Animal model for research on cancer immunotherapy agents, and preparation method therefor

Abstract

The present invention relates to an animal model for research on cancer immunotherapy agents, and a preparation method therefor. A nude mouse, which is a mouse model widely used for evaluating the effects of anticancer agents, can verify the efficacy of a cytotoxic anticancer agent, but cannot confirm the effect of an cancer immunotherapy agent because immune function is deficient. However, a cancer animal model according to the present invention has normal immune function, thereby enabling preclinical efficacy of a cancer immunotherapy agent to be accurately predicted, and thus is expected to contribute to improving the prognosis of a patient for whom surgery is not possible.

Description

Animal model for immunotherapy research and method for manufacturing the same

[0001] The present invention relates to an animal model for research on immuno-anticancer drugs and a method for manufacturing the same.

[0002] Biliary tract cancer is a cancer that occurs in the gallbladder and other bile ducts, which are the passages through which bile is discharged. Although it is a rare type of cancer compared to other cancers, the incidence rate (0.3 to 6 per 100,000 people) and mortality rate (1 to 6 per 100,000 people) are continuously increasing worldwide. In particular, mortality rates are high in some Asian countries such as Korea, China, and Thailand, and in the case of Korea, the incidence rate exceeds 6 per 100,000 people. In Korea, the incidence rate of biliary tract cancer is increasing every year, and based on the 2021 cancer incidence statistics, it is classified as a major cancer, ranking 9th among all types of cancer.

[0003] Despite advancements in diagnosis and treatment methods over the past decade, the 5-year survival rate (7–20%) and the rate of tumor recurrence after resection for biliary tract cancer have not significantly improved. In Korea, the 5-year survival rate has risen to 30%, but this remains low compared to most other cancer types, excluding pancreatic cancer (12.6%), and the improvement in survival rates has stagnated since 2011. This is due to the high rates of recurrence and distant metastasis after surgery, as well as the large disparity in survival rates by stage. In fact, the 5-year survival rate is 50% for surgically treatable localized biliary tract cancer and 34.2% for regional biliary tract cancer, whereas it is only 3.2% for surgically inoperable cases.

[0004] The primary treatment for biliary tract cancer is surgery, but surgical eligibility is limited to only about 40–50% of all patients, and there is a high risk of recurrence and metastasis even after surgery. In cases where surgery is not possible or the disease has recurred, systemic chemotherapy or radiation therapy is required, and the combination therapy of gemcitabine and cisplatin is used as a representative palliative chemotherapy. Although this combination therapy shows tumor size reduction or progression inhibition in about 50–60% of patients, the duration of effect is only an average of 8 months, and if the first-line treatment fails, the patient's condition deteriorates rapidly, meaning that only 15–25% are actually able to receive second-line treatment. Therefore, it is difficult to expect a cure for biliary tract cancer with palliative chemotherapy alone, and there are limitations to improving survival rates.

[0005] If lesions increase or resistance develops during anticancer treatment, the drug may be changed or immunotherapy may be considered. Recently, various new biopharmaceuticals, such as antibody-drug conjugates, immune checkpoint inhibitors, and immunotherapy cell therapies, have been developed and are showing excellent anticancer effects. In addition, their therapeutic efficacy has been proven through clinical trials of immunotherapy alone in bile duct cancer and gallbladder cancer, and they are being utilized in actual treatment. For example, the immune checkpoint inhibitor Keytruda (KEYTRUDA®, pembrolizumab) is available for use in patients with PD-L1-positive advanced bile duct cancer and gallbladder cancer, and has shown therapeutic effects in approximately 17% of patients with advanced and recurrent cancer.

[0006] Immune checkpoint inhibitors account for more than one-third of the global anticancer drug market, and in Korea, usage frequency has steadily increased since 2016, reaching a high level of 26 per 100,000 people in 2022. Global sales of PD-1 / PD-L1 anticancer drugs reached approximately $35 billion in 2020–2021, with an average annual growth rate of 21% and an estimated contribution of 34% to the overall anticancer drug market growth. As of 2021, the immune checkpoint inhibitor market was valued at approximately 44 trillion won, and it is projected to nearly double to approximately 84 trillion won by 2026.

[0007] For cancers with high prevalence, various alternative therapies have been developed in addition to standard chemotherapy, providing abundant treatment options; however, in the case of biliary tract cancer, additional treatment is often difficult due to resistance and side effects when standard therapy fails. Accordingly, there is an urgent need to develop alternative therapies, such as immunotherapy cell therapies, which have fewer resistances and side effects. In particular, to improve the survival rate of inoperable patients, it is necessary to verify the utility of new immunotherapies in biliary tract cancers that are refractory to palliative chemotherapy.

[0008] Preclinical studies of such immunotherapies require new animal models for biliary tract cancer. Existing animal models make it difficult to transplant human biliary tract cancer cells due to the influence of the immune system, and it is difficult to accurately evaluate the efficacy of immunotherapies in immune-deficient models. Therefore, a new animal model is needed that can transplant human biliary tract cancer cell lines resistant to palliative anticancer drugs without immune suppression or elimination; however, such models have been unavailable until now.

[0009] Against this backdrop, the present invention was conceived in response to the need to provide an animal model for evaluating the efficacy of immunotherapies in biliary tract cancer. Since the animal model for immunotherapy research according to the present invention can accurately predict the preclinical efficacy of immunotherapies for biliary tract cancer that is refractory to conventional treatment, it is expected to contribute to improving the prognosis of patients with inoperable biliary tract cancer.

[0010] The present invention relates to an animal model for research on immunotherapeutic agents and a method for manufacturing the same,

[0011] One objective of the present invention is to provide a method for producing a cancer animal model having normal immune function.

[0012] Another objective of the present invention is to provide a cancer animal model having normal immune function.

[0013] Another objective of the present invention is to provide a screening method for anticancer drug candidates using a cancer animal model with normal immune function.

[0014] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below.

[0015] Various embodiments described herein are described with reference to the drawings. In the following description, for a complete understanding of the invention, various specific details, such as specific forms, compositions, and processes, are described. However, specific embodiments may be practiced without one or more of these specific details, or in combination with other known methods and forms. In other examples, known processes and manufacturing techniques are not described as specific details so as not to unnecessarily obscure the invention. Reference throughout this specification to "one embodiment" or "an embodiment" means that the particular features, forms, compositions, or characteristics described in association with the embodiment are included in one or more embodiments of the invention. Accordingly, the context of "in one embodiment" or "an embodiment" expressed at various places throughout this specification does not necessarily represent the same embodiment of the invention. Additionally, particular features, forms, compositions, or characteristics may be combined in any suitable way in one or more embodiments.

[0016] Unless otherwise specifically defined in the specification, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains.

