screening an anticancer drug, a system for evaluating the efficacy of an anticancer drug, or a system for screening an anticancer drug. Specifically, the present invention relates to a method for evaluating the efficacy of an anticancer drug using a mixture of cytotoxic T cells differentiated from PBMCs and cancer organoids, a method for screening an anticancer drug, a system for evaluating the efficacy of an anticancer drug, or a system for screening an anticancer drug. Background Art

[0002] Organoids are organ analogs prepared by culturing cells derived from tissues or organs in three dimensions. Organoids have the advantages of long-term culture, low-temperature storage, and easy operation and observation. In addition, organoids are used as experimental models that can study physiological phenomena at a higher level than cells, by reproducing the hierarchical structure and tissue structure of cells that can be observed in vivo, and maintaining the original characteristics of cells without immortalization.

[0003] Due to these characteristics, organoids can evaluate drugs more accurately than immortalized cell lines with altered intrinsic properties or animal models with structures different from the human body. In particular, since patient-derived tissue is used, there is an advantage that the efficacy and safety of drugs can be examined before human clinical trials. However, cancer organoids previously developed as drug evaluation models have the limitation of not reflecting the tumor microenvironment, so there is a need to develop organoids with various tumor microenvironments and use them as drug evaluation models.

[0004] The tumor microenvironment is a cellular environment where cancer cells are surrounded by blood vessels, immune cells, fibroblasts, lymphocytes, signaling molecules, and extracellular matrix (ECM). In the tumor microenvironment, cancer cells affect the microenvironment by inducing the release of extracellular signals, promoting cancer cell angiogenesis, and peripheral immune tolerance. Therefore, cancer cells change the microenvironment, or the microenvironment affects the growth or metastasis of cancer cells. In particular, the tumor microenvironment associated with lymphocytes not only affects the occurrence and metastasis of cancer, but also affects the therapeutic response of anticancer drugs.

[0005] For example, CD8 + T cells differentiate into cytotoxic T cells, migrate into the tumor microenvironment, and play important roles in tumor immunity, such as killing tumor cells. Summary of the invention

[0006]

Technical issues

[0007] The inventors have conducted various studies to develop cancer organoids with a tumor microenvironment and use them as a platform for drug evaluation or drug screening. Thus, a system for co-culturing cancer organoids using cytotoxic T cells among various cells constituting the tumor microenvironment was established.

[0008] In particular, cytotoxic T cells are obtained by differentiating PBMCs from normal individuals into cancer patient-specific cytotoxic T cells, thereby reducing the burden of collecting biological samples such as blood and tissues from cancer patients. The present invention is accomplished by experimentally demonstrating that cytotoxic T cells can maintain survival even after long-term co-culture, thereby enabling long-term and high-precision evaluation of drug efficacy.

[0009]

Technical solution

[0010] Each description and embodiment disclosed in the present invention may also be applicable to other descriptions and embodiments. That is to say, all combinations of various elements disclosed in the present invention fall within the scope of the present invention. In addition, the scope of the present invention should not be considered as being limited by the following specific description.

[0011] In addition, terms not specifically defined in this specification should be understood to have meanings commonly used in the technical field to which the present invention belongs. In addition, unless specifically defined otherwise by the context, the singular includes the plural and the plural includes the singular.

[0012] One aspect of the present invention provides a method for evaluating the efficacy of an anticancer drug, the method comprising:

[0013] (a) Peripheral blood mononuclear cells (PBMCs) were mixed and co-cultured with cancer organoids to obtain cytotoxic T cells;

[0014] (b) mixing and co-culturing the cytotoxic T cells obtained in step (a) with the cancer organoids;

[0015] (c) treating the mixture of cytotoxic T cells and cancer organoids of step (b) with an anti-cancer drug; and

[0016] (d) if the group treated with the anti-cancer drug in step (c) shows increased growth inhibition or death of the cancer organoids compared to a group not treated with the anti-cancer drug or a positive control group, determining that the anti-cancer drug has anti-cancer efficacy.

[0017] Specifically, step (a) is to mix and co-culture PBMC with cancer organoids, differentiate PBMC into cytotoxic T cells, and obtain cytotoxic T cells. In addition, step (a) is a step of obtaining cytotoxic T cells by differentiating PBMCs derived from normal individuals into cancer patient-specific cytotoxic T cells. In addition, step (a) is a step in which cancer patient-specific cytotoxic T cells differentiated from PBMCs derived from normal individuals learn to recognize cancer cells or cancer organoids of cancer patients as their own cells.

[0018] The term "PBMC" used herein refers to "peripheral blood mononuclear cells" and refers to a cell population consisting of monocytes and lymphocytes (such as T cells, B cells, and NK cells). It is known that in the PBMC population, naive T cells account for the majority, accounting for about 45% to 70%, and helper T cells (CD4 + T cells) accounted for 25% to 60%, and cytotoxic T cells (CD8 + The proportion of T cells is 5% to 30%, the proportion of B cells is 5% to 10%, the proportion of NK cells is 10% to 30%, the proportion of monocytes is 5% to 10%, and the proportion of dendritic cells is 1% to 2%.

