Method for selecting drug to be used in combination with IDO1 inhibitor in cancer immunotherapy, and composition for cancer immunotherapy comprising drug to be used in combination with IDO1 inhibitor

Abstract

To provide a method for efficiently selecting another drug to be used in combination with an IDO1 inhibitor, since identifying an appropriate combination drug comprising an IDO1 inhibitor is more urgent from the viewpoint of combined cancer immunotherapy rather than using an IDO1 inhibitor as a single agent, in cancer immunotherapy.SOLUTION: Provided is a method for selecting a drug to be used in combination with an IDO1 inhibitor in cancer immunotherapy by performing transcriptome analysis on tumor cells derived from mouse colon cancer cell lines transfected with mouse Ido1 gene.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for selecting a drug to be used in combination with an IDO1 inhibitor in cancer immunotherapy, and also to a cancer immunotherapy composition comprising an IDO1 inhibitor and a drug selected by the method for selecting a drug to be used in combination with an IDO1 inhibitor in cancer immunotherapy.

Background Art

[0002] As a cancer treatment method, cancer immunotherapy that treats cancer by attacking cancer cells by utilizing the power of immunity against the immune escape mechanism of cancer cells, including methods using immune checkpoint inhibitors, has been variously developed. In the development of such cancer immunotherapy, kynurenine (Kyn), which is a metabolite of tryptophan (Trp), is an important regulator of mammalian immune responses such as cancer immune tolerance, and indoleamine-2,3-dioxygenase 1 (IDO1) is one of the main enzymes that regulate the first rate-limiting step of the Kyn pathway. Therefore, it has attracted attention as a new target for anti-cancer drugs in cancer immunotherapy.

[0003] So far, multiple mechanisms have been known to suppress the immune system and promote cancer progression in the tumor microenvironment. Among them, the activation of the Kyn pathway in the tumor microenvironment is an important mechanism of immune tolerance and is known to promote cancer progression. Kyn acts as an endogenous ligand for the aryl hydrocarbon receptor (AhR) and activates AhR. AhR suppresses dendritic cells (DC), macrophages, NK cells, natural lymphocytes, cytotoxic T lymphocytes (CTL), helper T cells of type 17, and helper T cells of type 22, and activates immunosuppressive cells such as regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC). That is, Kyn, through the activation of AhR, inhibits NK cells and effector CD4 +It is thought to suppress immunity by inhibiting the proliferation and function of T cells and CTLs and activating Tregs and MDSCs.

[0004] In addition, depletion of tryptophan due to overexpression of IDO1 in the tumor microenvironment activates GCN2 (general control non-derepressible 2), a sensor that senses amino acid deficiency, and suppresses mTOR (mammalian target of rapamycin) involved in intracellular signal transduction, thereby suppressing T cell proliferation and differentiation and inducing anergy. This has also been reported. Furthermore, in DCs expressing IDO1 by TGF-β, naive CD4 + T cells are induced to become Tregs and are reported to function in immunosuppression. From these findings, molecules related to the Kyn pathway including IDO1 are thought to be greatly involved in immunosuppression in the tumor environment and have attracted attention as new targets for cancer immunotherapy.

[0005] To date, IDO1 inhibitors with various inhibition modes have been discovered. Multiple IDO1 inhibitors, such as the competitive inhibitors of IDO1, epacadostat, navoximod, indoximod, BMS986205, and the non-competitive inhibitor PF-06840003, have been used in clinical trials, but IDO1 inhibitors have not been approved as single drugs. Furthermore, in the clinical development trial of the combination therapy of epacadostat and Keytruda, an anti-PD-1 antibody, for malignant melanoma, the clinical trial was terminated because no statistically significant difference was confirmed in the effectiveness against the risk of disease progression or death compared with Keytruda monotherapy in the phase 3 trial. Therefore, it is difficult to say that sufficient development has been carried out for the combination trials of IDO1 inhibitors and other drugs. For example, in claim 91 of Patent Document 1, a large number of small molecules as activators of the adaptive immune response are listed, including IDO1 inhibitors, but there is no description of other drugs to be combined with IDO1 inhibitors. In Patent Document 2, although the combination of a chromene compound and an IDO1 inhibitor is described, only a combination drug characterized by containing a chromene compound is described, and there is no description of other drugs to be combined with the IDO1 inhibitor.

