Novel Use of PBMC-Derived Cytotoxic T Cells

Abstract

The present invention relates to a method for evaluating the efficacy of an anticancer agent, a method for screening an anticancer agent, a system for evaluating the efficacy of an anticancer agent, or a system for screening an anticancer agent, which uses a mixture of PBMC (peripheral blood mononuclear cell) and cancer organoids. Specifically, the present invention relates to a method for evaluating the efficacy of an anticancer agent, a method for screening an anticancer agent, a system for evaluating the efficacy of an anticancer agent, or a system for screening an anticancer agent, which uses a mixture of cytotoxic T cells differentiated from PBMC and cancer organoids. The method for evaluating the efficacy of an anticancer agent, the screening method, the efficacy evaluation system, or the screening system according to the present invention can very similarly reproduce the tumor microenvironment, which is the form in which cancer cells exist in vivo. In addition, since PBMC derived from normal individuals are differentiated into cancer patient-specific cytotoxic T cells and used, the burden of collecting biological samples such as blood and tissues from cancer patients can be reduced, and the efficacy of anticancer agents can be evaluated with high accuracy.

Description

Technical Field

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

Background Art

[0002] An organoid is an organoid produced by three-dimensionally culturing cells derived from a tissue or an organ. Organoids have the advantages of being capable of long-term culture and cryopreservation, and being easy to operate and observe. At the same time, it does not require immortalization, and of course, the original characteristics of the cells are retained. By reproducing the cell layer and histological structure only seen in vivo, it is an experimental model that can study physiological phenomena at a higher dimension than cells. Due to such characteristics, organoids can evaluate drugs with higher accuracy compared to immortalized cell lines with changed intrinsic cell characteristics or animal models with different structures from the human body. In particular, since patient-derived tissues are used, prior to clinical trials involving humans, there is an advantage that not only the safety of the drug but also its efficacy can be confirmed in advance. However, existing cancer organoids developed as drug evaluation models had a limitation in that they could not reflect the tumor microenvironment, and thus efforts were needed to develop organoids with diverse tumor microenvironments and use them as drug evaluation models.

[0003] The tumor microenvironment is a cellular environment in which blood vessels, immune cells, fibroblasts, lymphocytes, signaling molecules, extracellular matrix (ECM), etc. surround cancer cells. In the tumor microenvironment, cancer cells induce extracellular signal release, promotion of cancer cell neovascularization, peripheral immune tolerance, etc., and affect the microenvironment. Therefore, cancer cells can change the microenvironment or the microenvironment can affect the growth or metastasis of cancer cells. In particular, the tumor microenvironment associated with lymphocytes, etc. is known to affect not only the occurrence and metastasis of cancer but also the therapeutic response to anticancer drugs.

[0004] For example, CD8 + T cells in the tumor microenvironment play an important role in tumor immunity such as differentiating into cytotoxic T cells, migrating into the tumor microenvironment, and killing tumor cells.

Summary of the Invention

Problems to be Solved by the Invention

[0005] The present inventors developed cancer organoids with a tumor microenvironment and conducted various studies for using them as a platform for drug evaluation or drug screening. As a result, among the various cells constituting the tumor microenvironment, a system of co-culturing cancer organoids with cytotoxic T cells was established.

[0006] In particular, since the cytotoxic T cells are those obtained by differentiating PBMCs derived from normal individuals into cancer patient-specific cytotoxic T cells, the burden of collecting biological samples such as blood and tissues from cancer patients can be reduced through this. Experimentally, it was demonstrated that the cytotoxic T cells can evaluate the efficacy of drugs with high accuracy over a long period as their survival is maintained even when co-culture is carried out for a long period, and thus the present invention was completed.

Means for Solving the Problems

[0007] Each of the descriptions and embodiments disclosed in the present invention can also be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present invention belong to the scope of the present invention. Also, it is not recognized that the scope of the present invention is limited by the specific descriptions described later.

[0008] In addition, terms not specifically defined in this specification should be understood to have the meanings usually used in the technical field to which the present invention pertains. Also, in the context, unless otherwise specifically defined, the singular includes the plural and the plural includes the singular.

[0009] One aspect of the present invention provides a method for evaluating the efficacy of an anticancer agent, including the following steps: (a) A step of mixing peripheral blood mononuclear cells (PBMCs) with cancer organoids and co-culturing them to obtain cytotoxic T cells; (b) A step of mixing the cytotoxic T cells obtained in step (a) with cancer organoids and co-culturing them; (c) A step of treating the mixture of cytotoxic T cells and cancer organoids in step (b) with an anticancer agent; and (d) A step of determining that the anticancer agent has anticancer efficacy when the growth inhibition or death of cancer organoids in the group treated with the anticancer agent in step (c) is increased compared to the group not treated with the anticancer agent or the positive control group.