[0017] In this specification, "normal immune function" refers to a state in which the immune system operates appropriately to protect the body from external pathogens or abnormal cells. Normal immune function is broadly composed of innate immunity and adaptive immunity. Innate immunity is a non-specific defense system possessed from birth, and includes physical barriers such as skin and mucous membranes, phagocytic cells such as macrophages and neutrophils, NK cells, the complement system, and inflammatory responses. It acts as the first line of defense by reacting immediately upon the invasion of pathogens. Adaptive immunity is a specific immune response that recognizes and remembers specific pathogens, with T cells and B cells playing central roles. B cells are responsible for humoral immunity by producing antibodies, while T cells are responsible for cellular immunity. CD4+ T cells act as helpers that regulate other immune cells, while CD8+ T cells directly eliminate infected or abnormal cells. Although the initial response of adaptive immunity is slow, it forms immune memory, enabling a rapid and powerful response upon re-exposure to the same pathogen. Normal immune function also includes the ability to distinguish between self and non-self. The immune system must selectively eliminate only external invaders or abnormal cells without attacking its own normal cells and tissues. If this immune tolerance is disrupted, autoimmune diseases occur; if the immune response is excessive, allergies or hypersensitivity reactions appear; and if immune function is insufficient, one becomes vulnerable to infection and the risk of cancer increases. Therefore, normal immune function refers to a state in which innate and adaptive immunity work in balance to effectively eliminate pathogens while protecting one's own tissues, and to maintain an appropriate level of immune response that is neither excessive nor insufficient.

[0018] In the present invention, the animal having normal immune function may be defined as not being an animal with artificially deprived immunity, and the animal with artificially deprived immunity may be defined as an experimental animal developed for research purposes by removing specific parts of the immune system through various genetic defects. Specifically, there are Nude mouse, Severe Combined Immunodeficiency Mouse (SCID mouse), Autoimmune SCID mouse (NOD-SCID mouse), NSG mouse, NOG mouse, Rag1 knockout mouse, Rag2 knockout mouse, Rag1 / 2 double knockout mouse, β2-microglobulin knockout mouse, CD4 knockout mouse, CD8 knockout mouse, IL2Rγ knockout mouse, Artemis knockout mouse, BALB / c SCID mouse, CB-17 SCID mouse, and BRG mouse (BAvLB / c Rag2- / - IL2Rγ- / - mouse). The above nude mouse is a mouse with a Foxn1 gene mutation that lacks a thymus and is deficient in T cells; it is hairless and lacks T-cell immunity, but B cells and NK cells function normally. The SCID mouse is a mouse with a Prkdc gene mutation that is deficient in both T cells and B cells, resulting in almost no adaptive immunity, but NK cell activity is maintained. The NOD-SCID mouse is a model with a SCID mutation on a NOD background; it has lower NK cell activity than a standard SCID, which reduces xenotransplant rejection.NSG mice are a model created by adding IL2Rγ deletion to NOD-SCID, with NK cells almost completely eliminated, making them the most widely used gold standard for human cell and tissue transplantation. NOG mice are similar to NSG mice but are a strain developed in Japan with a slightly different genetic background. Rag1 and Rag2 deletion mice have the respective genes required for V(D)J recombination removed, preventing the development of mature T and B cells, while Rag1 / 2 double deletion mice are models in which both genes are removed. β2-microglobulin deletion mice lack MHC class I molecular expression, preventing the development of CD8+ T cells, while CD4 and CD8 deletion mice selectively lack CD4+ or CD8+ T cells, respectively, and are utilized for studying the roles of specific T cell populations. IL2Rγ-deficient mice exhibit impaired development of T cells, B cells, and NK cells due to a common gamma chain deletion, while Artemis-deficient mice show a phenotype similar to human SCID due to a deficiency in T cells and B cells caused by a deletion of the DNA repair enzyme Artemis. BALB / c SCID mice and CB-17 SCID mice are SCID mice created from specific genetic backgrounds and exhibit slightly different characteristics depending on the background strain, while BRG mice are a model with a double deletion of Rag2 and IL2Rγ in the BALB / c background that exhibits high immunodeficiency characteristics similar to NSG and NOG mice. These immunodeficient mouse models are essential for various life science research fields, including cancer research, immunology research, cell therapy development, and xenotransplantation experiments.

[0019] In this specification, "cancer" refers to a condition characterized by uncontrolled cell growth, whereby a mass of cells called a tumor is formed due to such abnormal cell growth, infiltrates surrounding tissues, and in severe cases, metastasizes to other organs of the body. Academically, it is also referred to as a neoplasm. Cancer is an intractable chronic disease that, even with treatment through surgery, radiation, and chemotherapy, often fails to achieve a fundamental cure, causes suffering to the patient, and ultimately leads to death. While there are various factors contributing to the development of cancer, they are classified into internal and external factors. Although the exact mechanism by which normal cells transform into cancer cells has not been precisely elucidated, it is known that a significant number of cancers arise under the influence of external factors, such as environmental factors. Internal factors include genetic factors and immunological factors, while external factors include chemical substances, radiation, and viruses. Genes involved in the development of cancer include oncogenes and tumor suppressor genes, and cancer develops when the balance between them is disrupted by the internal or external factors described above.

[0020] In this specification, "immunotherapy" refers to an anticancer drug that helps attack cancer cells by activating the human immune system. As a treatment that complements the shortcomings of existing cancer treatments, while first-generation chemotherapy drugs directly attack cancer cells and second-generation targeted anticancer drugs attack cancer-related genes, immunotherapy drugs, referred to as third-generation anticancer drugs, treat cancer by strengthening immunity. Immunotherapy drugs include immune checkpoint inhibitors, immune cell therapies, and immunoviral therapies, but the most representative among them is immune checkpoint inhibitors, which are drugs that target PD-1, PD-L1, CTLA-4, TIGIT, etc. Among these, Pembrolizumab (Keytruda) and Nivolumab (Opdivo) have been developed and approved as PD-1 inhibitors, Atezolizumab (Tecentriq) and Durvalumab (Imfinzi) as PD-L1 inhibitors, and Ipilimumab (Yervoy) as a CTLA-4 inhibitor have been developed and approved and are in use.

[0021] As a third-generation anticancer drug, immunotherapy is a treatment method that activates the body's immune system to fight cancer cells. Therefore, compared to cytotoxic chemotherapy, it generally has relatively fewer side effects and offers advantages such as overcoming the limitations of existing drugs for metastatic cancer and resolving resistance issues. However, the biggest problem with immunotherapy is its low response rate. In cancer treatment, the "response rate" refers to the percentage of patients whose tumors shrink or disappear as a result of treatment. Monotherapy with immune checkpoint inhibitors generally shows a response rate of about 20%, although this varies depending on the type of cancer. While melanoma, which responds very well to treatment, shows a high response rate of around 40%, lung cancer shows a rate of about 20%, and gastric or biliary cancers show a low response rate of about 10%. Furthermore, a phenomenon called "acute progression" has been reported in a small number of patients, where tumor growth accelerates rather than the progression of the cancer being suppressed after immunotherapy. It is understood that these vastly different treatment response rates among individuals are due to the fact that even within the same type of cancer cells, immune responses can occur differently depending on the tumor microenvironment in which the cells grow.

[0022] If immunotherapy drugs fail to produce a therapeutic effect in cancer patients or if tumor growth actually accelerates, it creates a serious problem where not only is there a burden of extremely high treatment costs, but the "golden time"—when appropriate treatment could have been possible—is missed, making treatment more difficult. Therefore, there is an urgent and critical need for technology capable of predicting the therapeutic responsiveness to specific immunotherapies on an individual patient basis prior to administration.

[0023] In this specification, the term "immunosuppressant" refers to a drug that artificially reduces or suppresses the activity of the human immune system. Immunosuppressants regulate immune responses by inhibiting the proliferation, activation, or function of immune cells. These drugs act through various mechanisms, such as inhibiting the activity of various immune cells like T cells, B cells, and macrophages, blocking the production and action of immune signaling molecules like cytokines, and interfering with interactions between immune cells. Immunosuppressants are primarily used to prevent rejection after organ transplantation, helping the transplanted organ to engraft by suppressing the immune response that recognizes and attacks the transplanted organ as a foreign substance. They are also widely used in the treatment of autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease, and psoriasis, in which case they alleviate symptoms and inhibit disease progression by reducing abnormal immune responses that attack one's own tissues.

