Method for evaluating the effects of administering a substance

Abstract

Evaluate the efficacy of antitumor drugs. The effects of administering a substance are evaluated based on the results of screening using a non-human animal model of cancer to which the substance has been administered.

Description

[Technical Field]

[0001] The present invention relates to a method for evaluating the effect of administering a substance. [Background technology]

[0002] Mouse disease models play a very important role in studying the pathogenesis of human diseases and in drug screening, and various model mice have been produced. Mice are also used as an animal model for metabolic dysfunction-associated steatohepatitis with obesity (MASH). For example, Non-Patent Document 1 reports that liver hypertrophy was observed in mice fed a choline-deficient high-fat diet (CDAHFD). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Masayuki Sudo et al., "Development of a new NASH animal model for drug discovery: A drug discovery approach for nonalcoholic steatohepatitis (NASH), a liver metabolic disease," Japanese Pharmacology Journal, Vol. 144 (2014) No. 2, pp. 69-74 [Non-patent document 2] George G Schweitzer et al, "Liver-specific loss of lipin-1-mediated phosphatidic acid phosphatase activity does not mitigate intrahepatic TG accumulation in mice", Journal of lipid research, VOLUME 56, ISSUE 4, P848-858, APRIL 01, 2015 Summary of the Invention [Problem to be solved by the invention]

[0004] There has been a need for an animal model with a suitable pathological condition to evaluate the effects of administering substances, including the efficacy of antitumor drugs.

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

[0006] To achieve the object of the present invention, for example, a method according to one embodiment comprises the following steps: The STAM model mice are administered streptozotocin during the neonatal period and then fed a high-fat diet, which induces MASH and subsequently liver cancer. The substance is administered starting at 12 weeks of age. and evaluating the effect of administering the substance to the STAM model mice. based on tumor size measured by taking CT images, or based on the survival rate of the STAM model mice after administration of the substance. and a step of performing the steps. [Effects of the Invention]

[0007] It becomes possible to evaluate the effects of administering a substance. [Brief explanation of the drawings]

[0008] [Figure 1] A graph showing the difference in survival rate depending on whether or not anti-PD-L1 antibody was administered. [Figure 2] A graph showing the difference in tumor growth rate between the administration of anti-PD-L1 antibody and the non-administration. [Figure 3] A graph showing the difference in tumor counts between patients with and without administration of anti-PD-L1 antibody. [Figure 4] A graph showing the difference in CD8-positive areas between the administration of anti-PD-L1 antibody and the non-administration. DETAILED DESCRIPTION OF THE INVENTION

[0009] Hereinafter, the embodiments will be described in detail with reference to the accompanying drawings. Note that the following embodiments do not limit the scope of the invention as claimed, and not all combinations of features described in the embodiments are necessarily essential to the invention. Two or more of the features described in the embodiments may be combined in any desired manner. Furthermore, the same reference numerals are used to designate identical or similar components, and redundant descriptions will be omitted.

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

[0011] Mice can be used as an animal model for evaluating human diseases. Mice are relatively easy to handle as an animal model and have a high genomic homology with humans, so they are widely used as an animal model. The following explanation will be given assuming that mice (or rodents such as rats) are used as a non-human animal model, but this is only an example. Any animal model, such as rhesus monkeys or primates such as marmosets, can be used depending on the tumor therapeutic agent and disease to be evaluated.

[0012] In the present application, screening is performed using a non-human animal model of cancer. Examples of non-human animal models of cancer include non-human animal models of liver cancer, particularly non-human animal models of non-viral liver cancer. Examples of non-human animal models of non-viral liver cancer include non-human animal models of metabolic dysfunction-associated steatohepatitis (MASH), particularly STAM model mice. Below, the STAM model mice and other mice are 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 non-immune mice (such as nude mice, SCID mice, or NOG mice) (cell line derived xenograft models: CDX models), and models in which human cancer tissues are transplanted into non-immune mice (patient line derived xenograft models: PDX models).

[0014] (CDX model) CDX models are created by subcutaneously transplanting (heterotopic transplantation) human liver cancer cell lines into mice. Models created by transplanting cancer cells into an organ other than the organ of origin are suitable for screening and are widely used, but cannot be used to confirm tumor immunity because the mice are immune-naive.