[0010] Specifically, the a step is a step of mixing PBMC and cancer organoids for co-culture, which is a step of differentiating PBMC into cytotoxic T cells to obtain cytotoxic T cells. Also, the a step is a step of differentiating PBMC derived from a normal individual into cancer patient-specific cytotoxic T cells to obtain cytotoxic T cells. Further, the a step is a step of training cancer patient-specific cytotoxic T cells differentiated from PBMC derived from a normal individual to recognize cancer cells or cancer organoids of a cancer patient as self cells.

[0011] As used herein, the term "PBMC (peripheral blood mononuclear cell)" is called "peripheral blood mononuclear cell" and means a cell population composed of lymphocytes such as T cells, B cells, NK cells and monocytes. In the PBMC population, naive T cells account for about 45-70% and most of them, and in addition, helper T cells (helper T cell, CD4 + T cell) is 25-60%, cytotoxic T cell (cytotoxic T cell, CD8 + T cell) is 5-30%, B cell is 5-10%, NK cell is 10-30%, monocyte is 5-10%, and dendritic cell is 1-2%.

[0012] The PBMC according to the present invention may be derived from an individual with cancer (cancer patient), a normal individual without cancer, or a normal individual who has been cured after cancer onset. Preferably, it may be derived from a normal individual without cancer or a normal individual who has been cured after cancer onset, but is not limited thereto. In the present specification, the PBMC may be named interchangeably with "pre-differentiated T cells", and the "derived from" may be named interchangeably with "isolated" or "obtained".

[0013] As used herein, the term "individual" includes all individuals who have never developed cancer, individuals who may develop cancer, individuals who have developed cancer, or individuals who have been cured after developing cancer, and can include humans or any non-human animals without limitation. The non-human animals may be vertebrates, such as primates, dogs, cows, horses, pigs, rodents, such as mice, rats, hamsters, guinea pigs, etc. As used herein, the "individual" may be used interchangeably with "subject" or "patient".

[0014] As used herein, the term "biological sample" may be a tissue, cell, whole blood, serum, plasma or cell culture supernatant, preferably whole blood, but is not limited thereto.

[0015] As used herein, the term "cytotoxic T cell" means a lymphocyte that performs antigen-specific acquired immunity (adaptive immunity). Cytotoxic T cells kill cells infected with pathogens such as cancer cells, viruses, bacteria, etc., and produce cytokines such as TNF-α and IFN-γ. The cytotoxic T cells according to the present invention may be differentiated from PBMCs. As used herein, the cytotoxic T cells may be named interchangeably with "differentiated T cells", "post-differentiation T cells" or "CD8 + T cells".

[0016] As used herein, the term "organoid" means a mass of cells having a three-dimensional structure, and means an organoid produced through three-dimensional culture of cells separated from an organ or tissue. Organoids contain specific cell populations that make up an organ or tissue, and because they are structurally organized in a form similar to an actual tissue or organ, they can reproduce the special form and function of each tissue or organ.

[0017] As used herein, the term "cancer organoid" means an organoid produced from cells isolated from a tumor (tumor cells or cancer cells). The cancer organoid according to the present invention may be derived from an individual in whom cancer has developed (cancer patient), but is not limited thereto.

[0018] In the present invention, the PBMC and cancer organoid in the step a may be mixed at a cell number ratio of 15:1 to 25:1, preferably 20:1. Also, the co-culture in the step a may be carried out for 1 to 42 days. When the PBMC and cancer organoid are mixed at the above ratio and co-cultured during the above period, PBMC derived from a normal individual is differentiated into cancer patient-specific cytotoxic T cells, and the differentiation rate also becomes high, so that the burden of collecting biological samples such as blood and tissue from cancer patients can be reduced.

[0019] Further, the step b is a step of mixing and co-culturing the cytotoxic T cells obtained in the step a (that is, cytotoxic T cells differentiated from PBMC) with cancer organoids, and is a step of reproducing the in vivo tumor microenvironment in vitro. Also, the step b is a step in which cancer cells or cancer organoids of a cancer patient recognized as self cells by cancer patient-specific cytotoxic T cells differentiated from PBMC derived from a normal individual are attacked and killed.

[0020] At this time, the cancer organoid used in the step a and the cancer organoid used in the step b may be derived from the same cancer patient.

[0021] Also, the step a and the step b may be continuously carried out in the same culture space. For example, the step a and the step b may be continuously carried out in the same one culture plate.

[0022] In the present invention, the cytotoxic T cells and cancer organoids in the b step may be mixed at a cell number ratio of 15:1 to 25:1, preferably 20:1. Since the cytotoxic T cells do not die even during long-term culture, they can mimic the tumor microenvironment, in which cancer cells exist in vivo, for a long time, and thereby the efficacy of the anticancer agent can be evaluated with high accuracy for an even longer time.

[0023] In addition, the c step is a step of treating the mixture of the cytotoxic T cells and cancer organoids in the b step with an anticancer agent. Specifically, it is a step of treating the mixture of the cytotoxic T cells obtained in the a step (i.e., cytotoxic T cells differentiated from PBMC) and cancer organoids with an anticancer agent.