[0024] Representative immunosuppressants include cyclophosphamide, chlorambucil, melphalan, antimetabolites, azathioprine, methotrexate, mycophenolate mofetil, mycophenolic acid, calcineurin inhibitors, cyclosporine, tacrolimus (FK506), sirolimus (rapamycin), everolimus, dexamethasone, prednisone, prednisolone, methylprednisolone, and hydrocortisone. Examples include basiliximab (IL-2R antibody), daclizumab (IL-2R antibody), Muromob-CD3 (OKT3), rituximab (anti-CD20), alemtuzumab (anti-CD52), thalidomide, lenalidomide, abatacept (CTLA-4-Ig), and belatacept (CTLA-4-Ig).

[0025] In this specification, "screening" refers to selecting a substance having a specific desired property from a candidate group of various substances using a specific operation or evaluation method. For the purposes of the present invention, the screening of the present invention involves administering a candidate substance for cancer treatment to an animal model of cancer according to the present invention, and determining the candidate substance as a cancer preventive agent or therapeutic agent if cancer is prevented, treated, or the prognosis is improved compared to a control substance by said candidate substance.

[0026] In one aspect of the present invention, the present invention provides a method for preparing a cancer animal model comprising: (a) administering an immunosuppressant to an animal having normal immune function; (b) injecting cancer cells into the animal; and (c) further administering the immunosuppressant for a certain period and then stopping.

[0027] In the method for preparing a cancer animal model having normal immune function according to the present invention, the immunosuppressant is cyclophosphamide, chlorambucil, melphalan, antimetabolite, azathioprine, methotrexate, mycophenolate mofetil, mycophenolic acid, calcineurin inhibitors, cyclosporine, tacrolimus (FK506), sirolimus (rapamycin), everolimus, dexamethasone, prednisone, prednisolone, methylprednisolone, It may be one or more selected from the group consisting of hydrocortisone, basiliximab (IL-2R antibody), daclizumab (IL-2R antibody), Muromob-CD3 (OKT3), rituximab (anti-CD20), alemtuzumab (anti-CD52), thalidomide, lenalidomide, abatacept (CTLA-4-Ig), and belatacept (CTLA-4-Ig), and preferably cyclophosphamide or dexamethasone, but is not limited thereto.

[0028] Or, in the method for preparing a cancer animal model of the present invention, the cancer is a brain tumor, meningioma, schwannoma, pituitary tumor, spinal cord tumor, oral cancer, pharyngeal cancer, laryngeal cancer, tongue cancer, salivary gland cancer, ocular cancer, lung cancer, bronchial cancer, pleural cancer, heart cancer, mediastinal cancer, esophageal cancer, stomach cancer, small intestine cancer, colorectal cancer, colon cancer, rectal cancer, liver cancer, cholangiocarcinoma, gallbladder cancer, bile duct cancer, pancreatic cancer, anal cancer, leukemia, lymphoma, multiple myeloma, myelodysplastic syndrome, myeloproliferative tumor, thyroid cancer, adrenal cancer, parathyroid cancer, pancreatic endocrine tumor, cervical cancer, endometrial cancer, ovarian cancer, vaginal cancer, vulvar cancer, prostate cancer, testicular cancer, penile cancer, skin cancer, basal cell carcinoma, squamous cell carcinoma, melanoma, soft tissue sarcoma, liposarcoma, myosarcoma, angiosarcoma, cutaneous lymphoma, osteosarcoma, The animal may be one or more selected from the group consisting of chondrosarcoma, Ewing sarcoma, neuroblastoma, Wilms tumor, retinoblastoma, acute lymphoblastic leukemia, pediatric brain tumor, basement membrane carcinoma, Kaposi's sarcoma, gastrointestinal stromal tumor, and mesothelioma, and the animal having normal immune function may be a nude mouse, severe combined immunodeficiency mouse (SCID mouse), autoimmune SCID mouse (NOD-SCID mouse), NSG mouse, NOG mouse, Rag1 knockout mouse, Rag2 knockout mouse, Rag1 / 2 double knockout mouse, β2-microglobulin knockout mouse, CD4 knockout mouse, CD8 knockout mouse, IL2Rγ knockout mouse mouse), Artemis KO mouse, BALB / c SCID mouse, CB-17 SCID mouse (C.It may not be a B-17 SCID mouse), or a BRG mouse (BAvLB / c Rag2- / - IL2Rγ- / - mouse).

[0029] Alternatively, in the method for preparing a cancer animal model of the present invention, the administration of the immunosuppressant in step (a) may be administered once or twice, and the administration for a certain period in step (c) may be administered for 7 to 21 days.

[0030] Although not limited thereto, the method for preparing a cancer animal model of the present invention may preferably comprise: (a') administering 50 to 300 mg of cyclophosphamide once or twice to a mouse with normal immune function; (b') injecting cancer cells into the animal; and (c') administering 5 to 30 mg of cyclophosphamide for 7 to 21 days and then stopping; or, (a') administering 5 to 30 mg of dexamethasone once or twice to a mouse with normal immune function; (b') injecting cancer cells into the animal; and (c') administering 0.5 to 5 mg of dexamethasone for 7 to 21 days and then stopping.

[0031] In another aspect of the present invention, the present invention provides a cancer animal model having normal immune function, prepared by the method for preparing a cancer animal model described above.

[0032] The above cancer animal model is not a Nude mouse, Severe Combined Immunodeficiency Mouse (SCID mouse), Autoimmune SCID Mouse (NOD-SCID mouse), NSG mouse, NOG mouse, Rag1 knockout mouse, Rag2 knockout mouse, Rag1 / 2 double knockout mouse, β2-microglobulin knockout mouse, CD4 knockout mouse, CD8 knockout mouse, IL2Rγ knockout mouse, Artemis KO mouse, BALB / c SCID mouse, CB-17 SCID mouse, or BRG mouse. It may be characterized by that.

[0033] In another aspect of the present invention, the present invention provides a screening method for a candidate substance for cancer treatment, comprising: (a) preparing a cancer animal model having normal immune function prepared by the method for preparing a cancer animal model described above; and (b) administering a candidate substance for cancer treatment to the animal model. Herein, the screening method may determine that the candidate substance is effective for cancer treatment if the growth of the cancer tissue is delayed or its size is reduced after administration of the candidate substance.

[0034] Unlike conventional animal models using immunodeficient mice such as nude mice, the animal model of the present invention is formed in mice with normal immune function, thus enabling accurate evaluation of the preclinical efficacy of immunotherapeutic agents.

[0035] Figures 1 to 5 show a schematic diagram of the manufacturing process, tumor growth results, and tumor characteristics in a bile duct cancer xenograft animal model prepared in nude mice.

[0036] Figures 6 to 16 show the manufacturing process, tumor growth results, and tumor characteristics in an immunosuppressive-immune-recovering bile duct cancer xenograft animal model (pulse method using cyclophosphamide) prepared in a normal mouse.

[0037] FIGS. 17 to 24 show the manufacturing process, tumor growth results, and tumor characteristics in an immunosuppressive-immune-recovering bile duct cancer xenograft animal model (continuous injection method using cyclophosphamide) prepared in a normal mouse.