[0019] The PBMCs according to the present invention can be derived from individuals with cancer (cancer patients), normal individuals without cancer, or normal individuals completely cured after cancer, preferably normal individuals without cancer or normal individuals completely cured after cancer, but are not limited thereto. In this specification, PBMCs can be used interchangeably with "pre-differentiated T cells", and the term "derived" can be used interchangeably with "isolated" or "obtained".

[0020] The term "individual" as used herein includes all individuals who have never had cancer, individuals who may have cancer, individuals who have already had cancer, or individuals who have been completely cured after having cancer, and may include, but are not limited to, humans or any non-human animals. Non-human animals may be vertebrates, such as primates, dogs, cows, horses, pigs, rodents (e.g., mice, rats, hamsters, guinea pigs), etc. In this specification, the term "individual" may be used interchangeably with "subject" or "patient".

[0021] The term "biological sample" used herein may be tissue, cells, whole blood, serum, plasma or cell culture supernatant, preferably whole blood, but not limited thereto.

[0022] The term "cytotoxic T cell" as used herein refers to a lymphocyte that performs antigen-specific acquired immunity or adaptive immunity. Cytotoxic T cells kill cancer cells or cells infected by pathogens such as viruses and bacteria, and produce cytokines such as TNF-α and IFN-γ.

[0023] The cytotoxic T cells according to the present invention can be differentiated from PBMC. In this specification, cytotoxic T cells can be referred to as "differentiated T cells", "differentiated T cells" or "CD8 + "T cells" are used interchangeably.

[0024] The term "organoid" used herein refers to a cell mass having a three-dimensional structure and refers to an organ analog prepared by three-dimensionally culturing cells separated from an organ or tissue. Since an organoid includes a specific cell group constituting a tissue or organ and is organized in a structurally similar form to an actual tissue or organ, the specific function and shape of each tissue or organ can be reproduced.

[0025] The term "cancer organoid" used herein refers to an organoid prepared from cells isolated from a tumor (tumor cells or cancer cells). The cancer organoid according to the present invention can be derived from an individual suffering from cancer (cancer patient), but is not limited thereto.

[0026] In the present invention, the PBMCs and cancer organoids in step (a) can be mixed at a cell number ratio of 15:1 to 25:1, preferably 20:1. In addition, the co-culture in step (a) can be performed for 1 day to 42 days. When PBMCs and cancer organoids are mixed in a certain proportion and co-cultured for a period of time, PBMCs derived from normal individuals will differentiate into cancer patient-specific cytotoxic T cells, and the differentiation rate is improved, thereby reducing the burden of collecting biological samples such as blood and tissues from cancer patients.

[0027] In addition, step (b) is a step of co-culturing the cytotoxic T cells obtained in step (a) (i.e., cytotoxic T cells differentiated from PBMC) with cancer organoids, which reproduces the in vivo tumor microenvironment in vitro. In addition, step (b) is a step of attacking and killing cancer cells or cancer organoids of cancer patients that are recognized as self-cells by cancer patient-specific cytotoxic T cells differentiated from PBMCs derived from normal individuals.

[0028] The cancer organoids used in step (a) and the cancer organoids used in step (b) may be derived from the same cancer patient.

[0029] In addition, step (a) and step (b) can be performed continuously in the same culture space. For example, step (a) and step (b) can be performed sequentially in the same culture plate.

[0030] In the present invention, the cytotoxic T cells and cancer organoids in step (b) can be mixed at a cell number ratio of 15:1 to 25:1, preferably 20:1. Cytotoxic T cells will not die even if they are cultured for a long time, so the tumor microenvironment in which cancer cells exist in the body can be simulated for a long time. Therefore, the efficacy of anticancer drugs can be evaluated for a long time and with high accuracy.

[0031] In addition, step (c) is a step of treating the mixture of the cytotoxic T cells and cancer organoids of step (b) with an anticancer drug. Specifically, step (c) is a step of treating the mixture of the cytotoxic T cells obtained in step (a) (i.e., cytotoxic T cells differentiated from PBMC) and cancer organoids with an anticancer drug.

[0032] The term "anti-cancer drug" as used herein refers to a substance that has a preventive or therapeutic effect on cancer, specifically a substance that can kill cancer cells, cancer organoids or tumors, or inhibit their growth.

[0033] In the present specification, "anticancer drug" may be used interchangeably with "drug", and "treatment" may be used interchangeably with "addition" or "application".

[0034] Specifically, the anticancer drug can target PBMC, cytotoxic T cells differentiated from PBMC, cytotoxic T cells and / or cancer organoids. For example, the anticancer drug can target only cancer organoids, only cytotoxic T cells, or both cancer organoids and cytotoxic T cells. For example, the anticancer drug targeting cancer organoids can be atezolizumab, while the anticancer drug targeting cytotoxic T cells can be pembrolizumab.

[0035] Preferably, the anticancer drug includes, but is not limited to, compounds, proteins, fusion proteins, compound-protein conjugates, drug-protein conjugates, antibodies, compound-antibody conjugates, drug-antibody conjugates, amino acids, peptides, viruses, carbohydrates, lipids, nucleic acids, extracts and fractions.