[0024] As used herein, the term "anticancer agent" refers to a substance that exhibits a preventive or therapeutic effect against cancer, specifically, a substance that can kill cancer cells, cancer organoids, tumors, etc., or suppress their growth.

[0025] In this specification, the "anticancer agent" may be used interchangeably with the "drug", and the "treatment" may be used interchangeably with the "addition" or "administration".

[0026] Specifically, the anticancer agent may target PBMC, cytotoxic T cells differentiated from PBMC, cytotoxic T cells, and / or cancer organoids. For example, the anticancer agent may target only cancer organoids, only cytotoxic T cells, or may target cancer organoids and cytotoxic T cells simultaneously. For example, the anticancer agent targeting cancer organoids may be atezolizumab, and the anticancer agent targeting cytotoxic T cells may be Pembrolizumab.

[0027] Preferably, the anti-cancer agent includes, without limitation, compounds, proteins, fusion proteins, compound-protein complexes, drug-protein complexes, antibodies, compound-antibody complexes, drug-antibody complexes, amino acids, peptides, viruses, carbohydrates, lipids, nucleic acids, extracts, fractions, and the like.

[0028] For example, the anti-cancer agent can include, but is not limited to, compounds, peptides, peptidomimetics, fusion proteins, antibodies, aptamers, antibody-drug conjugates (ADCs), etc. Preferably, the anti-cancer agent can be an antibody or an immune anti-cancer agent.

[0029] As used herein, the term "antibody" includes monoclonal antibodies, polyclonal antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, and in addition to novel antibodies, antibodies already known in the art or commercially available. The antibody includes not only the full-length form containing two heavy chains and two light chains, but also functional fragments of the antibody molecule. The functional fragment of the antibody molecule means a fragment having at least an antigen-binding function, which may include, but is not limited to, Fab, F(ab´), F(ab´)2, Fv, etc. As used herein, the term "Peptide Mimetics" means a peptide or a modified peptide that biologically mimics an active ligand of a hormone, cytokine, enzyme substrate, virus, or other biological molecule. As used herein, the term "Aptamer" means a single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) that has a stable tertiary structure by itself and can bind to a target molecule with high affinity and specificity.

[0030] As another example, the anti-cancer agent can include, but is not limited to, antisense nucleic acids, siRNA, shRNA, miRNA, ribozymes, etc. that bind complementarily to DNA or mRNA.

[0031] As used herein, the term "antisense nucleic acid" means DNA, RNA, or fragments or derivatives thereof that contain a nucleic acid sequence complementary to the sequence of a specific mRNA, and acts to inhibit the translation of mRNA into protein by binding or hybridizing complementarily to the sequence of the mRNA. As used herein, the term "siRNA (small interfering RNA)" means a short double-stranded RNA that can induce RNAi (RNA interference) through cleavage of a specific mRNA. siRNA includes a sense RNA strand having a sequence homologous to the mRNA of the target gene and an antisense RNA strand having a sequence complementary thereto. Since siRNA can suppress the expression of the target gene, it is used in gene knockdown methods or gene therapy methods. As used herein, the term "shRNA (short hairpin RNA)" means a single-stranded RNA that is divided into a stem portion that forms a double-stranded portion by hydrogen bonding and a loop portion that is loop-shaped. It can be processed by a protein such as Dicer and converted into siRNA, and can perform the same function as siRNA. As used herein, the term "miRNA (micro RNA)" means a 21-23 nt non-coding RNA that regulates gene expression post-transcriptionally by promoting the degradation of target RNA or suppressing their translation. As used herein, the term "ribozyme" means an RNA molecule having a function like an enzyme that recognizes a specific base sequence and cleaves itself. A ribozyme is composed of a region that binds specifically to a complementary base sequence of a target messenger RNA strand and a region that cleaves the target RNA.

[0032] The anti-cancer agent whose efficacy is evaluated or screened by the method of the present invention may be, for example, one for preventing or treating biliary tract cancer, gastric cancer, lung cancer, liver cancer, colorectal cancer, colon cancer, small intestine cancer, pancreatic cancer, brain cancer, bone cancer, 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, blood cancer, leukemia, lymphoma, fibroadenoma, etc., but is not limited thereto.

[0033] Further, in the d step, when the growth inhibition or death of cancer organoids in the group treated with the anti-cancer agent in the c step is increased compared to the untreated group or the positive control group of the anti-cancer agent, the anti-cancer agent is determined to have anti-cancer efficacy.

[0034] The term "positive control group" means a group treated with a drug used as an anti-cancer agent in the art.

