Method for evaluating the effects of administering a substance

Abstract

We will evaluate the efficacy of cancer treatment drugs. [Solution] The effect of administering a substance is evaluated based on the screening results using a non-human animal model of cancer to which the substance has been administered.

Description

Technical Field

[0004] , , , ,

[0001] The present invention relates to a method for evaluating the effects of substance administration.

Background Art

[0002] Mouse disease models play a very important role in the study of the pathogenesis of human diseases and the screening of drugs, and various model mice have been created. Mice are also used as a pathological animal model for metabolic dysfunction-related steatohepatitis (MASH) associated with obesity. For example, in Non-Patent Document 1, liver hypertrophy has been confirmed in mice fed a choline-deficient high-fat diet (CDAHFD).

Prior Art Documents

Non-Patent Documents

[0003]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0004] To evaluate the effects of administering substances, including the efficacy of cancer treatment drugs, there was a need for animal models with suitable disease conditions.

[0005] The present invention aims to evaluate the effects of administering a substance. [Means for solving the problem]

[0006] To achieve the objectives of the present invention, for example, a method according to one embodiment comprises the following configuration: that is, the effect of administering the substance is evaluated based on screening results using a non-human animal model of cancer to which the substance has been administered. [Effects of the Invention]

[0007] This makes it possible to evaluate the effects of administering a substance. [Brief explanation of the drawing]

[0008] [Figure 1] A graph showing the difference in survival rates depending on whether or not anti-PD-L1 antibodies were administered. [Figure 2] A diagram showing the difference in tumor growth rates depending on whether or not anti-PD-L1 antibodies are administered. [Figure 3] A diagram showing the difference in tumor numbers depending on whether or not anti-PD-L1 antibodies were administered. [Figure 4] A figure showing the difference in CD8-positive regions depending on whether or not anti-PD-L1 antibodies were administered. [Modes for carrying out the invention]

[0009] The embodiments will be described in detail below with reference to the attached drawings. Note that the following embodiments do not limit the invention as defined in the claims, and not all combinations of features described in the embodiments are essential to the invention. Two or more of the features described in the embodiments may be combined in any way. Furthermore, identical or similar configurations will be given the same reference numeral, and redundant descriptions will be omitted.

[0010] In one embodiment of the present invention, the effect of administering a substance is evaluated based on screening results using a non-human animal model of cancer to which the substance has been administered.

[0011] Mice can be used as animal models to evaluate human diseases. Mice are widely used as animal models because they are relatively easy to handle and have high genome homology with humans. The following explanation will use mice (or rodents such as rats) as non-human animal models, but this is just one example. Any animal can be used as a model depending on the oncological drug and disease being evaluated, such as rhesus monkeys or primates such as marmosets.

[0012] In this application, screening for cancer is performed using non-human animal models. As non-human animal models for cancer, for example, non-human animal models for liver cancer, particularly non-viral liver cancer, can be used. As a non-human animal model for non-viral liver cancer, for example, non-human animal models of metabolic dysfunction-associated steatohepatitis (MASH), particularly STAM model mice, can be used. Below, STAM model mice and other mice will be described.

[0013] [Xenograft model] Xenograft models are models in which cells or tissues from different animal species are transplanted. Examples of xenograft models include models in which human cancer cell lines are transplanted into immune mice (such as nude mice, SCID mice, or NOG mice) (cell line derived xenograft models: CDX models), and models in which human cancer tissue is transplanted into immune mice (patient line derived xenograft models: PDX models).

[0014] (CDX model) CDX models are created, for example, by subcutaneously transplanting human hepatoma cell lines into mice (ectopic transplantation). Models created by transplanting cancer cell lines into organs different from their origin are suitable for screening and are widely used, but because the mice are immune, they cannot be used to confirm tumor immunity.