[0036] For example, the anticancer drug may include compounds, peptides, peptide mimetics, fusion proteins, antibodies, aptamers, and antibody-drug conjugates (ADCs), but are not limited thereto. Preferably, the anticancer drug may be an antibody or an immuno-oncology drug.

[0037] The term "antibody" used herein includes monoclonal antibodies, polyclonal antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, humanized antibodies and human antibodies, and in addition to novel antibodies, also includes antibodies known in the art or commercially available. It includes functional fragments of antibody molecules and full-length forms consisting of two heavy chains and two light chains. The functional fragment of an antibody molecule refers to a fragment that has at least antigen binding function, and may include but is not limited to Fab, F(ab'), F(ab')2, Fv, etc. The term "peptide mimetic" used herein refers to a peptide or modified peptide that biomimics the active ligand of a hormone, cytokine, enzyme substrate, virus or other biological molecule. The term "aptamer" used herein refers to a single-stranded nucleic acid (such as DNA, RNA or modified nucleic acid) that has a stable tertiary structure and binds to a target molecule with high affinity and specificity.

[0038] As another example, anticancer drugs may include, but are not limited to, antisense nucleic acids, siRNA, shRNA, miRNA, and ribozymes that bind to DNA or mRNA in a complementary manner.

[0039] The term "antisense nucleic acid" used herein refers to DNA, RNA or its fragment or derivative containing a nucleic acid sequence complementary to a specific mRNA sequence, which plays a role in inhibiting the translation of mRNA into protein by complementary binding or hybridization with the mRNA sequence. The term "siRNA (small interfering RNA)" used herein refers to a short double-stranded RNA that can induce RNA interference (RNAi) by cutting a specific mRNA. siRNA includes a sense RNA chain with a homologous sequence to the mRNA of the target gene and an antisense RNA chain with a complementary sequence thereto. Since siRNA can inhibit the expression of the target gene, it is used in gene knockout methods or gene therapy methods. The term "shRNA (short hairpin RNA)" used herein, as a single-stranded RNA, refers to an RNA composed of a stem portion forming a double-stranded portion by hydrogen bonds and a ring portion having a ring shape. It can be processed and converted into siRNA by proteins such as Dicer, and plays the same function as siRNA. The term "miRNA (micro RNA)" used herein refers to a non-coding RNA of 21nt to 23nt, which regulates post-transcriptional gene expression by promoting target RNA degradation or inhibiting target RNA translation. The term "ribozyme" used herein refers to an RNA molecule with enzyme-like functions that can recognize a specific nucleotide sequence and cut the sequence by itself. The ribozyme consists of a region that specifically binds to a complementary nucleotide sequence of a target messenger RNA chain and a region that cuts the target RNA.

[0040] The anticancer drugs to be evaluated or screened according to the method of the present invention can be, for example, for preventing or treating biliary tract cancer, gastric 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, kidney cancer, sarcoma, prostate cancer, urethral cancer, bladder cancer, hematological malignancies, leukemia, lymphoma and fibroadenoma, but are not limited thereto.

[0041] Furthermore, step (d) is a step of determining that the anticancer drug has anticancer efficacy when the growth inhibition or death of cancer organoids is increased in the group treated with the anticancer drug in step (c) compared to the positive control group or the group not treated with the anticancer drug.

[0042] The term "positive control group" used herein refers to a group treated with a substance used as an anticancer drug in the art.

[0043] Specifically, the growth inhibition or death of the cancer organoid can be confirmed by an increase or decrease in the volume, area, or number of cells of the cancer organoid. When the volume, area, or number of cells of the cancer organoid decreases, the growth inhibition or increased death of the cancer organoid can be determined. The increase or decrease in the volume, area, or number of cells of the cancer organoid can be confirmed by methods known in the art, such as, but not limited to, analysis methods such as high content screening (HCS) or flow cytometry.

[0044] Another aspect of the present invention provides a method for screening anticancer drugs, the method comprising:

[0045] (a) PBMCs were mixed and co-cultured with cancer organoids to obtain cytotoxic T cells;

[0046] (b) mixing and co-culturing the cytotoxic T cells obtained in step (a) with the cancer organoids;

[0047] (c) treating the mixture of cytotoxic T cells and cancer organoids of step (b) with an anti-cancer drug candidate; and

[0048] (d) if the growth inhibition or death of the cancer organoid is increased in the group treated with the anticancer drug candidate in step (c) compared to the group not treated with the anticancer drug candidate, then determining that the anticancer drug candidate is an anticancer drug.

[0049] In the method for screening anticancer drugs according to the present invention, unless explicitly stated otherwise, the relevant terms should be understood to have the same meanings as the above terms.

[0050] Specifically, step (a) is a step of mixing and co-culturing PBMC with cancer organoids to differentiate PBMC into cytotoxic T cells to obtain cytotoxic T cells. In addition, step (a) is a step of obtaining cytotoxic T cells by differentiating PBMCs derived from normal individuals into cancer patient-specific cytotoxic T cells. In addition, step (a) is a step in which cancer patient-specific cytotoxic T cells differentiated from PBMCs derived from normal individuals learn to recognize cancer cells or cancer organoids of cancer patients as their own cells.