[0035] Specifically, the growth inhibition or death of the cancer organoids may be confirmed through an increase or decrease in the volume, area, or number of cells of the cancer organoids. Also, when the volume, area, or number of cells of the cancer organoids decreases, it can be determined that the growth inhibition or death of the cancer organoids has increased. The increase or decrease in the volume, area, or number of cells of the cancer organoids can be confirmed by methods known in the art. For example, it can be confirmed through analysis methods such as HCS (High-Content Screening) and flow cytometry, but is not limited thereto.

[0036] Another aspect of the present invention provides a method for screening an anti-cancer agent, including the following steps: (a) A step of mixing PBMC with cancer organoids and co-culturing them to obtain cytotoxic T cells; (b) A step of mixing the cytotoxic T cells obtained in the a step with cancer organoids and co-culturing them; (c) Treating the cytotoxic T cell and cancer organoid mixture of step (b) with a candidate anti-cancer agent; and (d) Determining the candidate substance as an anti-cancer agent when growth inhibition or death of cancer organoids is increased in the group treated with the candidate anti-cancer agent of step (c) compared to the group untreated with the candidate anti-cancer agent.

[0037] In the method for screening an anti-cancer agent according to the present invention, unless otherwise specified, related terms are understood to have the same meaning as the terms described above.

[0038] Specifically, step (a) is a step of mixing PBMC and cancer organoids and co-culturing them, differentiating PBMC into cytotoxic T cells to obtain cytotoxic T cells. Also, step (a) is a step of differentiating PBMC derived from a normal individual into cancer patient-specific cytotoxic T cells to obtain cytotoxic T cells. Further, step (a) is a step of training cancer patient-specific cytotoxic T cells differentiated from PBMC derived from a normal individual to recognize cancer cells or cancer organoids of a cancer patient as self cells.

[0039] In the present invention, the PBMC and cancer organoids in step (a) may be mixed at a cell number ratio of 15:1 to 25:1, preferably 20:1. Also, the co-culture in step (a) may be performed for 1 to 42 days. When the PBMC and cancer organoids are mixed at the above ratio and co-cultured during the above period, even PBMC derived from a normal individual can be differentiated into cancer patient-specific cytotoxic T cells, and the differentiation ratio also becomes high, so the burden of collecting biological samples such as blood and tissue from cancer patients can be reduced.

[0040] Further, the b step is a step of mixing the cytotoxic T cells obtained in the a step (i.e., cytotoxic T cells differentiated from PBMC) with cancer organoids and co-culturing them, which is a step of reproducing the in vivo tumor microenvironment in vitro. Also, the b step is a step of attacking and killing the cancer cells or cancer organoids of a cancer patient that cancer patient-specific cytotoxic T cells differentiated from PBMC derived from a normal individual recognize as self cells.

[0041] At this time, the cancer organoids used in the a step and the cancer organoids used in the b step may be derived from the same cancer patient.

[0042] Also, the a step and the b step may be continuously performed in the same culture space. For example, the a step and the b step may be continuously performed in the same single culture dish.

[0043] In the present invention, the PBMC and cancer organoids in the b step may be mixed at a cell number ratio of 15:1 to 25:1, preferably 20:1. Since the cytotoxic T cells do not die even during long-term culture, the tumor microenvironment in which cancer cells exist in the living body can be simulated for a long time, and thereby the efficacy of the anticancer agent can be evaluated with high accuracy for an even longer time.

[0044] Also, the c step is a step of treating the cytotoxic T cell and cancer organoid mixture in the b step with an anticancer agent candidate substance, specifically, a step of treating the mixture of the cytotoxic T cells obtained in the a step (i.e., cytotoxic T cells differentiated from PBMC) and cancer organoids with an anticancer agent candidate substance.

[0045] As used herein, the term "anticancer agent candidate substance" means a substance expected to exhibit a cancer preventive or therapeutic effect, specifically, a substance expected to kill cancer cells, cancer organoids, tumors, etc., or to inhibit their growth.

[0046] In this specification, the "anticancer agent candidate substance" may be used interchangeably with the "drug", and the "treatment" may be used interchangeably with the "addition" or "administration".

[0047] Preferably, the anticancer agent candidate substance includes, without limitation, compounds, proteins, fusion proteins, compound-protein complexes, drug-protein complexes, antibodies, compound-antibody complexes, drug-antibody complexes, amino acids, peptides, viruses, carbohydrates, lipids, nucleic acids, extracts, fractions, etc.

[0048] By way of example, the anticancer agent candidate substance can include, but is not limited to, compounds, peptides, peptidomimetics, fusion proteins, antibodies, aptamers, antibody-drug conjugates (ADCs; Antibody Drug Conjugate), etc. Preferably, the anticancer agent candidate substance may be an antibody or an immunological anticancer agent.

[0049] Also, the d step is a step of determining the candidate substance as an anticancer agent when the growth inhibition or death of cancer organoids is increased in the group treated with the anticancer agent candidate substance in the c step compared to the group untreated with the anticancer agent candidate substance.