[0006] Furthermore, depending on the tumor, since tumors in non-human mammals are clinically, pathologically, and molecularly similar to tumors in humans, cancer immunotherapy is expected to be applicable not only to humans but also to non-human mammals such as dogs. Already, in Europe and the United States, the clinical application of cancer immunotherapy in the veterinary field has advanced, and an anti-PD-1 inhibitory antibody drug (Gilvetmab) has been launched, and clinical trials are also underway in Japan. Therefore, in the future, cancer immunotherapy is expected to become one of the pillars of cancer treatment for mammals such as dogs as well as for humans.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0008] Since the importance of Trp metabolism (Kyn pathway) in cancer immunotherapy has not been denied, rather than using an IDO1 inhibitor as a single agent, it is considered that a combination drug containing an IDO1 inhibitor may be more effective from the perspective of combined cancer immunotherapy, and the selection of drugs used in the combination drug has become an urgent task. Regarding other drugs to be used in combination with an IDO1 inhibitor, as described above, even if a number of small molecules listed in, for example, claim 91 of Patent Document 1 can be candidates, a method for efficiently selecting other drugs to be used in combination with an IDO1 inhibitor from among a number of candidates in a short time, efficiently, and with high accuracy has been demanded.

Means for Solving the Problems

[0009] The present inventor prepared a retroviral expression vector incorporating the mouse Ido1 gene, transfected mouse colon cancer cell line CT26 cells with a viral solution containing such a retroviral expression vector, and obtained a stable expression strain (CT26-IDO1) into which the mouse Ido1 gene was introduced, and established a technique for subcutaneously transplanting CT26-IDO1 into BALB / c mice. The present invention is based on the fact that, for tumor cells in a mouse transplantation model of CT26-IDO1 as a mouse colon cancer cell line into which the mouse Ido1 gene has been introduced, by performing transcriptome analysis, a method for efficiently selecting a drug to be used in combination with an IDO1 inhibitor was found by finding specific changes in the expression level in comparison with the wild type.

[0010] That is, the present invention is specified by the following matters. (1) A method for selecting a drug to be used in combination with an IDO1 inhibitor in cancer immunotherapy by performing transcriptome analysis on tumor cells derived from a mouse colon cancer cell line into which the mouse Ido1 gene has been introduced. (2) By performing transcriptome analysis, compare the expression level in tumor cells derived from a mouse colon cancer cell line transfected with the mouse Ido1 gene with the expression level in tumor cells derived from a mouse colon cancer cell line not transfected with the mouse Ido1 gene. The method according to (1) above is characterized by this. (3) The method according to (2) above, wherein the expression level is the expression level of cyclooxygenase 2 (COX-2). (4) A composition for cancer immunotherapy comprising an IDO1 inhibitor and a drug selected by the method according to (1) above. (5) The composition for cancer immunotherapy according to (4) above, wherein the subject is a mammal. (6) The composition for cancer immunotherapy according to (5) above, wherein the subject is a human. (7) The composition for cancer immunotherapy according to (5) above, wherein the subject is a dog.

Advantages of the Invention

[0011] By the method of the present invention, among a large number of drugs that may be used in combination with an IDO1 inhibitor in cancer immunotherapy, a drug to be used in combination with an IDO1 inhibitor can be selected efficiently, accurately, and in a short time.

Brief Description of the Drawings

[0012]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Mode for Carrying Out the Invention

[0013] The present invention relates to a method for selecting a drug contained in a composition for cancer immunotherapy together with an IDO1 inhibitor in cancer immunotherapy. Specifically, in a method for selecting a drug contained in a composition for cancer immunotherapy together with an IDO1 inhibitor, by performing transcriptome analysis of tumor cells in a mouse transplantation model of a mouse colon cancer cell line transfected with the mouse Ido1 gene, a method for efficiently selecting a drug to be used in combination with an IDO1 inhibitor by finding specific changes in expression levels in comparison with the wild type is provided.

[0014] The mouse Ido1 gene-introduced mouse colorectal cancer cell line was obtained by amplifying the mouse Ido1 gene using a commercially available mouse IDO1 expression vector, subcloning it into a retroviral expression vector, and transfecting it using a retroviral packaging cell line as described in the examples. After culturing, the virus solution was collected, transfected into a mouse colorectal cancer cell line, and a stable expression strain into which the mouse Ido1 gene was introduced was obtained. The mouse Ido1 gene-introduced mouse colorectal cancer cell line, that is, the stable expression strain into which the mouse Ido1 gene was introduced, can be obtained not only by the above method but also by conventional methods.