[0015] For example, a CDX model can be created by transplanting a human hepatic cancer cell line into a mouse liver (orthotopic transplantation). Even models created by transplanting the cancer cell line into the same organ as its original organ cannot be used to confirm tumor immunity because the mice are immune-naive. Therefore, the CDX model cannot be used to evaluate the effects of immune checkpoint inhibitors.

[0016] There are also models in which human PBMCs (peripheral blood mononuclear cells) are transferred (injected) into the heterotopic or orthotopic transplantation models described above. This model allows the observation of antitumor effects of T lymphocytes by transferring PBMCs containing human T lymphocytes into immune-naive mice. Because these models use mice lacking lymph nodes, it is not possible to evaluate endogenous responses. In other words, this model cannot evaluate how antitumor T lymphocytes are established, and therefore it is not possible to conduct tests such as "directly injecting immune checkpoint inhibitors into lymph nodes."

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

[0018] [Syngeneic model] A syngeneic model is a model in which cells or tissues from the same animal species are transplanted. Syngeneic models can be created, for example, by heterotopic or allotopic transplantation using mouse cells or tissues, similar to xenograft models. Because the mouse immune system is intact, syngeneic models can be used to evaluate tumor immunity. However, because the cancer cells are derived from mouse cell lines, they cannot be used to evaluate drugs that do not cross-react with humans or mice.

[0019] [Spontaneous and genetically engineered mice] Spontaneously developing mice, such as FLS (Fatty Liver Shionogi) mice, are an inbred strain of mice that spontaneously develop fatty liver from a young age under normal breeding conditions. There are naturally developing mice and mice that have been genetically modified to develop fatty liver. In both cases, the timing of onset is unclear, and the incidence rate varies. Therefore, it is difficult to consistently evaluate drug efficacy in a short period of time using these models.

[0020] [Stimulation-induced disease in mice] Examples of mice that develop the disease when stimulated with chemicals or the like include the STAM model, Gubra model, CDAHFD model, and DEN model.

[0021] Drug efficacy evaluations using xenograft and syngeneic models almost always involve the use of single tumors, limiting evaluation to stage I–II cancer (surgically resectable stages). While metastasis models, such as the lung metastasis model in which cancer cells are injected intravenously, exist, these models only mimic the process by which cancer cells enter the bloodstream and travel to distant organs, and do not allow for evaluation of drug efficacy in cases where true metastasis has occurred. In contrast, the STAM model allows for evaluation of drug efficacy in cases equivalent to stage IV cancer, where multiple tumors develop and spread, resulting in metastasis to other organs. Therefore, the STAM model, which allows for evaluation of drug efficacy in cases equivalent to stage IV cancer, can address the problem of efficacy in transplant models that cannot be used to evaluate stage IV cancer, where efficacy in transplant models does not achieve clinically meaningful results.

[0022] In this embodiment, a non-human animal model of MASH pathology is prepared. The non-human animal model of MASH pathology is mainly prepared by administering a high-fat diet, and examples of the non-human animal model include the STAM model and Gubra model described above.

[0023] The following describes the STAM model mouse, which is primarily used as an animal model in this embodiment. The STAM model can be produced, for example, by administering streptozotocin (e.g., 200 μg) to neonatal (e.g., 2-day-old) male mice (C57BL / 6J strain) to reduce their insulin secretory capacity and then feeding them a high-fat diet. Compared to other models, the STAM model shows extremely short progression of the disease (approximately 6 weeks until the onset of MASH), and liver cancer is reliably developed at approximately 20 weeks of age. Therefore, the STAM model can be efficiently used as a MASH-liver cancer pathology model.

[0024] Examples of the main components of the high-fat diet include crude fat, crude protein, crude fiber, crude ash, nitrogen-free soluble matter, and water. The high-fat diet used in the present invention is not particularly limited, but has a crude fat content of 20% or more, preferably 30% or more, and the proportion of calories derived from fat in total calories is usually 50% or more, preferably 60% or more. Ingredients that can be incorporated into the high-fat diet include powdered beef tallow, milk casein, powdered egg white, L-cystine, safflower oil, crystalline cellulose, maltodextrin, lactose, and sucrose. However, these substances are merely examples of components of the high-fat diet, and the diet does not necessarily need to contain these substances.