[0050] Specifically, the growth inhibition or death of the cancer organoids may be confirmed through the increase or decrease in the volume, area, or cell count of the cancer organoids. Further, when the volume, area, or cell count of the cancer organoids decreases, it can be determined that the growth inhibition or death of the cancer organoids has increased. The increase or decrease in the volume, area, or cell count of the cancer organoids can be confirmed by methods known in the art, and for example, can be confirmed through analysis methods such as HCS (High-Content Screening) and flow cytometry, but is not limited thereto.

[0051] Another aspect of the present invention is an anti-cancer agent efficacy evaluation system for using the anti-cancer agent efficacy evaluation method, and provides an anti-cancer agent efficacy evaluation system including PBMC and cancer organoids.

[0052] In the anti-cancer agent efficacy evaluation system according to the present invention, unless otherwise specified, related terms are understood to have the same meaning as the terms described above.

[0053] The anti-cancer agent efficacy evaluation system according to the present invention includes PBMC and cancer organoids, and the PBMC is derived from a normal individual and exhibits the characteristic 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 the PBMC do not die even during long-term culture with cancer organoids, so that the tumor microenvironment, which is the form in which cancer cells exist in the living body, can be mimicked for a long time. Therefore, the efficacy when the anti-cancer agent is administered in the living body can be accurately predicted, and further, the efficacy of the anti-cancer agent can be evaluated with high accuracy for a longer time, so that it can be usefully utilized for the evaluation of the efficacy of the anti-cancer agent.

[0054] Another aspect of the present invention is a screening system for an anticancer agent for use in the method of screening the anticancer agent, which provides a screening system for an anticancer agent containing PBMC and cancer organoids.

[0055] In the screening system for an anticancer agent according to the present invention, unless otherwise specified, related terms are understood to have the same meaning as the terms described above.

[0056] The screening system for an anticancer agent according to the present invention contains PBMC and cancer organoids. The PBMC is derived from a normal individual and exhibits the characteristic of differentiating into cancer patient-specific cytotoxic T cells. Therefore, the burden of collecting biological samples such as blood and tissue from cancer patients can be reduced. In addition, the cytotoxic T cells differentiated from the PBMC do not die even during long-term culture with cancer organoids, so the tumor microenvironment, in which cancer cells exist in the living body, can be mimicked for a long time. Therefore, the efficacy of the anticancer agent when administered in vivo can be accurately predicted, and the efficacy of the anticancer agent can be evaluated with high accuracy for a longer time, so it can be usefully utilized for the screening of anticancer agents.

Advantages of the Invention

[0057] The method for evaluating the efficacy of an anticancer agent, the screening method, the efficacy evaluation system, or the screening system according to the present invention can very similarly reproduce the tumor microenvironment in which cancer cells exist in the living body. In addition, since PBMC derived from a normal individual is differentiated into cancer patient-specific cytotoxic T cells and used, the burden of collecting biological samples such as blood and tissue from cancer patients can be reduced, and the efficacy of the anticancer agent can be evaluated with high accuracy.

Brief Description of the Drawings

[0058]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6a

Figure 6b

Best Mode for Carrying Out the Invention

[0059] Hereinafter, the present invention will be described in more detail through examples. These examples are for more specifically explaining the present invention, and the scope of the present invention is not limited by these examples.

[0060] Example 1. Evaluation of Anticancer Agent Efficacy and Establishment of Screening System An in vitro efficacy evaluation and screening system was established to accurately predict the efficacy when a drug is administered in vivo by similarly reproducing the tumor microenvironment in which cancer exists in the body.

[0061] Specifically, a co-culture system in which cancer organoids and T cells coexist was used. The cancer organoids and T cells can be derived from various individuals such as an individual (cancer patient) in whom cancer has developed, a normal individual in whom cancer has not developed, or a normal individual who has been cured after cancer has developed. Depending on these sources, the co-culture system can be classified into matching, non-matching, and normal conditions. The matching condition uses cancer organoids and T cells derived from the same cancer patient. For example, cancer organoids derived from cancer patient A and T cells derived from cancer patient A are used. The non-matching condition uses cancer organoids and T cells derived from different cancer patients. For example, cancer organoids derived from cancer patient A and T cells derived from cancer patient B are used. The normal condition uses cancer organoids derived from a cancer patient and T cells derived from a normal individual. For example, cancer organoids derived from cancer patient A and T cells derived from normal individual C are used.

[0062] The matching and non-matching conditions have the advantage of being able to reproduce the in-vivo tumor microenvironment more similarly in vitro in that both the cancer organoids and T cells are derived from an individual who has already developed cancer. However, in order to isolate T cells, a process such as collecting a blood sample from an individual is required, and collecting a blood sample from a cancer patient has a negative impact on the improvement of the cancer patient's condition.

[0063] Therefore, for the purpose of reducing the burden of collecting biological samples such as blood and tissue from cancer patients, a co-culture system under normal conditions that can reproduce the tumor microenvironment similarly to or even more excellently than the matching or non-matching conditions was established.