[0015] Transplantation of tumors into mice was performed by subcutaneously transplanting a certain number of mouse Ido1 gene-introduced mouse colorectal cancer cell lines into the ventral part, and the volume of the tumor was calculated by measuring the major axis and minor axis of the tumor after a certain period using a digital caliper.

[0016] In the present invention, transcriptome analysis is performed on tumor cells derived from a mouse Ido1 gene-introduced mouse colorectal cancer cell line. Transcriptome analysis is an analytical means capable of obtaining information on the functions of cells and genes because it reflects transcription and post-transcriptional regulation according to the functions of cells. In the examples, KEGG pathway analysis was performed as transcriptome analysis, but it is not limited to KEGG pathway analysis as long as changes in expression levels can be confirmed, and appropriate pathway analysis can be used. The genes to be analyzed are not particularly limited, but preferably, analysis of metabolism-related genes can be mentioned. Further, changes in expression levels can be confirmed by the fold change, and drugs that control the metabolism related to genes with a high fold change in expression may be considered candidates for drugs to be used in combination with IDO1 inhibitors.

[0017] Hereinafter, the present invention will be described in more detail in the examples, but the technical scope of the present invention is not limited thereto in any way.

Examples

[0018] [Generation of a mouse colon cancer cell line (CT26-IDO1 1G4 cells) transfected with the mouse Ido1 gene] To generate a retroviral vector carrying the mouse Ido1 gene, the mouse Ido1 gene was PCR-amplified from a mouse IDO1 expression vector (MC202318) purchased from OriGene and subcloned into the pMXs-Puro retroviral expression vector (Cell Biolabs). The retroviral expression vector incorporating mouse IDO1 was transfected into Platinum-A cells (Cell Biolabs), a retroviral packaging cell line, using X-tremeGENE9 DNA Transfection Reagent (Roche) and cultured for 48 hours. Then, the culture supernatant was collected and used as the virus solution. The collected virus solution was rapidly frozen in liquid nitrogen and stored in an ultra-low temperature freezer at -80°C until use. Mouse colon cancer cell line CT26 cells (ATCC) were transfected with the virus solution containing the retrovirus carrying the mouse Ido1 gene. After culturing for 10 days in a medium containing puromycin (InvivoGen) and 1 mM L-tryptophan (Sigma-Aldrich) to select cells into which the target gene had been introduced, CT26-IDO1 1G4 was obtained by the limiting dilution method. CT26-IDO1 1G4 cells were cultured at 37°C in the presence of 5% CO2 using a medium (tryptophan-added RPMI basal medium) prepared by adding 2 mM L-glutamine (Gibco), 1 mM L-tryptophan, 50 U / mL penicillin, and 50 μg / mL streptomycin (Gibco) to RPMI medium containing 10% FBS.

[0019] [Tumor transplantation into mice] Six-week-old female BALB / c mice (Japan SLC) were used in the experiment after being acclimated for 6 days or more. The backs and ventral sides of the BALB / c mice were shaved, and 5×10 5CT26-IDO1 1G4 cells or CT26 cells were subcutaneously transplanted. Five days after transplantation, the major axis (mm) and minor axis (mm) of the tumor were measured using a digital caliper (A&D), and the tumor volume was calculated. The formula for calculating the tumor volume was: tumor volume (mm 3 ) = 0.5 × (major axis mm) × (minor axis mm) 2 . The average of the tumor volumes on the left and right of each individual was calculated. The tumor diameter was measured twice a day until the tumor collection date. To measure the Trp concentration and Kyn concentration in the tumor, tumors derived from CT26-IDO1 1G4 cells and CT26 cells were excised 14 days after transplantation and immediately frozen in liquid nitrogen. Also, to perform transcriptome analysis of the tumor, tumors derived from CT26-IDO1 1G4 cells were excised 14 days after transplantation, and tumors derived from CT26 cells were excised 18 days after transplantation and immediately frozen in liquid nitrogen. The frozen tumors were stored in an ultra-low temperature freezer set at -80°C until each analysis after weighing. [Measurement of Trp Concentration and Kyn Concentration] For the tumors derived from CT26-IDO1 1G4 cells and CT26 cells obtained from the above mice, 1 mL of PBS was added per 0.1 g of the tumor to prepare a homogenate solution. 1.5 mL of the homogenate solution was added to a 2 mL tube and centrifuged (set at 4°C, 4000×g for 5 minutes), and 1 mL of the supernatant was collected. The obtained supernatant was stored in an ultra-low temperature freezer set at -80°C until measurement. An L-Tryptophan ELISA kit (immusmol, BA E-2700) was used for the measurement of the Trp concentration, and an L-Kynurenine ELISA kit (immusmol, BA E-2200) was used for the measurement of the Kyn concentration. The detailed operation procedures for ELISA measurement followed the attached materials of each kit. The results are shown in Figure 1. In the tumor derived from the mouse Ido1 gene-introduced mouse colorectal cancer cell line (CT26-IDO1 1G4 in the figure), a significant decrease in the Trp concentration and a significant increase in the Kyn concentration were observed compared to the tumor derived from the wild-type mouse colorectal cancer cell line (CT26) (CT26 in the figure). This result is considered to indicate the IDO1 activity in the tumor derived from the mouse Ido1 gene-introduced mouse colorectal cancer cell line.