[0015] Furthermore, CDX models can also be created by transplanting human hepatoma cell lines into mouse livers (orthotopic transplantation). However, models created by transplanting cancer cell lines into the same organ as their origin cannot be used to confirm tumor immunity because the mice used are immune. Therefore, the effects of immune checkpoint inhibitors cannot be evaluated using CDX models.

[0016] Furthermore, there are models in which human PBMCs (peripheral blood mononuclear cells) are transferred (injected) into the aforementioned ectopic or orthotopic transplantation models. This model allows observation of the antitumor effect of T lymphocytes by transferring PBMCs containing human T lymphocytes into immune mice. In such models, mice lacking lymph nodes are used, making it impossible to evaluate endogenous effects. In other words, this model cannot evaluate how antitumor T lymphocytes are established, and therefore it is not possible to perform tests such as "directly injecting immune checkpoint inhibitors into lymph nodes."

[0017] (PDX model) The PDX model is a variation of the CDX model in which patient-derived tissue is transplanted instead of cancer cell lines. Similar to the CDX model, this model uses unimmunized mice, making it impossible to evaluate tumor immunity.

[0018] [syngeneic model] A syngeneic model is a model in which cells or tissues of the same species of animal are transplanted. A syngeneic model can be created, for example, by performing heterotopic transplantation or allogeneic transplantation using mouse cells or tissues, similar to a xenograft model. Since the immune system of the mouse exists in a complete state in a syngeneic model, it can be used for evaluating tumor immunity. However, since cancer cells are a mouse-derived cell line, it is not possible to evaluate drugs that do not cross between humans and mice.

[0019] [Spontaneously occurring mice, mice with disease onset due to genetic modification] Spontaneously occurring mice are, for example, FLS (Fatty Liver Shionogi) mice, which are inbred mice that spontaneously develop fatty liver from a young age under normal breeding conditions. There are also mice with genetic modifications that develop fatty liver, and in both cases, the onset time is unclear and the incidence rate is uneven. Therefore, it is difficult to evaluate the drug efficacy without variation in a short period using these models.

[0020] [Mice with disease onset due to stimulation] Examples of mice that develop disease by being stimulated with chemicals or the like include mice of the STAM model, Gubra model, CDAHFD model, or DEN model.

[0021] Evaluating drug efficacy using xenograft or syngeneic models mostly involves single tumors and is limited to evaluating cancer at stages I-II (surgically resectable stages). While metastasis models such as lung metastasis models, which involve intravenous injection of cancer cells, exist, these only mimic the process of cancer cells entering distant organs from the bloodstream and do not allow for evaluation of drug efficacy in cases where true metastasis has occurred. In contrast, using the STAM model allows for evaluation of drug efficacy in conditions equivalent to stage IV, where multiple tumors occur and the tumors invade and metastasize to other organs. Therefore, the problem that arose with conventional transplant models that could not evaluate stage IV cancer—where efficacy was observed in transplant models but not achieved in clinical settings—can be resolved by using the STAM model, which can evaluate drug efficacy in conditions equivalent to stage IV.

[0022] In this embodiment, a non-human animal model of MASH disease is created. The non-human animal model of MASH disease is mainly created by feeding it a high-fat diet, and for example, the STAM model and Gubra model mentioned above are used.

[0023] The following describes the STAM model mouse, which is mainly used as an animal model in this embodiment. The STAM model can be created, for example, by administering streptozotocin (e.g., 200 μg) to male mice (C57BL / 6J strain) in the neonatal period (e.g., 2 days old) to reduce insulin secretion ability, and then feeding them a high-fat diet. The STAM model has an extremely short disease progression compared to other models (the period until MASH onset is about 6 weeks), and reliably develops liver cancer at around 20 weeks of age, making it possible to efficiently utilize it as a MASH-liver cancer disease model.