[0064] Specifically, the system under normal conditions was produced through a first step of co-culturing PBMC (peripheral blood mononuclear cell) derived from a normal individual and cancer organoids derived from a cancer patient to differentiate PBMC into T cells; and a second step of co-culturing the differentiated T cells with cancer organoids derived from the patient.

[0065] More specifically, PBMC isolated from a normal individual mostly consists of naive T cells, and cytotoxic T cells in a differentiated form that can attack cancer cells exist at a very low ratio. Therefore, in order to mimic the in-vivo tumor microenvironment, PBMC was differentiated into cytotoxic T cells by providing cancer organoids as an antigen for PBMC. At the same time, for PBMC derived from a normal individual, PBMC was differentiated into cytotoxic T cells by providing cancer organoids derived from a cancer patient as an antigen, and the cytotoxic T cells derived from the normal individual were made to recognize the cancer organoids derived from the cancer patient as self cells.

[0066] This was done through the first step of co - culturing cancer organoids and PBMC. The cancer organoids and PBMC were manufactured and prepared in the following ways.

[0067] As the type of cancer organoids, lung cancer organoids were used. Lung cancer tissues obtained from patients were separated into single cells, and these were cultured together with ECM (extracellular matrix) to produce organoids with a 3D structure. The formed lung cancer organoids were confirmed to be tumors through pathological analysis, and after being separated into single cells through the use of Tryp - LE, they were used.

[0068] PBMC were separated from the blood obtained from donors using Ficoll, stored in a nitrogen tank until use after separation, thawed immediately before use, and used after one - day stabilization.

[0069] Thereafter, single - cell lung cancer organoids and PBMC were mixed at a cell number ratio of 1:20 (7.5×10 3 cells / well: 1.5×10 5 cells / well), and the first step was carried out by culturing for 0 - 42 days. The culture period was advanced while confirming using a Flow cytometer until the expression of cytotoxic T cells exceeded 10%. Thereafter, the second step was carried out by additionally culturing by mixing cancer organoids derived from cancer patients with cytotoxic T cells at a cell number ratio of 1:20 in the above - mentioned method, and through this, a system was established that mimics the in - vivo tumor microenvironment in which cytotoxic T cells kill cancer organoids.

[0070] Example 2. Evaluation of Anticancer Agent Efficacy and Verification of Screening System

[0071] Example 2.1. Confirmation of Cytotoxic T Cell Marker Expression To verify whether the system established in Example 1 can be appropriately used for drug efficacy evaluation and screening, it was analyzed whether the differentiation of PBMC into cytotoxic T cells by the performance of the first step was carried out well.

[0072] Specifically, the percentages of cells expressing CD56, CD3, and CD8 proteins as cytotoxic T cell markers on the cell surface were analyzed. FACS analysis was performed on PBMCs co-cultured in the first step under each of the matching, non-matching, and normal conditions, and the percentages of CD56 + CD3 + CD8 + T cells in the PBMC population were analyzed.

[0073]

Table 1

[0074] As a result, as seen from Table 1 and Figure 1,

[0075] Under the matching condition, cytotoxic T cells increased rapidly from day 0 to day 28 of the start of co-culture in the first step, and about 60.4% of cytotoxic T cells were present on day 28. However, it was confirmed that during long-term co-culture after day 42, most of the cytotoxic T cells died and returned to about 13.1%, the same level as on day 0 of the start of co-culture.

[0076] Under the non-matching condition, cytotoxic T cells increased rapidly from day 0 to day 28 of the start of co-culture in the first step, and about 53.7% of cytotoxic T cells were present on day 28. It was also confirmed that during long-term co-culture after day 42, cytotoxic T cells maintained a relatively high differentiation level of about 44.5%.

[0077] Under the normal condition, almost no cytotoxic T cells were present from day 0 to day 14 of the start of co-culture in the first step. However, cytotoxic T cells increased rapidly from day 28 and maintained a high level of about 50%. In particular, during long-term co-culture after day 42, cytotoxic T cells showed a significantly high level of about 62.4%, which was higher than the maximum value (60.4% on day 28) under the matching condition and about 4.7 times higher than 13.1% on day 42. It was confirmed that this was the case.

[0078] Such results suggest that when PBMCs are differentiated into cytotoxic T cells by the method of Example 1 (first step), and an in vitro system that mimics the tumor microenvironment is produced using the cytotoxic T cells (second step), PBMCs isolated from normal individuals can also be differentiated into cytotoxic T cells at levels similar to those of PBMCs isolated from cancer patients, suggesting that a system used for drug screening, efficacy evaluation, etc. can be produced in a more efficient manner to be even more similar to the in vivo tumor microenvironment.

[0079] Example 2.2. Confirmation of ICP (Interferon-Producing Cell) Marker Expression To verify whether the system established in Example 1 can be appropriately used for drug efficacy evaluation and screening, it was analyzed whether the differentiation of PBMCs into cytotoxic T cells by the performance of the first step was successfully carried out.