[0051] In the present invention, the PBMCs and cancer organoids in step (a) can be mixed at a cell number ratio of 15:1 to 25:1, preferably 20:1. In addition, the co-culture in step (a) can be performed for 1 day to 42 days. When PBMCs and cancer organoids are mixed in a certain proportion and co-cultured for a period of time, PBMCs derived from normal individuals will differentiate into cancer patient-specific cytotoxic T cells, and the differentiation rate is improved, thereby reducing the burden of collecting biological samples such as blood and tissues from cancer patients.

[0052] In addition, step (b) is a step of co-culturing the cytotoxic T cells obtained in step (a) (i.e., cytotoxic T cells differentiated from PBMC) with cancer organoids, which reproduces the in vivo tumor microenvironment in vitro. In addition, step (b) is a step of attacking and killing cancer cells or cancer organoids of cancer patients that are recognized as self-cells by cancer patient-specific cytotoxic T cells differentiated from PBMCs derived from normal individuals.

[0053] The cancer organoids used in step (a) and the cancer organoids used in step (b) may be derived from the same cancer patient.

[0054] In addition, step (a) and step (b) can be performed continuously in the same culture space. For example, step (a) and step (b) can be performed sequentially in the same culture plate.

[0055] In the present invention, the PBMC and the cancer organoid in step (b) can be mixed at a cell number ratio of 15:1 to 25:1, preferably 20:1. Cytotoxic T cells will not die even if they are cultured for a long time, so the tumor microenvironment in which cancer cells exist in the body can be simulated for a long time. Therefore, the efficacy of anticancer drugs can be evaluated for a long time and with high accuracy.

[0056] In addition, step (c) is a step of treating the mixture of the cytotoxic T cells and cancer organoids of step (b) with an anticancer drug candidate. Specifically, step (c) is a step of treating the mixture of the cytotoxic T cells obtained in step (a) (i.e., cytotoxic T cells differentiated from PBMCs) and cancer organoids with an anticancer drug candidate.

[0057] The term "anti-cancer drug candidate" as used herein refers to a substance that is expected to have a preventive or therapeutic effect on cancer, specifically a substance that is expected to kill cancer cells, cancer organoids or tumors, or inhibit their growth.

[0058] In the present specification, "anti-cancer drug candidate" may be used interchangeably with "drug", and "treatment" may be used interchangeably with "addition" or "administration".

[0059] Preferably, the anti-cancer drug candidates include, but are not limited to, compounds, proteins, fusion proteins, compound-protein conjugates, drug-protein conjugates, antibodies, compound-antibody conjugates, drug-antibody conjugates, amino acids, peptides, viruses, carbohydrates, lipids, nucleic acids, extracts and fractions.

[0060] For example, the anticancer drug candidate may include compounds, peptides, peptide mimetics, fusion proteins, antibodies, aptamers, and antibody-drug conjugates (ADCs), but is not limited thereto. Preferably, it may be an antibody or an immuno-oncology drug.

[0061] Furthermore, step (d) is a step of determining that the anticancer drug candidate is an anticancer drug if the growth inhibition or death of cancer organoids is increased in the group treated with the anticancer drug candidate in step (c) compared to the group not treated with the anticancer drug candidate.

[0062] Specifically, the growth inhibition or death of the cancer organoid can be confirmed by an increase or decrease in the volume, area, or number of cells of the cancer organoid. When the volume, area, or number of cells of the cancer organoid decreases, the growth inhibition or increased death of the cancer organoid can be determined. The increase or decrease in the volume, area, or number of cells of the cancer organoid can be confirmed by methods known in the art, such as, but not limited to, analysis methods such as high content screening (HCS) or flow cytometry.

[0063] Another aspect of the present invention is an anticancer drug efficacy evaluation system to use a method for evaluating the efficacy of an anticancer drug, and provides an anticancer drug efficacy evaluation system comprising PBMCs and cancer organoids.

[0064] In the anticancer drug efficacy evaluation system according to the present invention, unless otherwise explicitly stated, the relevant terms should be understood to have the same meanings as the above terms.

[0065] The anticancer drug efficacy evaluation system according to the present invention includes PBMC and cancer organoids. Although PBMCs are derived from normal individuals, they show the characteristics of differentiating into cancer patient-specific cytotoxic T cells. Therefore, the burden of collecting biological samples such as blood and tissues from cancer patients can be reduced. In addition, the cytotoxic T cells differentiated from PBMCs will not die even if they are cultured for a long time with cancer organoids, so they can simulate the tumor microenvironment in which cancer cells exist in the body for a long time. Therefore, the efficacy of anticancer drugs when administered in vivo can be accurately predicted, and the efficacy of anticancer drugs can be evaluated with high accuracy over a long period of time. Therefore, the anticancer drug efficacy evaluation system can be used to evaluate the efficacy of anticancer drugs.