[0024] The main components of the high-fat diet described above include, for example, crude fat, crude protein, crude fiber, crude ash, soluble non-nitrogenous substances, and water. The high-fat diet used in the present invention is not particularly limited, but it has a crude fat content of 20% or more, preferably 30% or more, and the ratio of calories derived from fat to total calories is usually 50% or more, preferably 60% or more. In addition, components that may be included in the high-fat diet include, for example, powdered beef tallow, milk casein, egg white powder, L-cystine, safflower oil, crystalline cellulose, maltodextrin, lactose, and sucrose, but these substances are just examples of components of a high-fat diet and do not necessarily have to be included.

[0025] The above-mentioned high-fat diets are not particularly restricted, but examples include those with a crude fat content higher than that of a normal diet (for example, about 30% or more higher). Examples include High Fat Diet 32 ​​(manufactured by CLEA Japan) and D12492 (manufactured by Research Diet), which are commercially available as research animal feed.

[0026] The high-fat diet described above is usually administered to mice starting at 2 to 6 weeks of age, preferably 3 to 5 weeks of age, and more preferably 4 weeks of age. The amount of high-fat diet per dose is approximately 3 to 6 grams for mice. It is generally preferable to feed the mice with the high-fat diet for at least one week. Those skilled in the art can adjust the amount of high-fat diet as appropriate, taking into account the type, size, and weight of the experimental animals used. Fatty liver can be induced by feeding the animals with a high-fat diet.

[0027] Furthermore, it is possible to create MASH disease model mice with different characteristics by feeding them different samples. Here, a model fed a fructose-containing high-cholesterol diet (Gubra Amylin NASH-diet (D09100310), Research Diet Co., Ltd.) is referred to as the Gubra model. In addition, a model of mice that develop fatty liver disease, fed a choline-deficient high-fat diet (CDAHFD (A06071302), Research Diet Co., Ltd.), is referred to as the CDAHFD model.

[0028] The Gubra model is a model in which male mice (C57BL / 6J strain) are fed the aforementioned fructose-containing high-cholesterol diet to induce the pathology of MASH. For example, the Gubra model can be created by feeding mice a fructose-containing high-cholesterol diet for 30 weeks. The fructose-containing high-cholesterol diet D09100310 has been used, for example, in the creation of model mice in Non-Patent Document 2, and the Gubra model may be created in a similar manner (by feeding Alb-Lpin1- / - mice D09100310 for 10 weeks). Compared to other models (e.g., the CDAHFD model), the Gubra model exhibits obesity symptoms, but the degree of obesity is milder, and hyperlipidemia is not observed.

[0029] The CDAHFD model is a model in which male mice (C57BL / 6J strain) are fed the aforementioned CDAHFD to induce fatty liver disease. For example, a CDAHFD model can be created by feeding mice the CDAHFD for 12 weeks. As mentioned above, the CDAHFD model exhibits a more severe degree of fibrosis than other models and, like STAM, does not show obesity symptoms.

[0030] Thus, the non-human animal model of MASH pathology according to this embodiment can be prepared by any method. In the following, the STAM model, one of the non-human animal models of MASH pathology, will be used as the non-human animal model of cancer according to this embodiment.

[0031] In this embodiment, a prepared STAM model mouse is administered a substance to be screened. The substance administered to the non-human animal model according to this embodiment is not particularly limited, and any substance that is thought to have an effect on the pathogenesis of cancer can be used. For example, an oncology drug may be used as the substance. The oncology drug to be screened can be arbitrarily selected, and the screening method after administration can be any known technique depending on the oncology drug. The substance according to this embodiment can be administered, for example, orally or by injection. Here, the oncology drug used as the substance is assumed to be used to treat MASH, but the target disease for screening the drug's efficacy is not limited in this way, and the oncology drug can be arbitrarily selected according to the type of disease or the severity of the disease being treated.