[0025] The high-fat diet is not particularly limited, but examples thereof include those with a higher crude fat content than normal diets (e.g., 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 laboratory animal feeds.

[0026] In the case of mice, for example, the administration of the high-fat diet is usually initiated 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 feeding is, for example, about 3 to 6 grams in the case of mice. It is usually preferable to raise the animals on a high-fat diet for at least one week. Those skilled in the art can adjust (adjust) the amount of high-fat diet as appropriate, taking into consideration the type, size, weight, etc. of the experimental animal used. Fatty liver can be induced by raising the animals on a high-fat diet.

[0027] It is also possible to generate MASH pathology model mice with different characteristics by feeding them different diets. Here, a model fed a fructose-containing high-cholesterol diet (Gubra Amylin NASH-diet (D09100310), Research Diet) is referred to as the Gubra model. Furthermore, a mouse model that develops steatohepatitis and is fed a choline-deficient high-fat diet (CDAHFD (A06071302), Research Diet) is referred to as the CDAHFD model.

[0028] The Gubra model is a model in which male mice (C57BL / 6J strain) are fed the above-mentioned fructose-containing high-cholesterol diet to develop MASH pathology. For example, the Gubra model can be generated by feeding mice the fructose-containing high-cholesterol diet for 30 weeks. The fructose-containing high-cholesterol diet D09100310 has been used to generate model mice, for example, in Non-Patent Document 2, and the Gubra model may be generated 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 modest, and hyperlipidemia is not observed.

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

[0030] As described above, the non-human animal model of MASH pathology according to this embodiment can be produced by any method. In the following, the non-human animal model of cancer according to this embodiment will be described assuming that the STAM model, which is one of the non-human animal models of MASH pathology, is used.

[0031] In this embodiment, a substance to be screened is administered to a prepared STAM model mouse. The substance to be administered to the non-human animal model of this embodiment is not particularly limited, and any substance thought to be effective against the pathology of cancer can be used. For example, a tumor therapeutic drug may be used as the substance. The tumor therapeutic drug to be screened can be selected arbitrarily, and known techniques can be used for screening after administration depending on the tumor therapeutic drug. The substance of this embodiment can be administered orally or by injection, for example. Here, the tumor therapeutic drug used as the substance is assumed to be one used to treat MASH. However, the target disease for which efficacy screening is performed is not limited in this way, and the tumor therapeutic drug can also be selected arbitrarily depending on the type of disease or the severity of the condition to be treated.

[0032] Drugs used in drug therapy can be used as tumor therapeutic agents. For example, anticancer drugs can be used as tumor therapeutic agents. As tumor therapeutic agents, drugs for treating liver cancer, drugs for treating non-viral liver cancer, or drugs for treating MASH liver cancer can be used. Here, drugs targeting the tumor microenvironment (TME) can be used as tumor therapeutic agents. As TME-targeted drugs, molecular targeted drugs that act on specific target molecules, such as TET1 inhibitors or SIRT2 inhibitors, can be used. Furthermore, immune checkpoint inhibitors can be used as TME-targeted drugs. As tumor therapeutic agents, drugs for treating non-viral liver cancer or drugs for treating MASH liver cancer can be used. The type of tumor therapeutic agent to be evaluated is not limited to these, and can be selected as appropriate depending on the animal model used, the expected pathology, and the like. Here, an anti-PD-L1 antibody, an immune checkpoint inhibitor, is used as the anti-cancer agent to be screened.

[0033] For example, a therapeutic agent used in combination with an anticancer agent may be used as a tumor therapeutic agent. Furthermore, an adjuvant (including those used in chemotherapy, hormone therapy, etc.) administered in combination with a vaccine may also be used as a tumor therapeutic agent. Furthermore, functional foods, supplements, or intestinal bacteria that are expected to be effective in treating or preventing cancer may also be used as the administered substance. In the following description, a tumor therapeutic agent will be used as the administered substance.