[0066] Another aspect of the present invention is an anticancer drug screening system and a method for using the anticancer drug screening system, and provides an anticancer drug screening system comprising PBMC and cancer organoids.

[0067] In the anticancer drug screening system according to the present invention, unless otherwise explicitly stated, the relevant terms should be understood to have the same meanings as the above terms.

[0068] The anticancer drug screening system according to the present invention includes PBMC and cancer organoids. Although PBMCs are derived from normal individuals, they show the characteristics of differentiating into cancer patient-specific cytotoxic T cells. Therefore, the burden of collecting biological samples such as blood and tissues from cancer patients can be reduced. In addition, the cytotoxic T cells differentiated from PBMCs will not die even if they are cultured for a long time with cancer organoids, so they can simulate the tumor microenvironment in which cancer cells exist in the body for a long time. Therefore, the efficacy of anticancer drugs when applied in vivo can be accurately predicted, and the efficacy of anticancer drugs can be evaluated with high accuracy over a long period of time. Therefore, the anticancer drug screening system can be used to screen anticancer drugs.

[0069] The method, screening method, efficacy evaluation system or screening system for evaluating the efficacy of anticancer drugs according to the present invention can closely reproduce the tumor microenvironment, that is, the form in which cancer cells exist in an organism. In addition, since PBMCs derived from normal individuals are used and differentiated into cancer patient-specific cytotoxic T cells, the burden of collecting biological samples such as blood and tissue samples from cancer patients can be reduced, and the efficacy of anticancer drugs can be evaluated with high accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 The FACS analysis results of peripheral blood mononuclear cells (PBMCs) co-cultured with cancer organoids according to the first step are shown, wherein A is a chromatogram under matching conditions, B is a chromatogram under non-matching conditions, and C is a chromatogram under normal conditions. + 、CD3+ After sorting, CD8 + Carry out sorting.

[0071] Figure 2 The growth rate of cancer organoids after treatment with atezolizumab as a drug on day 14 after the start of co-culture in the first step is shown. The control group refers to an untreated group, and the experimental group refers to a group treated with 10 nM atezolizumab.

[0072] Figure 3 The growth rate of cancer organoids after treatment with atezolizumab as a drug on day 28 after the start of co-culture in the first step is shown. The control group refers to an untreated group, and the experimental group refers to a group treated with 10 nM atezolizumab.

[0073] Figure 4 The growth rate of cancer organoids after treatment with atezolizumab as a drug on day 42 after the start of co-culture in the first step is shown. The control group refers to an untreated group, and the experimental group refers to a group treated with 10 nM atezolizumab.

[0074] Figure 5 The overall efficacy of Atezolizumab following treatment with Atezolizumab on day 14, day 28 or day 42 after the start of the first step of co-cultivation is shown.

[0075] Figure 6a The overall efficacy of Atezolizumab after treatment with Atezolizumab on day 14, 28 or 42 after the start of the co-culture in the first step in the control group and experimental groups 1 to 3, respectively, is shown.

[0076] Figure 6b The overall efficacy of Atezolizumab after treatment with Atezolizumab on day 14, 28 or 42 after the start of the co-culture in the first step in the control group and experimental groups 4 to 6, respectively, is shown. DETAILED DESCRIPTION

[0077] [Example]

[0078] The present invention will be described in more detail below by way of examples. These examples are used to explain the present invention in more detail, and the scope of the present invention is not limited by these examples.

[0079] Example 1 Establishment of an anticancer drug efficacy evaluation and screening system

[0080] By similarly reproducing the tumor microenvironment in which cancer exists in vivo, the inventors have established an in vitro efficacy evaluation and screening system that can accurately predict the efficacy of a drug when it is administered to a subject.

[0081] Specifically, a co-culture system in which cancer organoids coexist with T cells is used. Cancer organoids and T cells can be derived from different individuals, such as individuals with cancer (cancer patients), normal individuals without cancer, or normal individuals cured after suffering from cancer. Depending on the source, the co-culture system can be divided into matching conditions, non-matching conditions, and normal conditions. Matching conditions use cancer organoids and T cells derived from the same cancer patient, such as cancer organoids derived from cancer patient A and T cells derived from cancer patient A. Non-matching conditions use cancer organoids and T cells derived from different cancer patients, such as cancer organoids derived from cancer patient A and T cells derived from cancer patient B. Normal conditions use cancer organoids derived from cancer patients and T cells derived from normal individuals, such as cancer organoids derived from cancer patient A and T cells derived from normal individual C.

[0082] The advantage of matched and non-matched conditions is that they can more closely reproduce the in vivo tumor microenvironment in vitro, because both cancer organoids and T cells are derived from individuals who already have cancer. However, in order to isolate T cells, a process of collecting blood samples from individuals is required, and collecting blood samples from cancer patients will have a negative impact on the improvement of cancer patients' conditions.

[0083] Therefore, to alleviate the burden of collecting biological samples such as blood and tissues from cancer patients, we established a co-culture system under normal conditions that can recapitulate the tumor microenvironment to a degree similar to or better than matched or non-matched conditions.