[0032] As a tumor treatment drug, drugs used in pharmacotherapy can be used. For example, anticancer drugs may be used as tumor treatment drugs. As a tumor treatment drug, drugs for liver cancer may be used, drugs for nonviral liver cancer may be used, and drugs for MASH liver cancer may be used. Here, as a tumor treatment drug, a drug that targets the tumor microenvironment (TME) may be used. As a TME-targeting drug, molecularly targeted drugs that act on specific target molecules, such as TET1 inhibitors or SIRT2 inhibitors, can be used. In addition, immune checkpoint inhibitors may be used as TME-targeting drugs. Furthermore, as a tumor treatment drug, drugs for nonviral liver cancer may be used, and drugs for MASH liver cancer may be used. The types of tumor treatment drugs to be evaluated are not limited in this way and can be arbitrarily adopted depending on the animal model used or the assumed disease state. Here, an immune checkpoint inhibitor, an anti-PD-L1 antibody, is used as the anticancer drug to be screened.

[0033] For example, a drug used in combination with an anticancer drug may be used as a tumor treatment drug. Adjuvants (including those administered through chemotherapy, hormone therapy, etc.) administered in combination with vaccines may also be used as tumor treatment drugs. Furthermore, functional foods, supplements, or intestinal bacteria that are expected to have an effect in treating or preventing cancer may be used as administered substances. In the following explanation, tumor treatment drugs will be used as the administered substances.

[0034] The effects of administering a substance can be evaluated, for example, as the effect of a tumor drug (therapeutic effect). The effect of a tumor drug is evaluated, for example, as an antitumor effect. The antitumor effect in this embodiment may be, for example, a tumor immune regulatory effect, and in particular, a lymphocyte regulatory effect, or a T cell regulatory effect. For example, as a T cell regulatory effect, the degree of infiltration of CD8+ T cells into tumor tissue may be evaluated. This evaluation of the degree of CD8+ T cell infiltration can be performed by CD8 immunostaining, for example, as explained with reference to Figure 4 later. Here, the degree of infiltration of CD8+ T cells into tumor tissue may be in the tissue inside the tumor, or in the peripheral tissue of the tumor. Furthermore, the therapeutic effect of the tumor drug described in this embodiment can be evaluated according to whether or not the survival rate of the animal model after administration of the tumor drug or the pathological morphology of the disease has improved (or the degree of improvement). In addition, any effect that occurs as a result of administration can be evaluated, such as changes in the disease state after administration of the functional food.

[0035] The duration of administration of anti-cancer drugs is not particularly limited and can be arbitrarily set according to the type of anti-cancer drug or the patient's condition. For example, administration of anti-cancer drugs to non-animal models may be started at 1 week of age, 2 weeks of age, 4 weeks of age, 5 weeks of age, 8 weeks of age, 10 weeks of age, 12 weeks of age, 15 weeks of age, 16 weeks of age, or 18 weeks of age. Furthermore, administration of anti-cancer drugs to non-animal models may be continued up to 5 weeks of age, 8 weeks of age, 10 weeks of age, 15 weeks of age, 16 weeks of age, 18 weeks of age, 20 weeks of age, 22 weeks of age, or 24 weeks of age. The administration interval of anti-cancer drugs can also be arbitrarily set according to the type of anti-cancer drug or the patient's condition.

[0036] The inventors of this application have found that STAM model mice exhibit more natural liver tissue changes, similar to the liver formation process in humans, compared to other models. For this reason, STAM model mice are suitable for evaluating the efficacy of oncological drugs for the treatment of human liver cancer. In particular, the inventors have found that administering tumor immune response promoters, such as immune checkpoint inhibitors, to STAM model mice not only suppresses the progression of liver tumors but also increases the infiltration of CD8+ T cells into liver tumor tissue. This result indicates that, in STAM model mice, a mechanism for suppressing tumor immunity functions similarly to that in humans, and therefore, the efficacy of tumor immune response promoters can be evaluated.