[0080] Specifically, cytotoxic T cells express ICP (Immune Check Point Protein), and it is known that the expression level varies among individuals. It was analyzed whether the ICP marker was expressed identically under the conditions where the expression in the cytotoxic T cells matched the conditions of differentiation in the matching and normal conditions. FACS analysis was performed on the cytotoxic T cells that had been differentiated and separated according to Example 1.1, and the proportion (shown in % in Table 2 below) and number (described in parentheses in Table 2 below) of cytotoxic T cells expressing LAG3, TIM3, PD-1, Light, 4-1BB, or BTLA, known as ICP markers, on the cell surface among the cytotoxic T cells were analyzed.

[0081]

Table 2

[0082] As a result, as seen from Table 2 above, in each of the matching, non-matching, and normal conditions, the cytotoxic T cells all expressed the ICP marker at similar levels.

[0083] Such results suggest that when PBMCs are differentiated into cytotoxic T cells by the method of Example 1 (the first step) and a system is produced using the cytotoxic T cells (the second step), PBMCs isolated from normal individuals can also be differentiated into cytotoxic T cells at a level similar to that of PBMCs isolated from cancer patients, thereby suggesting that a system used for drug screening, efficacy evaluation, etc. can be produced in a more efficient way to be more similar to the in vivo tumor microenvironment.

[0084] Example 2.3. Evaluation of Immunotherapy Anticancer Agent Efficacy

[0085] Example 2.3.1. Efficacy Evaluation by Culture Period To verify whether the system established in Example 1 can be appropriately used for efficacy evaluation and drug screening, atezolizumab was treated as an exemplary drug on the system produced by performing the first step and the second step to confirm its cancer organoid killing ability. Atezolizumab is a drug currently commercially available and used as a lung cancer therapeutic agent.

[0086] Specifically, for the system, the co-culture in the first step was carried out for 42 days, and 10 nM of atezolizumab was treated on the 14th day, 28th day, and 42nd day from the start of co-culture respectively, and the efficacy by drug treatment was confirmed for 72 hours. Thereafter, the growth rate of cancer organoids was measured at 0 hour, 12 hours, 24 hours, and 72 hours after atezolizumab treatment, and the growth inhibition (cancer organoid killing) effect and overall efficacy of atezolizumab on cancer organoids were analyzed. The overall efficacy was evaluated by calculating the area change of the drug-treated group compared to the organoid area of the drug-untreated group 72 hours after drug treatment.

[0087] As a result, as can be seen from FIGS. 2 to 4,

[0088] On the 14th day from the start of the co-culture in the first step, the growth inhibitory effect (cancer organoid death) of atezolizumab on cancer organoids was shown to be the best under the matching conditions, showed a lower effect than the matching conditions under the non-matching conditions, showed almost no effect under the normal conditions, and showed a growth rate similar to that of the control group (Figure 2).

[0089] In contrast, on the 28th day from the start of the co-culture in the first step, the growth inhibitory effect (cancer organoid death) of atezolizumab on cancer organoids was shown to be the best under the matching conditions, but the non-matching or normal conditions also showed an effect similar to that of the matching conditions (Figure 3).

[0090] In particular, on the 42nd day from the start of the co-culture in the first step, the growth inhibitory effect (cancer organoid death) of atezolizumab on cancer organoids was the lowest under the matching conditions and showed a significantly excellent effect under the normal conditions. The effect under the non-matching conditions was lower than that under the normal conditions (Figure 4).

[0091] Taking this all together, as can be seen from Figure 5,

[0092] Under the matching conditions, the overall efficacy of atezolizumab was maintained at about 35-36% from the 0th day to the 28th day from the start of the co-culture in the first step, but decreased rapidly to about 16%, which is half of that level, during long-term culture after the 42nd day.

[0093] Under the non-matching conditions, the overall efficacy of atezolizumab was similarly maintained at about 26-33% from the 0th day to the 42nd day from the start of the co-culture in the first step.

[0094] Under normal conditions, from the 0th day to the 14th day of the start of co-culture in the first step, the overall efficacy of atezolizumab was hardly shown at about 3%, but it increased rapidly to about 32% on the 28th day, and the effect further improved to about 35% during long-term co-culture after the 42nd day. Also, such an effect was superior to the maximum effect (about 33%) under non-matching conditions and was similar to the maximum effect (about 36%) under matching conditions.

[0095] Such results indicate that the system manufactured by the method according to Example 1 can be usefully used for drug screening, efficacy evaluation, etc. because it mimics the tumor microenvironment.

[0096] In particular, under normal conditions, PBMCs from normal individuals are differentiated into cancer patient-specific cytotoxic T cells for use. This has the advantage that additional biological samples do not need to be collected from cancer patients, and the efficacy of drugs can be evaluated with high accuracy even during long-term co-culture. This suggests that it can be usefully utilized for screening and efficacy evaluation of new drugs with an increased half-life.