[0038] FIGS. 25 to 36 show the manufacturing process, tumor growth results, and tumor characteristics in an immunosuppressive-immune-recovering bile duct cancer xenograft animal model (pulse therapy using Dexamethasone) prepared in a normal mouse.

[0039] Figures 37 to 48 show the manufacturing process, tumor growth results, and tumor characteristics in an immunosuppressive-immune-recovering bile duct cancer xenograft animal model prepared in a normal mouse (confirmation of recovery period after pulse therapy using Dexamethasone).

[0040] FIGS. 49 to 52 show the results of a comparison of differentially expressed genes (DEGs) between a nude mouse animal model and an immunosuppressed mouse animal model prepared in the present invention.

[0041] FIGS. 53 to 58 show the results of confirming the patterns of immune-related pathway regulation in a nude mouse model, a dexamethasone pulse mouse model, and a dexamethasone maintain mouse model prepared in the present invention.

[0042] We compared the expression patterns of immune-related target genes in a biliary tract cancer transplantation model between normal mice with induced immunosuppression using dexamethasone and immunodeficient nude mice. In nude mice, T cells are congenitally deficient, so the expression of genes involved in the immune system was hardly observed. Consequently, the expression of genes that induce immune responses, particularly PD-1-related genes corresponding to immune checkpoint pathways, was not confirmed within the tumor tissue. On the other hand, in normal mice with induced immunosuppression using dexamethasone, the expression of immune target genes, including the PD-1 pathway, was clearly observed within the tumor tissue, suggesting that the immune response was activated as the tumor grew while immune function was partially maintained.

[0043] The present invention will be described in more detail below through examples. These examples are intended solely to explain the present invention more specifically, and it will be obvious to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the invention.

[0044] Example.

[0045] 1. Preparation of a Biliary Tract Cancer Xenograft Model in Nude Mice

[0046] A xenograft model of biliary tract cancer was established in Balb / c nude mice using a human biliary tract cancer cell line (SCK cell line), and tumor growth patterns and physiological changes were observed. 5-week-old Balb / c nude mice were subjected to SCK cell line 1 x 10⁶ 6 Cells / 100 µl were injected into the dorsal subcutaneous tissue, and it took about 1 to 2 weeks for tumor formation to occur. Tumor size measurements were initiated based on the tumor being hard upon palpation, and subsequently, the mouse body weight was measured twice a week and tumor size was measured at 3-day intervals (Fig. 1).

[0047] As a result of the experiment, the body weight of the mice continued to increase without decrease even under immunosuppression, and no systemic side effects were observed. The tumors grew gradually over time, and a rapid increase in size was observed particularly starting from day 17 of the experiment (Fig. 2). This is considered to reflect the period of active tumor proliferation. After the experiment ended, tumor and liver tissues were excised from each individual and their weights were compared; the results showed no significant differences between individuals (Table 1), suggesting that physiological uniformity was ensured among the models.

[0048] Weight of liver and tumor excised from mouse N Liver (g) Tumor (g) SCK - 11.5 1.7 SCK - 2 1.3 1.5 SCK - 3 1.1 0.7

[0049] On day 24, tumor tissue was excised from individuals with sufficiently grown tumors. The excised tumors were localized to the dorsal side, and connectivity with surrounding blood vessels was observed (Fig. 3). The tumor tissue and liver tissue were separated to measure their size and weight (Fig. 4), and the histological characteristics of the tumors were confirmed through pathological analysis. Necrosis was present within the tumors, and a high cell density and numerous spindle-shaped cells were observed (Fig. 5, from left: Х8, Х100, Х200, Х400). The significance of this experiment lies in providing a foundation for the preclinical evaluation of immunotherapies with secured tumor growth and physiological stability.

[0050] 2. Preparation of an immunosuppressive-immunorecovery biliary tract cancer xenograft model in normal mice - Pulse method using cyclophosphamide

[0051] A biliary tract cancer transplantation model was established in standard mice (C57BL / 6J) using the pulse method of immunosuppression followed by immune recovery with cyclophosphamide, and tumor growth and physiological responses were evaluated according to immune status. The mice used in the experiment were 5-week-old C57BL / 6J individuals, and the human biliary tract cancer cell line (SCK cell line, 1 X 10⁶ 6 Tumors were induced by injecting cells (100 µl / 100 µl) into the dorsal subcutaneous tissue.

[0052] Mice were divided into a total of four groups and administered cyclophosphamide according to the immunosuppression and maintenance phases (Table 2 and Fig. 6). It took approximately 1 to 2 weeks for tumor formation to occur, and size measurements began from the point when the tumors had hardened. Mouse body weight was measured twice a week, and tumor size was measured at 3-day intervals (Fig. 7). Through this, the tumor growth patterns according to the recovery period after immunosuppression were compared.

[0053] Group Drug Administration Group 1 (Control Group) Inducted phase: Immunosuppressant (Cyclophosphamide 100 mg) injected intraperitoneally (IP) on days 1 and 5. Maintain phase: Saline injected IP on days 8–28. Group 2 (Experimental Group) Inducted phase: Immunosuppressant (Cyclophosphamide 100 mg) injected IP on days 1 and 5. Maintain phase: Cyclophosphamide 10 mg IP injection on days 8–14 (7 days). Group 3 (Experimental Group) Inducted phase: Immunosuppressant (Cyclophosphamide 100 mg) injected IP on days 1 and 5. Maintain phase: Cyclophosphamide 10 mg IP injection on days 8–21 (14 days). Group 4 (Experimental Group) Inducted phase: Immunosuppressant (Cyclophosphamide 100 mg) IP on days 1 and 5. Injection Maintain phase Days 8–28 (21 days) Cyclophosphamide 10 mg IP injection

[0054] As a result of the experiment, mouse body weight temporarily decreased immediately after the administration of a high dose of cyclophosphamide but recovered within 2–3 days and showed a sustained overall increase, with no systemic side effects observed (Fig. 8). Tumors grew stably in Group 1 (control group) (Fig. 9). In addition, the histological characteristics of the tumors were confirmed through pathological analysis (Fig. 10, from left: X8, X100, X200, X400). Conversely, in the experimental groups, the shorter the maintenance administration period, the more often tumor traces were minimal or only blood vessels were observed. In Group 2, only tumor traces were observed in most cases (Fig. 11), and in Group 3, tumors grew in some individuals, while only microvessels were identified in the others (Fig. 12). Pathological findings of tumor tissue excised from Group 3 mice showed high tumor cell density accompanied by necrosis (Fig. 13, from left: X8, X100, X200, X400). In Group 4 mice, the tumor attached to and grew in the dorsal subcutaneous region in Case 3 as well, and in Case 1, the tumor morphology and surrounding blood vessels were observed more clearly compared to other experimental groups. In Case 2, only traces of microvessels and the tumor were observed (Fig. 14). Pathological findings of the tumor tissue excised from Group 4 mice revealed high-density tumor cells accompanied by necrosis (Fig. 15, from left: Х8, Х100, Х200, Х400). Additionally, CBC tests using peripheral blood and pathological analysis of the excised tumor tissue were performed to evaluate the effects of immunosuppression and recovery processes on tumor growth and histological characteristics (Table 3).