[0037] Furthermore, the Gubra model has a long incubation period (68 weeks to 100% cancer development), resulting in delayed data acquisition and high trial costs. The CDAHFD model does not involve metabolic abnormalities and is not a MASH disease model. The DEN model has a genetic background different from clinical hepatocellular carcinoma. In contrast, the STAM model mouse has a background of MASH disease, develops cancer in 100% of mice in a short period of 20 weeks, and is genetically the most similar to hepatocellular carcinoma patients, making it the most suitable model for evaluating drug efficacy.

[0038] The following describes an example of screening for anti-PD-L1 antibodies using a STAM model mouse, with reference to Figures 1-4.

[0039] [evaluation] In this study, we prepared two groups of STAM model mice: one group administered with anti-PD-L1 antibody (anti PD-L1) and a control group that did not receive anti-PD-L1 antibody (Control IgG). We then evaluated each of the items described below for both groups.

[0040] First, we will explain the procedure for creating the treatment groups described above. As STAM model mice, we used male mice that were 2 days old and administered 200 μg of streptozotocin, and fed a high-fat diet. These STAM model mice were administered anti-PD-L1 antibody at a concentration of 5 mg / kg intraperitoneally twice a week from 16 to 20 weeks of age to create STAM model mice for the treatment group. The control group consisted of mice raised in the same manner except that they were not administered anti-PD-L1 antibody.

[0041] Figure 1 shows a comparison of the survival rates of STAM model mice in the treatment group and the control group. From 8 days after the start of measurement, the survival rate of the treatment group was consistently higher, and at 20 days, the survival rate of the treatment group was over 80% compared to approximately 60% in the control group. Thus, it can be concluded that administration of anti-PD-L1 antibodies significantly reduces the mortality rate of STAM model mice.

[0042] Furthermore, the tumor growth rate was measured in both the treatment group and the control group from a few days before the start of the study at 16 weeks of age to a few days before necropsy at 20 weeks of age. Data at these timings were obtained by administering iopamilon (Bayer HealthCare AG) as a contrast agent via the tail vein of the mice and acquiring CT images immediately after administration. The imaging was performed by capturing tomographic images of the entire liver. Subsequently, the imaging data was analyzed using CT analysis software (Horos Project) to measure the number of tumors and the maximum diameter length of each tumor in the axial and coronal sections. The longer side of the axial and coronal sections was defined as the major axis, and the shorter side as the minor axis. The growth rate was calculated using the SOL (space occupied lesion) volume calculated using the following formula (1). SOL(mm3)=(minor axis)2×(major axis)×π / 6 Equation (1)

[0043] The tumor growth rate calculated in this way is shown in Figure 2, and the number of tumors (tumor nodules) at 20 weeks of age is shown in Figure 3. The number of tumor nodules was recorded by visually measuring nodules with a maximum diameter of 2 mm or more. Compared to the control group, the number of tumor nodules observed in the treatment group was reduced by approximately 80% in percentage terms, and by an average of 5 in number of nodules. Therefore, it can be concluded that administration of anti-PD-L1 antibody reduces liver tumors in STAM model mice.

[0044] Furthermore, autopsies were performed at 20 weeks of age, and the extent of CD8+ T cell infiltration into the tumor tissue was evaluated by CD8 immunostaining. Here, immunostaining was performed using paraffin sections in which the water of the autopsy tissue had been replaced with paraffin. The procedure for CD8 immunostaining is described below.