[0084] Specifically, the system under normal conditions is prepared by, in a first step, co-culturing peripheral blood mononuclear cells (PBMCs) from normal individuals and cancer organoids from cancer patients to differentiate the PBMCs into T cells; and in a second step, co-culturing the differentiated T cells with cancer organoids from patients.

[0085] More specifically, most of the PBMCs isolated from normal individuals are naive T cells, and the presence of differentiated cytotoxic T cells that can attack cancer cells is very low. Therefore, in order to simulate the in vivo tumor microenvironment, PBMCs are differentiated into cytotoxic T cells by providing cancer organoids as antigens to PBMCs. For PBMCs derived from normal individuals, cancer organoids derived from cancer patients are provided as antigens to differentiate PBMCs into cytotoxic T cells, and cytotoxic T cells derived from normal individuals can recognize cancer organoids derived from cancer patients as their own cells.

[0086] This was achieved by a first step of co-culturing cancer organoids and PBMCs. The cancer organoids and PBMCs were made and prepared as follows.

[0087] Lung cancer organoids were used as a type of cancer organoid. Organoids with a 3D structure were prepared by isolating lung cancer tissue obtained from a patient into single cells and culturing them with an extracellular matrix (ECM). The formed lung cancer organoids were confirmed as tumors by pathological analysis, and the formed lung cancer organoids were separated into single cells using Tryp-LE before use.

[0088] PBMCs were isolated from donor blood using Ficoll. After isolation, the PBMCs were stored in a nitrogen tank until use. PBMCs were thawed immediately before use, stabilized for one day, and then used.

[0089] Subsequently, the first step was to mix single-cell lung cancer organoids and PBMCs at a cell number ratio of 1:20 (7.5x10 3 Cells / well: 1.5x10 5 Cells / well) and cultured for 0 to 42 days. Continue to culture while checking with flow cytometry until the expression of cytotoxic T cells is greater than 10%. Subsequently, a second step is performed in which the cytotoxic T cells obtained by the above method are mixed with cancer organoids derived from cancer patients at a cell number ratio of 1:20 and further cultured. In this way, we established a system that simulates the in vivo tumor microenvironment, in which cytotoxic T cells kill cancer organoids.

[0090] Example 2 Validation of the Anticancer Drug Efficacy Evaluation and Screening System

[0091] Example 2.1 Confirmation of cytotoxic T cell marker expression

[0092] In order to verify whether the system established in Example 1 is suitable for drug efficacy evaluation and screening, it was analyzed whether the process of differentiating PBMCs into cytotoxic T cells by performing the first step proceeds well.

[0093] Specifically, the proportion of cells expressing CD56, CD3, and CD8 proteins as cytotoxic T cell markers on their cell surfaces was analyzed. FACS analysis was performed on the PBMCs co-cultured in the first step under matched conditions, non-matched conditions, and normal conditions, and the CD56 + 、CD3 + 、CD8 + The proportion of T cells.

[0094] Table 1

[0095] Day 0 Day 14 Day 28 Day 42 Matching conditions 12.8％ 42.5％ 60.4％ 13.1％ Non-matching conditions 12.6％ 46.4％ 53.7％ 44.5％ Normal conditions 0.3％ 9.6％ 50.8％ 62.4％

[0096] The results are shown in Table 1 and Figure 1 shown.

[0097] Under matching conditions, in the first stage of co-culture, cytotoxic T cells increased rapidly from day 0 to day 28, with approximately 60.4% of cytotoxic T cells present on day 28. However, during long-term co-culture after day 42, it was confirmed that most cytotoxic T cells had died, recovering to approximately 13.1%, the same level as on day 0 after the start of co-culture.

[0098] Under non-matching conditions, in the first stage of co-culture, cytotoxic T cells increased rapidly from day 0 to day 28, with approximately 53.7% of cytotoxic T cells present on day 28. It was confirmed that even during long-term co-culture after day 42, cytotoxic T cells maintained a high level of differentiation, approximately 44.5%.

[0099] Under normal conditions, in the first stage of co-culture, cytotoxic T cells were almost absent from day 0 to day 14. However, starting from day 28, cytotoxic T cells increased rapidly and were maintained at a high level of about 50%. In particular, during the long-term co-culture after day 42, cytotoxic T cells showed a significantly high level of about 62.4%, and it was confirmed that this level was higher than the maximum value under matching conditions (60.4% on day 28) and about 4.7 times higher than 13.1% on day 42.

[0100] These results indicate that when PBMCs are differentiated into cytotoxic T cells according to the method of Example 1 (first stage), and the cytotoxic T cells are used to prepare an in vitro system that simulates a tumor microenvironment (second stage), PBMCs isolated from normal individuals can be differentiated into cytotoxic T cells at levels similar to those of PBMCs isolated from cancer patients, and thus a system for drug screening and efficacy evaluation can be efficiently prepared to more closely simulate the in vivo tumor microenvironment.

[0101] Example 2.2 Confirmation of interferon-producing cell (ICP) marker expression

[0102] In order to verify whether the system established in Example 1 is suitable for drug efficacy evaluation and screening, it was analyzed whether the process of differentiating PBMCs into cytotoxic T cells by performing the first step proceeds well.