[0055] normal rangeGroup 1(n=6)Group 2(n=3)Group 3(n=3)Group 4(n=3)p-valueWBC (10^3 / uL), median (IQR)3.0-14.24.22(3.29-4.83)3.40(3.06-3.80)3.25(2.62-4.93)3.97(2.57-4.07)0.690RBC (10^6 / uL), median (IQR)5.0-9.58.6(8.6- 8.6)8.6(8.6- 8.6)8.6(8.6- 8.6)8.6(8.6- 8.6)1.000Hct (%),median (IQR)42.1-68.353.4(50.8-54.0)54.6(54.6-56.6)54.8(53.1-57.2)51.5(51.4-53.3)0.057Hb (g / dL),median (IQR)10.9-16.315.9(15.4-16.2)16.4(16.4-16.6)16.3(16.0-16.6)15.7(15.2-16.3)0.046MCHC (g / dL),median (IQR)23.3-32.729.8(29.2-30.8)30.0(29.3-30.0)30.1(28.5-30.3)30.1(29.5-30.6)0.732MCV (fL),median (IQR)44.0-55.052.0(51.9-53.8)53.4(53.3-53.5)54.0(53.8-54.4)54.9(54.8-55.2)0.023MPV (fL),median (IQR)4.6-5.97.0(6.9-7.1)7.0(7.0-7.1)7.1(7.0-7.2)7.1(6.9-7.4)0.642Lymphocyte (%),median (IQR)48.8-83.184.7(59.9-95.4)80.6(79.7-83.2)81.7(81.2-85.6)77.4(67.3-82.6)0.753

[0056] Synthesizing the pathological findings of the excised tumor tissues, the experimental groups (Groups 3 and 4), administered cyclophosphamide continuously after pulse, had smaller tumor sizes and exhibited necrosis compared to the control group (Group 1), which was administered cyclophosphamide only by pulse. However, the morphological findings of the tumor cells were similar to those of the control group (Fig. 16, left X8, right X400). In Group 2, no tumor tissue grew. This model is significant in that it establishes a foundation for providing conditions suitable for the preclinical evaluation of immunotherapies by inducing tumors using immunosuppressants in normal mice with preserved immune function and subsequently regulating the immune recovery process.

[0057] 3. Preparation of an immunosuppressive-immunorecovery biliary tract cancer xenograft model in normal mice - Continuous injection method using cyclophosphamide

[0058] A biliary tract cancer transplantation model was established in standard mice (C57BL / 6J) using a continuous injection method with cyclophosphamide, and tumor growth and physiological responses were evaluated according to immune status. The mice used in the experiment were 5-week-old C57BL / 6J individuals, and the human biliary tract cancer cell line (SCK cell line, 1 X 10⁶ 6 Tumors were induced by injecting cells (100 µl / 100 µl) into the dorsal subcutaneous tissue.

[0059] The mice were divided into a total of two groups for the experiment (Table 4 and Fig. 17). It took approximately 1 to 2 weeks for tumor formation to occur, and size measurements began from the point when the tumors had hardened. The body weight of the mice was measured twice a week, and the tumor size was measured at 3-day intervals (Fig. 18).

[0060] Group Drug Administration Group 1 (Control Group) Inducted phase: IP injection of saline on days 1–10 (10 days) Group 2 (Experimental Group) Inducted phase: IP injection of an immunosuppressant (Cyclophosphamide 50 mg) on ​​days 1–10 (10 days)

[0061] As a result of the experiment, only minor changes were observed in the mouse body weight during the cyclophosphamide administration period, showing a continuous overall increase, and no systemic side effects were observed (Fig. 19). Tumors grew stably in the control group; in some individuals, tumors were observed attached to the dorsal side, while in others, only microvascular and tumor traces were identified (Fig. 20). Pathological findings of tumor tissue excised from Group 1 mice showed high tumor cell density and the presence of some necrotic areas (Fig. 21, from left: X8, X100, X200, X400). In the experimental group, after continuous administration of cyclophosphamide for 10 days, tumors grew in some individuals, but most retained only microvascular traces, and clear tumor tissue formation was limited (Fig. 22). Pathological findings of tumor tissue excised from Group 2 mice showed low cell density within the tissue and no clear tumor formation was observed (Fig. 23, from left: X8, X100, X200, X400). Additionally, a CBC test using peripheral blood (Table 5) and a pathological analysis of the excised tumor tissue were performed to evaluate the effects of immunosuppressive conditions on tumor growth and histological characteristics.

[0062] normal rangeGroup (n=3)Group 2 (n=3)p-valueWBC (10^3 / uL), median (IQR)3.0-14.23.49 (3.07-3.58)3.38 (3.11-3.64)1.000RBC (10^6 / uL), median (IQR)5.0-9.58.60(8.60 - 8.60)8.60(8.60 - 8.60)1.000Hct (%),median (IQR)42.1-68.353.8 (49.9-54.3)51.7 (51.7-52.2)0.700Hb (g / dL),median (IQR)10.9-16.316.0 (15.6-16.2)15.6 (15.5-15.6)0.100MCHC (g / dL),median (IQR)23.3-32.729.8 (29.7-31.3)30.0 (29.9-30.0)0.700MCV (fL),median (IQR)44.0-55.051.7 (51.6-52.9)53.0 (53.0-53.0)0.100MPV (fL),median (IQR)4.6-5.97.1 (7.1-7.2)7.0 (6.9-7.0)0.100Lymphocyte (%),median (IQR)48.8-83.192.7 (79.5-96.6)93.7 (82.6-93.7)1.000

[0063] Synthesizing the pathological findings of the excised tumor tissues, there were no significant morphological differences between the control group (Group 1) and the experimental group (Group 2). Tumor cell density was similar in both groups, and some necrotic areas were observed (Fig. 24, left X8, right X400). This model is significant in that it establishes a foundation for providing conditions suitable for the preclinical evaluation of immunotherapies by continuously inducing immunosuppression in normal mice.

[0064] 4. Preparation of an Immunosuppressive-Immune-Recovering Biliary Tract Cancer Xenograft Model in Standard Mice - Pulse Therapy Using Dexamethasone

[0065] Immunosuppression-immune recovery conditions were induced in standard mice (C57BL / 6J) using the steroid dexamethasone, and based on this, a biliary tract cancer transplantation model was constructed to evaluate tumor growth and physiological responses. The mice used in the experiment were 5-week-old C57BL / 6J individuals, and the human biliary tract cancer cell line (SCK cell line, 1 X 10⁶ 6 Tumors were induced by injecting cells (100 µl / 100 µl) into the dorsal subcutaneous tissue.

[0066] Mice were divided into a total of four groups for the experiment (Table 6 and Fig. 25). It took approximately 1 to 2 weeks for tumor formation to occur, and size measurements began from the point when the tumors had hardened. Mouse body weight was measured twice a week, and tumor size was measured at 3-day intervals (Fig. 26). Through this, the tumor growth patterns according to the recovery period after immunosuppression were compared.