[0020] [Transcriptome analysis] As a transcriptome analysis of tumors derived from CT26-IDO1 1G4 cells obtained from the above mice, the KEGG pathway was performed for metabolism-related genes. It was confirmed that the enhancement of the Kyn pathway due to IDO1 expression brought about changes in the expression of metabolism-related genes. The results are shown in FIGS. 3 and 4. From FIG. 3, an increase (upregulation) in the expression of related genes in the tryptophan metabolism system and the arachidonic acid metabolism system was confirmed, suggesting the enhancement of these pathways. Furthermore, from FIG. 4, in the arachidonic acid metabolism system (cascade), in tumor cells derived from a mouse colon cancer cell line transfected with the mouse Ido1 gene (shown as CT26-IDO1 1G4 in FIG. 4), the expression of Ptgs2 (encoding COX-2) was shown to be significantly increased compared to the wild type (shown as CT26 in FIG. 4). In addition, in tumor cells derived from a mouse colon cancer cell line transfected with the mouse Ido1 gene, the metabolism-related genes whose expression is increased to the same extent or more than that of Ptgs2 compared to the wild type are shown in Table 1 below. Drugs that control the metabolism related to these genes are considered to be candidates for drugs with expected effects when used in combination with IDO1 inhibitors.

[0021]

Table 1

Example

[0022] As a result of the transcriptome analysis of tumors derived from CT26-IDO1 1G4 cells obtained from mice, as shown in Table 1, the expression of Ptgs2 was significantly increased, and as shown in FIG. 4, it was also confirmed from qPCR analysis that the expression of Ptgs2 was significantly increased. Therefore, the combined effect on CT26-IDO1 tumor growth when a COX-2 inhibitor was selected as a drug for cancer immunotherapy in combination with an IDO1 inhibitor was confirmed by the following method.

[0023] [Method for preparing administration solution] Epacadostat was used as an IDO1 inhibitor, and Celecoxib was used as a COX-2 inhibitor. Both Epacadostat and Celecoxib were administered orally. For the combination administration group, the 2-fold concentrated suspensions of each were mixed at a ratio of 1:1 immediately before administration and administered at once. The specific preparation of the administration solution for mice weighing 20 g was carried out as follows. (1) Each compound was weighed, and a suspension was prepared with a 0.5 w / v% methylcellulose 400 solution (manufactured by Fujifilm Wako Pure Chemical Corporation) to obtain a 2-fold concentration at the time of administration. · For Epacadostat (50 mg / kg), a 10 mg / mL suspension, which is 2 times the concentration, was prepared so that it would be 1 mg / 200 μL / mouse (5 mg / mL suspension) at the time of administration. · For Celecoxib (5 mg / kg), a 1 mg / mL suspension, which is 2 times the concentration, was prepared so that it would be 0.1 mg / 200 μL / mouse (0.5 mg / mL suspension) at the time of administration. (2) For the single-agent administration group, the 2-fold concentrated suspension and a 0.5 w / v% methylcellulose 400 solution (manufactured by Fujifilm Wako Pure Chemical Corporation) were mixed at a ratio of 1:1 immediately before administration. (3) For the combination administration group, the 2-fold concentrated suspensions of each were mixed at a ratio of 1:1 immediately before administration.