[0045] First, paraffin sections were deparaffinized and hydrophilized using xylene, 100%-70% alcohol, and RO water, and then surrounded with a liquid blocker. Next, endogenous peroxidase was inhibited with 0.3% hydrogen peroxide, and then antigen retrieval was performed using antigen retrieval solution H (Mitsubishi Medience Corporation) at 121°C for 10 minutes. After washing with PBS(-), the sections were treated with Phosphate Buffered Saline with Tween 20 (PBST), and then reacted with blocking solution (Block Ace, KAC Corporation, Cat No.: UK-B80) at room temperature for 10 minutes. After washing with PBS, Normal Goat Serum, 2.5% was reacted at room temperature for 20 minutes. Then, CD8 antibody (abcam, ab209775) was reacted overnight at 4°C. After washing with PBS, ImmPRESS Polymer Reagent was reacted at room temperature for 30 minutes. The sections were washed with PBS, treated with PBST, and then stained with a chromogenic substrate (Simple Stain DAB, Nichirei Bioscience Co., Ltd., Cat No.: 41572). After washing with RO water, they were passed through an 8-fold diluted hematoxylin for 1 minute and immediately washed with RO water. The sections were then left to stand in running water for 15 minutes and mounted in Aquatex (Merck KGaA, Cat No.: 108562) to prepare the specimens. The specimens were observed using a bright-field microscope (Leica Microsystems).

[0046] Figure 4 shows the percentage of CD8-positive areas in the tumor tissue. In the control group, the percentage of positive areas was less than 0.25% in all but one individual, while in the treatment group, several individuals were found to have a percentage exceeding 0.5%. Furthermore, the largest CD8-positive area in the control group was approximately 0.7%, while the largest CD8-positive area in the treatment group was approximately 1.7%. This suggests that administration of anti-PD-L1 antibody increases the infiltration of CD8+ T cells into tumors in STAM model mice.

[0047] Thus, by screening for anti-tumor drugs using STAM model mice, it becomes possible to evaluate whether the disease state in the STAM model is improved by anti-tumor drugs. In particular, since the mechanism that suppresses tumor immunity functions in STAM model mice as in humans, the STA model is useful for evaluating the efficacy of anti-tumor immune response promoters. Note that the items used for evaluation here are just examples, and anti-tumor drugs may also be screened by observing other pathological morphologies observed in the MASH disease model.

Claims

1. A method for evaluating the effects of administering a substance based on screening results using a non-human animal model of cancer to which the substance has been administered.

2. The method according to claim 1, characterized in that the non-human animal model of cancer is a non-human animal model of liver cancer.

3. The method according to claim 2, characterized in that the non-human animal model of liver cancer is a non-human animal model of non-viral liver cancer.

4. The method according to claim 3, characterized in that the non-human animal model of non-viral liver cancer is a non-human animal model of MASH pathology.

5. The method according to claim 4, characterized in that the non-human animal model of MASH pathology is a mouse model of the STAM model.

6. The method according to claim 1, characterized in that the substance is a tumor treatment drug.

7. The method according to claim 6, characterized in that the tumor treatment drug is a drug used in pharmacotherapy.

8. The method according to claim 7, characterized in that the tumor therapeutic agent is a therapeutic agent that targets the tumor microenvironment.

9. The method according to claim 8, characterized in that the tumor treatment drug is a molecularly targeted drug or an immune checkpoint inhibitor.

10. The method according to claim 9, characterized in that the tumor treatment drug is an immune checkpoint inhibitor.

11. The method according to claim 10, characterized in that the effect of the tumor drug is evaluated by CD8 immunostaining using tissue from inside a tumor of a STAM model mouse.

12. The method according to claim 6, characterized in that the tumor treatment drug is a treatment drug for nonviral liver cancer.

13. The method according to claim 12, characterized in that the tumor treatment drug is a treatment drug for liver cancer with MASH pathology.

14. The method according to claim 1, characterized in that the antitumor effect of the substance is evaluated.

15. The method according to claim 14, characterized in that the tumor immunomodulatory effect is evaluated as an effect of the substance.

16. The method according to claim 15, characterized in that the effect of the substance is evaluated on the control of lymphocytes.

17. The method according to claim 16, characterized in that the regulatory effect of the substance is evaluated as an effect of the substance on T cells.

18. The method according to claim 17, characterized in that the effect of the substance is to evaluate the degree of infiltration of CD8+ T cells into tumor tissue.

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