[0067] Group Drug Administration Group 1 (Control Group) Inducted phase: Immunosuppressant (Dexamethasone 10 mg) injected intraperitoneally (IP) on days 1 and 5 Maintain phase: Saline injected IP on days 8–28 Group 2 (Experimental Group) Inducted phase: Immunosuppressant (Dexamethasone 10 mg) injected IP on days 1 and 5 Maintain phase: Dexamethasone 2 mg IP injection on days 8–14 (7 days) Group 3 (Experimental Group) Inducted phase: Immunosuppressant (Dexamethasone 10 mg) injected IP on days 1 and 5 Maintain phase: Dexamethasone 2 mg IP injection on days 8–21 (14 days) Group 4 (Experimental Group) Inducted phase: Immunosuppressant (Dexamethasone 10 mg) on ​​days 1 and 5 IP injection Maintain phase Days 8–28 (21 days) Dexamethasone 2 mg IP injection

[0068] As a result of the experiment, the body weight of the mice continuously increased without decrease during the dexamethasone administration period, and no systemic side effects were observed (Fig. 27). In the control group, tumors attached and grew in the dorsal subcutaneous region in Cases 1 and 3, while a fine tumor was observed in Case 2 (Fig. 28) (Fig. 29, from left: Х8, Х100, Х200, Х400). In the experimental group, the shorter the maintenance administration period, the more frequently tumor traces were minimal or only blood vessels were observed (Fig. 30). In Group 2 mice, Cases 1 and 2 had small tumor sizes accompanied by necrosis, and the morphology of the tumor cells was similar to that of the control group. The Case 3 sample was lost during the staining process (Fig. 31, from left: Х8, Х100, Х200, Х400). In Group 3 mice, tumors grew in the dorsal subcutaneous region (Fig. 32), the tumors were small and accompanied by necrosis, and the tumor cell morphology was similar to that of the control group (Fig. 33, from left: Х8, Х100, Х200, Х400). In Group 4 mice, tumors grew in the dorsal subcutaneous region (Fig. 34), the tumors were larger than those of the other groups, accompanied by necrosis, and the tumor cell morphology was similar (Fig. 35, from left: Х8, Х100, Х200, Х400). Additionally, CBC tests using peripheral blood and pathological analysis of excised tumor tissues were performed to evaluate the effects of steroid-based immunosuppressive conditions on tumor growth and histological characteristics (Table 7).

[0069] normal rangeGroup 1(n=3)Group 2(n=2)Group 3(n=2)Group 4(n=2)p-valueWBC (10^3 / uL), median (IQR)3.0-14.23.594.153.393.320.707RBC (10^6 / uL), median (IQR)5.0-9.58.608.608.608.601.000Hct (%),median (IQR)42.1-68.350.3053.4550.9052.850.261Hb (g / dL),median (IQR)10.9-16.316.3015.6015.6015.950.225MCHC (g / dL), median (IQR)23.3-32.730.4030.5030.9030.200.308MCV (fL),median (IQR)44.0-55.054.0053.8552.9054.450.171MPV (fL),median (IQR)4.6-5.96.907.207.207.200.111Lymphocyte (%),median (IQR)48.8-83.183.6096.8071.1059.500.077

[0070] Synthesizing the pathological findings of the excised tumor tissues, compared to the control group, the tumors in the 7-day and 14-day administration groups were smaller and accompanied by necrosis, while the morphology of the tumor cells was similar. In the 21-day administration group, the tumors were larger than in the other groups and accompanied by necrosis, and the morphology of the tumor cells was observed to be identical (Fig. 36, left X8, right X400). This model is significant in that it establishes a foundation for providing conditions suitable for the preclinical evaluation of immunotherapies through immunomodulation using dexamethasone in normal mice.

[0071] 5. Preparation of an Immunosuppressive-Immune-Recovering Biliary Tract Cancer Xenograft Model in Standard Mice - Verification of Recovery Period After Pulse Therapy Using Dexamethasone

[0072] Immunosuppression-immune recovery conditions were induced in standard mice (C57BL / 6J) using the steroid dexamethasone, and the growth and physiological response of biliary tract cancer were evaluated during the recovery period. The mice used in the experiment were 5-week-old C57BL / 6J individuals, and the human biliary tract cancer cell line (SCK cell line, 1 X 10⁶ 6 Tumors were induced by injecting cells (100 µl / 100 µl) into the dorsal subcutaneous tissue.

[0073] Mice were divided into a total of four groups for the experiment (Table 8 and Fig. 37). It took approximately 1 to 2 weeks for tumor formation, and size measurements began from the point when the tumors had hardened. Mouse body weight was measured twice a week, and tumor size was measured at 3-day intervals. After a 2-week immune recovery period, the mice were sacrificed for analysis (Fig. 38). Although observation for 3 weeks was planned after the end of administration, sampling was conducted at the 2-week mark due to the rapid increase in tumor size.

[0074] Group Drug Administration Group 1 (Control Group) Inducted phase: Immunosuppressant (Dexamethasone 10 mg) injected intraperitoneally (IP) on days 1 and 5 Maintain phase: Saline injected IP on days 8–28 Group 2 (Experimental Group) Inducted phase: Immunosuppressant (Dexamethasone 10 mg) injected IP on days 1 and 5 Maintain phase: Dexamethasone 2 mg IP injection on days 8–14 (7 days) Group 3 (Experimental Group) Inducted phase: Immunosuppressant (Dexamethasone 10 mg) injected IP on days 1 and 5 Maintain phase: Dexamethasone 2 mg IP injection on days 8–21 (14 days) Group 4 (Experimental Group) Inducted phase: Immunosuppressant (Dexamethasone 10 mg) on ​​days 1 and 5 IP injection Maintain phase Days 8–28 (21 days) Dexamethasone 2 mg IP injection

[0075] As a result of the experiment, the body weight of the mice continuously increased without decrease during the period of dexamethasone administration, and no systemic side effects were observed (Fig. 39). In the control group, tumors attached to the dorsal subcutaneous area and grew significantly (Fig. 40) (Fig. 41, from left: X8, X100, X200, X400). In the case of Group 2 mice, tumors attached to the dorsal subcutaneous area and grew significantly (Fig. 42) (Fig. 43, from left: X8, X100, X200, X400), and in Group 3 mice, tumors attached to the dorsal subcutaneous area and grew significantly (Fig. 44) (Fig. 45, from left: X8, X100, X200, X400). In Group 4 mice as well, tumors attached to the dorsal subcutaneous region and grew distinctly (Fig. 46) (Fig. 47, from left: X8, X100, X200, X400). CBC results using peripheral blood showed that white blood cell (WBC) counts in the experimental group tended to increase slightly compared to the control group, but there was no statistically significant difference (p=0.151). Most hematological indicators, such as red blood cells (RBC), hematocrit (Hct), hemoglobin (Hb), mean corpuscular volume (MCV), and mean platelet volume (MPV), remained within the normal range, and no significant differences were observed between the groups. The lymphocyte ratio increased slightly in the experimental group, but some groups also showed a decreasing trend, suggesting variability in the immune recovery process (p=0.064) (Table 9).

[0076] normal rangeGroup 1(n=6)Group 2(n=4)Group 3(n=6)Group 4(n=5)p-valueWBC (10^3 / uL), median (IQR)3.0-14.23.46(2.90-3.72)4.44(4.03-5.69)4.23(3.56-5.26)3.40(3.20-3.60)0.151RBC (10^6 / uL), median (IQR)5.0-9.58.60(8.60-8.60)8.60(8.60-8.60)8.60(8.60-8.60)8.60(8.60-8.60)1.000Hct (%),median (IQR)42.1-68.350.25(49.22-51.05)51.85(50.25-53.00)52.45(49.92-54.22)52.85(51.92-53.80)0.505Hb (g / dL),median (IQR)10.9-16.315.25(14.85-15.58)16.00(15.42-16.35)16.20(15.62-16.77)15.95(15.90-16.27)0.219MCHC (g / dL),median (IQR)23.3-32.730.20(29.95-30.38)30.75(30.60-30.85)31.00(30.82-31.1)30.80(30.35-31.15)0.054MCV (fL),median (IQR)44.0-55.054.10(54.02-54.62)53.55(53.48-53.78)53.00(52.75-53.25)53.70(53.25-54.28)0.169MPV (fL),median (IQR)4.6-5.97.00(6.90-7.18)7.15(6.98-7.32)7.20(7.12-7.28)7.20(7.18-7.22)0.224Lymphocyte (%),median (IQR)48.8-83.182.15(60.15-91.85)85.65(73.12-96.15)92.95(76.42-93.95)62.35(60.08-64.88)0.064

[0077] Synthesizing the pathological findings of the excised tumor tissues, there was no significant difference in tumor size between the control group and the experimental group, and necrosis was present in most tumors. The morphology of the tumor cells was observed to be similar between the groups (Fig. 48, left X8, right X400). This model can be utilized as an effective preclinical platform to evaluate the biological response of biliary tract cancer during the immune recovery period through dexamethasone-based immunomodulation.