[0097] Example 2.3.2. Efficacy Evaluation under Normal Conditions It was verified whether the co-culture system under the normal conditions established in Example 1 can be appropriately used for drug efficacy evaluation and drug screening.

[0098] Specifically, cancer organoids derived from different cancer patients (Patient A, Patient B) were used, and PBMCs from a normal person (Normal Individual C) were each differentiated into cytotoxic T cells (first step). If the origins of the PBMCs and cancer organoids used in each experimental group are sorted out, they are as shown in Table 3 below.

[0099]

Table 3

[0100] Cytotoxic T cells differentiated in each experimental group were mixed with cancer organoids derived from Patient A and co-cultured (second step). The co-culture was treated with atezolizumab by the method according to Example 2.3.1., and its efficacy was evaluated.

[0101] As a result, as seen from Fig. 6a, In the case of Experimental Groups 1 to 3 (using cancer organoids derived from the same cancer patient A in the first and second steps), it was confirmed that the overall efficacy of atezolizumab was up to about 5 times higher than that of the control group with only cancer organoids. In particular, in the case of Experimental Group 3 (normal condition in which PBMC derived from normal individual C was differentiated into cytotoxic T cells against Patient A), the overall efficacy of atezolizumab increased rapidly from the 15th day of the start of co-culture in the first step to about 35%, and the effect was maintained at about 30% even during long-term co-culture after the 42nd day.

[0102] On the other hand, as seen from Fig. 6b, In the case of Experimental Groups 4 to 6 (using cancer organoids derived from different cancer patients in the first and second steps. That is, the first step uses those derived from cancer patient B, and the second step uses those derived from cancer patient A), it was confirmed that the overall efficacy of atezolizumab was similar to that of the control group with only cancer organoids. In particular, in the case of Experimental Group 6 (normal condition in which PBMC derived from normal was differentiated into cytotoxic T cells), it was confirmed that there was no significant difference in the overall efficacy of atezolizumab when compared with the control group.

[0103] Such results suggest that the system can be usefully used for drug screening, efficacy evaluation, etc. only when the cancer organoids used in the first and second steps are derived from the same cancer patient even under co-culture conditions using PBMC from the same normal individual.

Claims

1. A method for evaluating the efficacy of an anticancer agent, comprising the following steps: (a) A step of mixing peripheral blood mononuclear cells (PBMC) with cancer organoids and co-culturing them to obtain cytotoxic T cells; (b) A step of mixing the cytotoxic T cells obtained in step (a) with cancer organoids and co-culturing them; (c) A step of treating the mixture of the cytotoxic T cells and cancer organoids in step (b) with an anticancer agent; and (d) A step of determining that the anticancer agent has anticancer efficacy when growth inhibition or death of the cancer organoids increases in the group treated with the anticancer agent in step (c) compared to the group not treated with the anticancer agent or the positive control group.

2. A method for screening an anticancer agent, comprising the following steps: (a) A step of mixing PBMC with cancer organoids and co-culturing them to obtain cytotoxic T cells; (b) A step of mixing the cytotoxic T cells obtained in step (a) with cancer organoids and co-culturing them; (c) A step of treating the mixture of the cytotoxic T cells and cancer organoids in step (b) with a candidate anticancer substance; and (d) A step of determining the candidate substance as an anticancer agent when growth inhibition or death of the cancer organoids increases in the group treated with the candidate anticancer substance in step (c) compared to the group not treated with the candidate anticancer substance.

3. The method according to claim 1 or claim 2, wherein the PBMC in step (a) is derived from a normal individual.

4. The method according to claim 1 or claim 2, wherein the cancer organoids in step (a) are derived from a cancer patient.

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

6. The method according to claim 1 or claim 2, wherein the co-culture in step (a) is carried out for 14 to 42 days.

7. The anticancer agent according to the method of claim 1 or claim 2 is one or more selected from the group consisting of a compound, a peptide, a peptidomimetic, a fusion protein, an antibody, an aptamer, an antibody-drug conjugate (ADC), an antisense nucleic acid that binds complementarily to DNA or mRNA, siRNA, shRNA, miRNA, and a ribozyme.

8. The cancer according to the method of claim 1 or claim 2 is one or more 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, bone cancer, 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, blood cancer, lymphoma, and fibroadenoma.

9. The growth inhibition or death of the cancer organoid in the d step is to be confirmed through an increase or decrease in the volume, area, or cell number of the cancer organoid, according to the method of claim 1 or claim 2.

10. An anticancer agent efficacy evaluation system for using the anticancer agent efficacy evaluation method according to claim 1, the anticancer agent efficacy evaluation system comprising PBMC and a cancer organoid.

11. An anticancer agent screening system for using the anticancer agent screening method according to claim 2, the anticancer agent screening system comprising PBMC and a cancer organoid.

Images