[0103] Specifically, cytotoxic T cells express immune checkpoint proteins (ICPs), and it is known that their expression levels vary from person to person. We analyzed whether the ICP markers in cytotoxic T cells under matching conditions and under normal differentiation conditions are expressed the same. It was analyzed to see if it is feasible. FACS analysis was performed on the cytotoxic T cells that were differentiated and separated according to Example 1.1. Then, in the cytotoxic T cells, the proportion (expressed in % in Table 2) and quantity (expressed in brackets in Table 2) of cytotoxic T cells expressing LAG3, TIM3, PD-1, Light, 4-1BB or BTLA (referred to as ICP markers) on the cell surface were analyzed.

[0104] Table 2

[0105] Matching conditions Non-matching conditions Normal conditions LAG3 96.0％(499) 94.5％(431) 95.7％(367) TIM3 98.1％(986) 98.0％(1007) 97.7％(976) PD-1 94.5％(465) 92.2％(172) 92.8％(92) Light 99.1％(2062) 98.3％(2378) 98.8％(1875) 4-1BB 97.8％(301) 95.5％(405) 96.8％(186) BTLA 98.8％(202) 98.2％(250) 98.8％(181)

[0106] The results are shown in Table 2 . Under matched conditions, unmatched conditions, and normal conditions, cytotoxic T cells expressed ICP markers at similar levels.

[0107] These results indicate that when PBMCs are differentiated into cytotoxic T cells according to the method of Example 1 (first stage) and the cytotoxic T cell preparation system is used (second stage), PBMCs isolated from normal individuals can be differentiated into cytotoxic T cells at a level similar to that of PBMCs isolated from cancer patients, and thus a system for drug screening and efficacy evaluation can be efficiently prepared to more closely simulate the in vivo tumor microenvironment.

[0108] Example 2.3 Evaluation of the efficacy of immuno-oncology drugs

[0109] Example 2.3.1 Efficacy evaluation according to culture period

[0110] In order to verify whether the system established in Example 1 is suitable for drug efficacy evaluation and screening, the system prepared according to the first step and the second step was treated with atezolizumab as a drug, and then its ability to kill cancer organoids was evaluated. Atezolizumab is a drug currently on sale and used to treat lung cancer.

[0111] Specifically, the system performs the first step of co-cultivation for a period of 42 days. 10nM atezolizumab was given for treatment on the 14th, 28th and 42nd day of co-cultivation, and the efficacy of 72-hour drug treatment was evaluated. Subsequently, the growth rate of cancer organoids was measured at 0H, 12H, 24H and 72H after atezolizumab treatment, respectively, to analyze the inhibitory effect of atezolizumab on the growth of cancer organoids (killing cancer organoids) and the overall efficacy. The overall efficacy was evaluated by calculating the change in the organoid area of ​​the drug-treated group 72 hours after drug treatment and the organoid area of ​​the untreated group.

[0112] The results are as follows Figures 2 to 4 shown.

[0113] On day 14 from the start of the first step of co-culture, the effect of atezolizumab in inhibiting the growth of cancer organoids (killing cancer organoids) was best under matched conditions, and the effect under non-matched conditions was lower than that under matched conditions. Under normal conditions, almost no effect was observed, showing a growth rate similar to that of the control group ( Figure 2 ).

[0114] On the other hand, at 28 days from the start of the first step of co-culture, the effect of atezolizumab on the growth inhibition (killing of cancer organoids) of cancer organoids was best under the matched condition, but also showed an effect similar to that under the non-matched condition or normal condition ( Figure 3 ).

[0115] In particular, on day 42 from the start of co-culture in the first step, the effect of atezolizumab on growth inhibition (killing of cancer organoids) of cancer organoids was the lowest under matched conditions, while the effect was significantly superior under normal conditions. The effect under non-matched conditions was lower than that under normal conditions ( Figure 4 ).

[0116] In view of this, if Figure 5 As shown,

[0117] Under matched conditions, the overall efficacy of atezolizumab was maintained at about 35% to 36% from day 0 to day 28 of the first step of co-culture. However, in long-term culture after day 42, the effect dropped sharply to about 16%, equivalent to half the effect.

[0118] Under non-matched conditions, the overall efficacy of atezolizumab remained similar, approximately 26% to 33%, from day 0 to day 42 of the first step of co-culture.

[0119] Under normal conditions, the overall efficacy of atezolizumab was barely shown, about 3%, from day 0 to day 14 of the first step of co-culture. However, it rapidly increased to about 32% from day 28, and the effect was further improved to about 35% during the long-term co-culture after day 42. This effect was better than the maximum effect under non-matching conditions (about 33%) and close to the maximum effect under matching conditions (about 36%).

[0120] These results indicate that the system prepared according to the method of Example 1 simulates the tumor microenvironment and can be effectively used for drug screening and efficacy evaluation.

[0121] In particular, it was shown that under normal conditions, PBMCs from normal individuals differentiated into cancer patient-specific cytotoxic T cells, which has the advantage that no additional biological samples need to be collected from cancer patients, and drug efficacy can be evaluated with high accuracy even during long-term co-culture. Therefore, this suggests that it can be used for the screening and efficacy evaluation of new drugs with extended half-life.