[0078] 6. Comparison of Differentially Expressed Genes (DEGs) Between Nude Mice and Immunosuppressed Mice

[0079] Transcriptome analysis was performed to compare differences in gene expression between nude mice and immunosuppressive mice in a biliary tract cancer transplantation model. Through this, the patterns of gene expression within the tumor and the activity of immune-related target genes were evaluated according to immune status.

[0080] When comparing the genes expressed in tumor tissues between mice with immunosuppression induced using cyclophosphamide and nude mice, almost no expression of genes involved in the immune system was observed in nude mice. On the other hand, in cyclophosphamide-induced immunosuppressed mice, various immune-related genes, including T cells, were found to be activated and expressed (red box) (Fig. 49). This suggests that an immune response was induced as the tumor grew while immune function was partially maintained.

[0081] The same analysis was performed on immunosuppressed mice using dexamethasone, and while there was almost no expression of immune-related genes similar to nude mice, it was confirmed that T cell-related genes and other immune response genes were expressed in dexamethasone-induced mice (red box) (Fig. 50). This demonstrates that the immune response within the tumor is partially maintained even under steroid-based immunomodulation conditions.

[0082] Additionally, the activity of immune checkpoint-related genes, including the PD-1 immune checkpoint pathway, was evaluated by comparing the expression of immune target genes. In cyclophosphamide-induced mice, immune target genes including the PD-1 pathway were expressed, which indicates the presence of a target for the immunotherapy drug (red box) (Fig. 51). A similar trend was observed in dexamethasone-induced mice, which is interpreted as a result suggesting the applicability of immune checkpoint inhibitors.

[0083] Additionally, the expression patterns of immune-related target genes in a biliary tract cancer transplantation model were compared between normal mice with induced immunosuppression using dexamethasone and immunodeficient nude mice (Fig. 52). In nude mice, T cells are congenitally deficient, so the expression of genes involved in the immune system was hardly observed. Consequently, the expression of genes that induce immune responses, particularly PD-1 related genes corresponding to the immune checkpoint pathway, was not confirmed within the tumor tissue. On the other hand, in normal mice with induced immunosuppression using dexamethasone, the expression of immune target genes, including the PD-1 pathway, was clearly observed within the tumor tissue, suggesting that the immune response was activated as the tumor grew while immune function was partially maintained.

[0084] 7. Comparison of nude mice, dexamethasone pulse mice, and dexamethasone maintain mice

[0085] (1) KEGG Pathway Analysis

[0086] In the dexamethasone pulse group, immune-related pathways were generally upregulated compared to the xenograft model of biliary tract cancer. The major upregulated pathways included antigen presentation, T-cell receptor signaling, and cytokine-cytokine receptor interaction, reflecting a state where both adaptive and innate immunity were activated. Additionally, an increase in natural killer cell-mediated cytotoxicity and cell adhesion molecules (CAMs) pathways was also associated with the intensification of the inflammatory response (Fig. 53). On the other hand, in the dexamethasone maintenance group, overall inhibition of immune-related pathways was observed due to long-term exposure to dexamethasone. Pathways such as antigen presentation, T-cell receptor signaling, cytokine-cytokine receptor interaction, and hematopoietic cell lineage were significantly reduced compared to the dexamethasone pulse group. This shows that long-term exposure to dexamethasone inhibits the activation and differentiation of immune cells, thereby inducing immune tolerance (Fig. 54).

[0087] (2) TF Motif Activity Analysis and Immune Checkpoint Gene Expression

[0088] Transcriptomics motif analysis revealed that the motif activities of NF-κB-related transcription factors (Nfkb1, Nfkb2, Rela) and T-cell activation-related transcription factors (Tcf21, Runx1) were significantly elevated in the dexamethasone pulse group. This is consistent with the immune pathway activation observed in the KEGG analysis and suggests that short-term dexamethasone exposure temporarily activates inflammation-related transcriptional networks (Fig. 55). Conversely, the activity of these transcription factors was distinctly reduced in the dexamethasone maintenance group, indicating that long-term dexamethasone exposure reprograms the immunosuppressive program at the transcriptional level. Additionally, in the immune checkpoint gene heatmap analysis, genes related to immunosuppression and T-cell exhaustion, such as Pdcd1 (PD-1), Ctla4, Lag3, Tigit, Tox, and Eomes, were distinctly increased in the dexamethasone maintenance group. These results indicate that the dexamethasone maintenance model features an immunosuppressive environment with an enhanced PD-1 / CTLA-4 / LAG-3 axis (Fig. 56).

[0089] (3) T-cell Activation and Signaling Gene Heatmap

[0090] Genes related to T-cell receptor signaling and cytokine-mediated activation were significantly increased in the dexamethasone pulse group. The increased expression of Cd3d, Cd3e, Zap70, Lck, Nfatc1, Ifng, Il2ra, and Cd69 indicates that T-cell activation via the NF-κB-TCR axis is enhanced upon dexamethasone pulse treatment. Conversely, in the dexamethasone maintenance group, the expression of these genes was suppressed, showing a transition to a state of immune tolerance (Fig. 57).

[0091] (4) Transcription Factor Information Content and Motif Distribution

[0092] In motifs such as Rela_Nfkb1, Nfkb2, Tcf21, and Runx1, the dexamethasone pulse group showed higher information content and a wider distribution of motif activity compared to the xenograft model. On the other hand, in the dexamethasone maintenance group, the motif information content remained generally low, and transcriptional repression was observed. This implies that continuous exposure to dexamethasone induces a state of transcriptional immunosuppression (Fig. 58).

[0093] The results of Examples 1 to 7 of the present invention are combined and summarized as follows:

[0094] 1. Nude mice, commonly used as mouse models to verify the effects of anticancer drugs, are useful for evaluating the efficacy of cytotoxic agents, but they cannot evaluate the effects of immunotherapies because they are in an immunodeficient state. On the other hand, while normal mice have a preserved immune system, transplanted cancer cells cannot grow due to the immune response; therefore, syngeneic mice lacking specific immunity have been developed. However, these models have limitations, such as being specific to cancer types and having high production costs.

[0095] 2. In this study, to induce the growth of cancer tissue in normal mice, we temporarily suppressed immunity using cyclophosphamide and dexamethasone and transplanted cancer cells under these conditions. Subsequently, by stopping the administration of immunosuppressants after the cancer tissue had grown, we established a new model in which the cancer tissue is maintained even after immunity is restored.

[0096] 3. The tumors formed in this immunosuppression-immune recovery mouse model were pathologically (H&E staining) similar to nude mouse tumors, but there were distinct differences in the expression of immune-related genes at the transcriptome level. In particular, immune checkpoint-related genes, including PD-L1, were expressed only in the immunosuppression-immune recovery model, suggesting that this model may reflect responsiveness to immunotherapies.