[0034] The effect of the administration of a substance is evaluated, for example, by the effect (therapeutic effect) of a tumor therapeutic drug. The effect of a tumor therapeutic drug is evaluated, for example, as an antitumor effect. The antitumor effect according to this embodiment may be, for example, a tumor immunosuppressive effect, particularly a lymphocyte suppressive effect, or a T cell suppressive effect. For example, the T cell suppressive effect may be evaluated by assessing the degree of CD8+ T cell infiltration into tumor tissue. This assessment of the degree of CD8+ T cell infiltration can be performed, for example, by CD8 immunostaining, as described below with reference to FIG. 4. Here, the degree of CD8+ T cell infiltration into tumor tissue may be in tissue within the tumor or in the tumor margin tissue. Furthermore, the therapeutic effect of the tumor therapeutic drug described in this embodiment can be evaluated based on the survival rate of an animal model after administration of the tumor therapeutic drug, or whether or not the pathological morphology of the disease has improved (or the degree of improvement). Note that the effect of the administration of a substance can be evaluated based on any effect resulting from administration, such as changes in the pathological condition after administration of a functional food.

[0035] The administration period of the tumor therapeutic agent is not particularly limited and can be set arbitrarily depending on the type of tumor therapeutic agent, the condition of the disease, etc. For example, administration of the tumor therapeutic agent to a non-animal model may be initiated 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 the tumor therapeutic agent to a non-animal model may be continued until 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. Furthermore, the administration interval of the tumor therapeutic agent can also be set arbitrarily depending on the type of tumor therapeutic agent, the condition of the disease, etc.

[0036] The present inventors have found that the STAM model mouse exhibits more natural liver tissue changes, more similar to the process of liver formation in humans, compared with other models. Therefore, the STAM model mouse is suitable for evaluating the efficacy of tumor therapeutic drugs for the treatment of human liver cancer. In particular, the present inventors have found that administering tumor immune response promoters, such as immune checkpoint inhibitors, to the STAM model mouse not only suppresses liver tumor progression but also increases CD8+ T cell infiltration into liver tumor tissue. These results indicate that the same tumor immune suppression mechanism functions in the STAM model mouse as in humans, making it possible to evaluate the efficacy of tumor immune response promoters.

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

[0038] An example of screening for anti-PD-L1 antibodies using the STAM model mouse will be described below with reference to Figures 1 to 4.

[0039] [evaluation] Here, STAM model mice were divided into an anti-PD-L1 antibody treatment group (anti-PD-L1) and a control group (Control IgG) that was not administered the anti-PD-L1 antibody, and these groups were evaluated for each of the items described below.

[0040] First, we will explain the procedure for generating the above-mentioned treatment groups. STAM model mice were prepared by administering 200 μg of streptozotocin to 2-day-old male mice and feeding them on a high-fat diet. These STAM model mice were then intraperitoneally administered anti-PD-L1 antibody at a concentration of 5 mg / kg twice a week from 16 to 20 weeks of age to generate the treatment group of STAM model mice. The control group consisted of mice reared in the same manner, except that they were not administered anti-PD-L1 antibody.

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

[0042] Tumor growth rates were measured in the treatment and control groups from a few days before the start of the study at 16 weeks of age until a few days before autopsy at 20 weeks of age. Data for these timings were obtained by administering iopamiron (Bayer HealthCare AG) as a contrast agent via the tail vein of the mice and capturing CT images immediately after administration. Images were captured by capturing cross-sectional images of the entire liver. The captured data were then analyzed using CT analysis software (Horos Project), and tumor counts and the maximum diameters of each tumor were measured in axial and coronal sections. The growth rate was calculated from the space occupied lesion volume (SOL) calculated using the following formula (1), where the longer axis of the axial and coronal sections was the major axis and the shorter axis was the minor axis. SOL(mm3)=(minor axis)2×(major axis)×π / 6 Equation (1)

[0043] The calculated tumor growth rate 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 counting 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%, and the number of nodules was reduced by an average of 5. Therefore, it can be said that administration of anti-PD-L1 antibodies reduces liver tumors in STAM model mice.