[0024] [Administration Schedule] After tumor transplantation into mice, the drugs were administered daily from day 6 to day 13. A 0.5 w / v% methylcellulose 400 solution (manufactured by Fujifilm Wako Pure Chemical Corporation) was administered as a vehicle to the group without drug administration (Control group). After tumor transplantation into mice, the major axis (mm) and minor axis (mm) of the tumor were measured using a digital caliper on days 6, 8, 10, 12, 13, and 14, and the tumor volume was calculated. The formula for calculating the tumor volume was tumor volume (mm 3 ) = 0.5 × (major axis mm) × (minor axis mm) 2 and the average tumor volume in each group was calculated. The evaluation item was the average of each tumor volume on the last day. To evaluate the effect of drug administration on tumor volume, a Dunnett test was performed on the evaluation items of the drug administration group with respect to those of the Control group. When P < 0.05 or P < 0.01, a significant difference was considered to exist, and it was determined that there was an effect on tumor growth due to drug administration.

[0025] The results are shown in Fig. 5. The volume of tumors derived from CT26-IDO1 1G4 cells decreased when the IDO1 inhibitor and the COX-2 inhibitor were used in combination, showing a decrease in increase that was not seen when the IDO1 inhibitor or the COX-2 inhibitor was used alone. Also, since it was larger than the decrease amount obtained by adding up the decrease amounts when the IDO1 inhibitor and the COX-2 inhibitor were used alone respectively, a synergistic effect due to the combination was confirmed. When a Dunnett test was performed on the evaluation items of the drug administration group with respect to those of the Control group, since P < 0.01 in the combined use group, a significant difference was considered to exist, and it was determined that there was an effect on tumor growth due to combined administration. This indicates the effectiveness of the COX-2 inhibitor as a combined agent with the selected IDO1 inhibitor based on transcriptome analysis.

Example

[0026] When a COX-2 inhibitor was selected as a drug for cancer immunotherapy to be used in combination with an IDO1 inhibitor, the effect of combined administration on dogs was confirmed by the following method. The subjects were canine urothelial cancer cases with measurable target lesions, expected to have a remaining life of more than 2 weeks, excluding cases to which anticancer drugs, molecular target drugs, radiotherapy, and surgical therapy had been applied within 2 weeks and cases severely suffering from other diseases. The cases in Table 2 below were used as subjects.

Table 2

[0027] Based on physical examination, blood test, urine test, chest X-ray examination, and abdominal ultrasound examination, the effects and side effects were evaluated. For the effects, imaging diagnosis (ultrasound examination, chest X-ray examination if there is metastasis) was performed on the target lesion, and evaluation was carried out according to the guidelines for the determination of the treatment effect of solid cancer in dogs (RECIST; ver. 1.0). For side effects, physical examination, blood test, and urine test were performed, and evaluation was carried out according to the method for evaluating adverse events after investigational treatment in dogs and cats (VCOG-CTCAE; ver. 2). As shown in Fig. 6, as for the effects, in the case of single administration of Epacadstat, partial response (PR) was 1 out of 4 cases (25%), and stable disease (SD) was 3 out of 4 cases (75%). In the case of combined administration of Epacadstat and NSAIDs, partial response (PR) was 2 out of 2 cases (100%). Therefore, the effect of combined administration was also confirmed in dogs. Regarding side effects, no grade 3 or higher side effects were observed in all evaluation items, and no side effects leading to dose reduction, extension of the administration interval, or discontinuation were observed.

Industrial Applicability

[0028] By using Ido1 gene-introduced cancer cell lines and performing transcriptome analysis on tumors derived from the Ido1 gene-introduced cancer cell lines, other drugs to be used in combination with IDO1 inhibitors can be selected efficiently, accurately, and in a short time, contributing to the development of future combination drugs containing IDO1 inhibitors. Furthermore, by using stable expression cell lines of specific genes, it may be easier to select other drugs to be used in combination with drugs that inhibit the expression of specific genes.

Claims

1. A method for selecting a drug to be used in combination with an IDO1 inhibitor in cancer immunotherapy by performing transcriptome analysis on tumor cells derived from a mouse colon cancer cell line transfected with the mouse Ido1 gene.

2. The method according to claim 1, characterized in that, by performing transcriptome analysis, a comparison is made between the expression level in tumor cells derived from a mouse colon cancer cell line transfected with the mouse Ido1 gene and the expression level in tumor cells derived from a mouse colon cancer cell line not transfected with the mouse Ido1 gene.

3. The method according to claim 2, wherein the expression level is the expression level of cyclooxygenase 2 (COX-2).

4. A composition for cancer immunotherapy comprising an IDO1 inhibitor and a drug selected by the method according to claim 1.

5. The composition for cancer immunotherapy according to claim 4, wherein the subject is a mammal.

6. The composition for cancer immunotherapy according to claim 5, wherein the subject is a human.

7. The composition for cancer immunotherapy according to claim 5, wherein the subject is a dog.

Images