[0097] 4. There are various types of immunosuppressants, and although not all drugs were tested in this study, it is highly likely that the same response would be obtained for any drug capable of inducing immunosuppression. In addition, tumor tissue growth was possible in both transient high-dose administration and low-dose maintenance therapy following high dose. This study is the first to establish such an immunosuppression-immune recovery model and presents a new preclinical model capable of evaluating the effects of immune-related anticancer drugs in normal mice.

[0098] 5. Although this study focused on biliary tract cancer, it was confirmed that immune-preserving tumors can be formed in a pancreatic cancer model using the same immunosuppression-immune recovery strategy. This implies that it can be utilized in the future to evaluate the efficacy of immunotherapies in various solid tumors, not just biliary tract and pancreatic cancers.

[0099] 6. The immunosuppression-immune recovery-based model established in this study is a novel model in which cancer cells are transplanted after the immune system is temporarily suppressed by administering cyclophosphamide or dexamethasone to normal mice, and then the immune system is restored so that the tumor exists while the immune system is functioning. This is an original approach that enables the coexistence of the immune system and the tumor.

[0100] 7. The tumors grown in the model were pathologically similar to nude mouse tumors, but transcriptome analysis showed that PD-1 and T-cell activation-related gene expression was maintained, exhibiting immune activation. This provides a preclinical advantage of being able to mimic the responsiveness of immunotherapies.

[0101] 8. Dexamethasone exhibited the characteristic of regulating immunity in a time-dependent manner. Short-term administration stimulated the NF-κB TCR axis to induce immune activation, but long-term administration enhanced the PD-1, CTLA-4, and LAG-3 axes, leading to immune tolerance and T-cell exhaustion. This result demonstrates the time-dependent immunomodulation of dexamethasone at the molecular level.

[0102] 9. In the long-term dexamethasone exposure group, the activity of transcription factors such as NF-κB, Runx1, and Tcf21, as well as the amount of motif information, were generally reduced, and a "transcriptionally immune-repressed state" in which immune-related transcriptional programs are suppressed was identified. This supports the fact that the long-term dexamethasone maintenance model is a valid preclinical model that reproduces an environment of immune tolerance.

[0103] 10. In conclusion, the immunosuppression-immune recovery mouse model established in this study overcomes the limitations of existing nude mice and presents a new preclinical platform capable of evaluating the efficacy of immunotherapies in normal mice. In particular, since the dexamethasone maintenance model forms an immunosuppressive environment with an enhanced PD-1 / CTLA-4 axis, it can be utilized as important basic data for evaluating the responsiveness of anti-PD-1 class immune checkpoint inhibitors and designing combination therapy studies.

[0104] Specific parts of the present invention have been described in detail above. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

[0105] The animal model for immunotherapy research of the present invention is expected to contribute to improving the prognosis of patients with inoperable biliary tract cancer, as it can accurately predict the preclinical efficacy of immunotherapy for biliary tract cancer that is refractory to conventional treatment.

Claims

1. (a) A step of administering an immunosuppressant to an animal with normal immune function; (b) a step of injecting cancer cells into the animal; and, (c) a step of discontinuing the administration of an immunosuppressant after a certain period; comprising a method for preparing an animal model of cancer.

2. In Paragraph 1, The above-mentioned immunosuppressants are cyclophosphamide, chlorambucil, melphalan, antimetabolite, azathioprine, methotrexate, mycophenolate mofetil, mycophenolic acid, calcineurin inhibitors, cyclosporine, tacrolimus (FK506), sirolimus (rapamycin), everolimus, dexamethasone, prednisone, prednisolone, methylprednisolone, hydrocortisone, A method of manufacturing, wherein one or more selected from the group consisting of basiliximab (IL-2R antibody), daclizumab (IL-2R antibody), Muromoab-CD3 (OKT3), rituximab (anti-CD20), alemtuzumab (anti-CD52), thalidomide, lenalidomide, abatacept (CTLA-4-Ig), and belatacept (CTLA-4-Ig).

3. In Paragraph 1, A method of manufacturing in which the administration of the immunosuppressant in step (a) above is administered once or twice.

4. In Paragraph 1, A manufacturing method wherein the administration of the specified period in step (c) above is administered for 7 to 21 days.

5. In Paragraph 1, The above cancers include brain tumors, meningiomas, schwannomas, pituitary tumors, spinal cord tumors, oral cancer, pharyngeal cancer, laryngeal cancer, tongue cancer, salivary gland cancer, ocular cancer, lung cancer, bronchial cancer, pleural cancer, heart cancer, mediastinal cancer, esophageal cancer, stomach cancer, small intestine cancer, colorectal cancer, colon cancer, rectal cancer, liver cancer, cholangiocarcinoma, gallbladder cancer, bile duct cancer, pancreatic cancer, anal cancer, leukemia, lymphoma, multiple myeloma, myelodysplastic syndrome, myeloproliferative tumors, thyroid cancer, adrenal cancer, parathyroid cancer, pancreatic endocrine tumors, cervical cancer, endometrial cancer, ovarian cancer, vaginal cancer, vulvar cancer, prostate cancer, testicular cancer, penile cancer, skin cancer, basal cell carcinoma, squamous cell carcinoma, melanoma, soft tissue sarcoma, liposarcoma, myosarcoma, angiosarcoma, cutaneous lymphoma, osteosarcoma, chondrosarcoma, Ewing sarcoma, A method of manufacturing, wherein one or more selected from the group consisting of neuroblastoma, Wilms tumor, retinoblastoma, acute lymphoblastic leukemia, pediatric brain tumor, basement membrane carcinoma, Kaposi's sarcoma, gastrointestinal stromal tumor, and mesothelioma.

6. In Paragraph 1, The above-mentioned animals with normal immune function are Nude mouse, Severe Combined Immunodeficiency Mouse (SCID mouse), Autoimmune SCID Mouse (NOD-SCID mouse), NSG mouse, NOG mouse, Rag1 knockout mouse, Rag2 knockout mouse, Rag1 / 2 double knockout mouse, β2-microglobulin knockout mouse, CD4 knockout mouse, CD8 knockout mouse, IL2Rγ knockout mouse, Artemis KO mouse, BALB / c SCID mouse, CB-17 SCID mouse, or BRG mouse (BALB / c Rag2- / - IL2Rγ- / - A manufacturing method that is not a mouse.

7. In Paragraph 1, The above manufacturing method is, (a') a step of administering 50 to 300 mg of cyclophosphamide once or twice to a mouse with normal immune function; (b') a step of injecting cancer cells into the animal; and, (c') a step of additionally administering 5 to 30 mg of cyclophosphamide for 7 to 21 days and then stopping; comprising a method for preparing an animal model of cancer.

8. In Paragraph 1, The above manufacturing method is, (a') a step of administering 5 to 30 mg of dexamethasone once or twice to a mouse with normal immune function; (b') a step of injecting cancer cells into the animal; and, (c') a step of additionally administering 0.5 to 5 mg of dexamethasone for 7 to 21 days and then stopping; comprising a method for preparing an animal model of cancer.

9. A cancer animal model having normal immune function, manufactured by the method of any one of claims 1 to 8. 10.(a) Step of preparing the cancer animal model of Paragraph 9; and, (b) a step of administering a candidate substance for cancer treatment to the animal model; comprising a screening method for a candidate substance for cancer treatment.

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