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

[0045] First, paraffin sections were deparaffinized and hydrophilized using xylene, 100%–70% alcohol, and RO water. Then, the sections were surrounded by liquid blocker. Endogenous peroxidase was then inhibited with 0.3% hydrogen peroxide, and 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 incubated with blocking solution (Block Ace, KAC Corporation, Cat. No. UK-B80) for 10 minutes at room temperature. After washing with PBS, the sections were incubated with 2.5% normal goat serum for 20 minutes at room temperature. Then, CD8 antibody (Abcam, ab209775) was incubated overnight at 4°C. After washing with PBS, the sections were incubated with ImmPRESS Polymer Reagent for 30 minutes at room temperature. After washing with PBS and treating with PBST, the sections were stained with a chromogenic substrate (Simple Stain DAB, Nichirei Biosciences, Cat. No. 41572). The sections were washed with RO water, passed through 8x diluted hematoxylin for 1 minute, and immediately rinsed with RO water. The sections were then placed in running water for 15 minutes and mounted in Aquatex (Merck, Cat. No. 108562) to prepare specimens. The specimens were observed under a bright-field microscope (Leica Microsystems).

[0046] The percentage of CD8-positive areas in tumor tissue is shown in Figure 4. In the control group, the percentage of CD8-positive areas was less than 0.25% in all but one mouse, whereas in the treatment group, several mice had a percentage of CD8-positive areas exceeding 0.5%. Furthermore, the largest percentage of CD8-positive areas in the control group was approximately 0.7%, while the largest percentage of CD8-positive areas in the treatment group was approximately 1.7%. This indicates that administration of anti-PD-L1 antibodies increases the infiltration of CD8+ T cells into tumors in the STAM model mice.

[0047] In this way, by screening tumor therapeutic drugs using STAM model mice, it becomes possible to evaluate whether the pathology in the STAM model is improved by tumor therapeutic drugs. In particular, since the mechanism of tumor immunity suppression functions in STAM model mice similar to that in humans, the STA model is useful for evaluating the efficacy of tumor immune response promoters. Note that the items used for evaluation here are only examples, and tumor therapeutic drugs may also be screened by observing other pathological forms observed in the MASH pathological model.

Claims

1. A method for the treatment of liver cancer in a mouse model (STAM model) in which streptozotocin is administered to the neonatal period and then a high-fat diet is administered to induce liver cancer through the development of MASH, comprising the steps of: starting the administration of a substance from 12 weeks of age; Evaluating the effect of administering the substance to the STAM model mice based on the size of the tumor measured by taking a CT image or based on the survival rate of the STAM model mice after administration of the substance; A method comprising:

2. 10. The method of claim 1, wherein the substance is a tumor therapeutic agent.

3. The method according to claim 2, wherein the tumor treatment agent is a drug used in drug therapy.

4. The method of claim 3, wherein the tumor therapeutic agent is a therapeutic agent that targets the tumor microenvironment.

5. The method according to claim 2, wherein the tumor therapeutic agent is a molecular targeted drug or an immune checkpoint inhibitor.

6. 6. The method of claim 5, wherein the tumor therapeutic agent is an immune checkpoint inhibitor.

7. The method according to claim 6, characterized in that the degree of infiltration of CD8+ T cells into tumor tissue is evaluated by CD8 immunostaining using tissue inside the tumor of a STAM model mouse.

8. The method according to claim 2, wherein the tumor therapeutic agent is a therapeutic agent for non-viral liver cancer.

9. The method according to claim 8, wherein the tumor therapeutic agent is a therapeutic agent for liver cancer in the MASH pathology.

10. The method according to claim 1, wherein the effect of administering the substance is evaluated as an antitumor effect.

11. The method according to claim 10, wherein the effect of administering the substance is evaluated as a tumor immunosuppressive effect.

12. The method according to claim 11, wherein the effect of administering the substance is evaluated by evaluating the effect of regulating lymphocytes.

13. The method according to claim 12, wherein the effect of administering the substance is evaluated by evaluating the regulatory effect on T cells.

14. The method according to claim 13, wherein the effect of administering the substance is evaluated by evaluating the degree of infiltration of CD8+ T cells into tumor tissue.

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