[0122] Example 2.3.2 Efficacy evaluation under normal conditions

[0123] It was verified whether the co-culture system under normal conditions established in Example 1 is suitable for drug efficacy evaluation and drug screening.

[0124] Specifically, each cancer organoid derived from a different cancer patient (patient A, patient B) was used to differentiate PBMCs from a normal individual (normal subject C) into cytotoxic T cells (first step). The sources of PBMCs and cancer organoids used in each experimental group are listed in Table 3.

[0125] Table 3

[0126]

[0127] The cytotoxic T cells differentiated from each experimental group were mixed and co-cultured with cancer organoids from patient A (second step). The co-cultures were treated with atezolizumab according to the method of Example 2.3.1, and then its efficacy was evaluated.

[0128] The results are as follows Figure 6a shown.

[0129] In Experimental Groups 1 to 3 (the first and second steps used cancer organoids derived from the same cancer patient A), the overall efficacy of atezolizumab was confirmed to be about 5 times higher than that of the control group with only cancer organoids. In particular, in Experimental Group 3 (normal conditions, in which PBMCs derived from normal subject C were differentiated into cytotoxic T cells for patient A), it was confirmed that the overall efficacy of atezolizumab increased rapidly from the 15th day of co-culture in the first step to about 35%, and even during the long-term co-culture after the 42nd day, the effect was still maintained at a high level of about 30%.

[0130] On the other hand, Figure 6b As shown,

[0131] It was confirmed that in Experimental Groups 4 to 6 (the first and second steps used cancer organoids derived from different cancer patients, i.e., the first step used cancer organoids derived from cancer patient B, and the second step used cancer organoids derived from cancer patient A), the overall efficacy of atezolizumab was similar to that of the control group with only cancer organoids. In particular, in Experimental Group 6 (normal conditions, in which PBMCs derived from normal subjects were differentiated into cytotoxic T cells), it was confirmed that the overall efficacy of atezolizumab was not significantly different from that of the control group.

[0132] These results suggest that this system can be used for drug screening and efficacy evaluation under co-culture conditions using PBMCs derived from the same normal individual only when the cancer organoids used in the first and second steps are derived from the same cancer patient.

Claims

1. A method for evaluating the efficacy of an anticancer drug, the method comprising: (a) Peripheral blood mononuclear cells (PBMCs) were mixed and co-cultured with cancer organoids to obtain cytotoxic T cells; (b) mixing and co-culturing the cytotoxic T cells obtained in step (a) with cancer organoids; (c) treating the mixture of cytotoxic T cells and cancer organoids of step (b) with an anti-cancer drug; and (d) if the group treated with the anticancer drug in step (c) shows increased growth inhibition or death of cancer organoids compared to a group not treated with the anticancer drug or a positive control group, determining that the anticancer drug has anticancer efficacy.

2. A method for screening anticancer drugs, comprising: (a) PBMCs were mixed and co-cultured with cancer organoids to obtain cytotoxic T cells; (b) mixing and co-culturing the cytotoxic T cells obtained in step (a) with cancer organoids; (c) treating the mixture of cytotoxic T cells and cancer organoids of step (b) with an anti-cancer drug candidate; and (d) if the group treated with the anticancer drug candidate in step (c) shows increased growth inhibition or death of cancer organoids compared to a group not treated with the anticancer drug candidate, determining that the anticancer drug candidate is an anticancer drug.

3. The method according to claim 1 or 2, wherein: The PBMCs in step (a) are derived from normal individuals.

4. The method according to claim 1 or 2, wherein: The cancer organoids of step (a) are derived from cancer patients.

5. The method according to claim 1 or 2, wherein: The cancer organoid in step (a) and the cancer organoid in step (b) are derived from the same cancer patient.

6. The method according to claim 1 or 2, wherein: The co-cultivation in step (a) is performed for 14 to 42 days.

7. The method according to claim 1 or 2, wherein: The anticancer drug is at least one selected from the group consisting of compounds, peptides, peptide mimetics, fusion proteins, antibodies, aptamers, antibody-drug conjugates (ADCs), and antisense nucleic acids, siRNAs, shRNAs, miRNAs, and ribozymes that bind to DNA or mRNA in a complementary manner.

8. The method according to claim 1 or 2, wherein: The cancer is at least one selected from the group consisting of biliary tract cancer, gastric 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, kidney cancer, sarcoma, prostate cancer, urethral cancer, bladder cancer, hematological malignancies, lymphoma and fibroadenoma.

9. The method according to claim 1 or 2, wherein: The growth inhibition or death of the cancer organoid in step (d) is confirmed by an increase or decrease in the volume, area or cell number of the cancer organoid. 10 . A system for evaluating the efficacy of an anticancer drug, the system being used to use the method for evaluating the efficacy of an anticancer drug according to claim 1 , the system comprising PBMCs and cancer organoids.

11. A system for screening anticancer drugs, the system being used to use the method for screening anticancer drugs according to claim 2, the system comprising PBMCs and cancer organoids.

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