Application of gilteritinib to various variants

Abstract

It was found from analysis of various kinase inhibitors on tumors with acquired ALK-TKI resistance that gilteritinib, a therapeutic agent for FLT3-positive acute myeloid leukemia, was effective on the tumors. Gilteritinib directly inhibits kinase activity of ALK to exhibit an effect on ALK fusion-positive tumors with multiple duplications for which there has been no effective therapeutic agent. Gilteritinib alone can also overcome resistance of ALK-TKI resistant cancer via gene fusions with NTRK, ROS1 or LTK or via AXL, and combination therapy thereof can overcome ALK-TKI resistance mechanisms via bypass pathways such as KRAS, BRAF and EGFR.

Description

[Technical Field]

[0001] This invention relates to a drug effective against ALK fusion gene-positive tumors that have acquired resistance to multiple drugs, and to its scope of application. [Background technology]

[0002] Cancer drug therapy has seen improved efficacy with the development of molecularly targeted drugs tailored to various gene mutations. Molecularly targeted drugs target driver oncogenes and treat cancer by controlling their function. As a result, they often have relatively milder side effects than conventionally used cytotoxic anticancer drugs. However, even when the drug is effective and the tumor shrinks, resistance eventually develops, leading to the problem of drug resistance, where the drug becomes ineffective.

[0003] Lung cancer is a common cancer, with approximately 125,000 new cases reported annually in Japan, making it the third most common type of cancer. Globally, lung cancer is also prevalent, with over 2 million new cases reported each year. Lung cancer is broadly classified into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), with non-small cell lung cancer being the majority.

[0004] While various driver gene mutations have been reported in non-small cell lung cancer, representative genetic abnormalities often include EGFR and K-RAS gene mutations, as well as ALK, ROS1, and NTRK fusion genes. ALK fusion genes are said to be found in 3-5% of non-small cell lung cancer patients, making them the most common cause of lung cancer caused by fusion gene mutations. The ALK fusion gene is an oncogene that arises from the fusion of the ALK gene, a receptor tyrosine kinase, with a gene encoding a protein with a multimerizing domain, such as EML4. The ALK fusion protein can cause cancer by multimerizing through the function of the partner protein it fuses with, and by constitutively possessing kinase activity.

[0005] For ALK-positive lung cancer, molecularly targeted drugs that specifically inhibit ALK tyrosine kinase have been clinically demonstrated to be effective. To date, five ALK-TKIs (ALK-TKIs) have been clinically applied, starting with crizotinib, the first ALK tyrosine kinase inhibitor (ALK-TKI) developed. However, as mentioned above, the problem remains that cancer cells acquire drug resistance after treatment, leading to recurrence.

[0006] Alectinib, which has been shown to have approximately three times longer progression-free survival (PFS) than crizotinib, is used as a first-line treatment for ALK-positive lung cancer, but the problem is that drug resistance develops in most cases. Kinase domain mutations such as G1202R and I1171N / S / T mutations have been detected as alectinib-resistant mutations. It has been reported that lorlatinib, developed as a third-generation ALK-TKI, is effective against about half of these mutations. However, it has been reported that compound mutations occur in the kinase domain of ALK, leading to resistance to lorlatinib as well. Some compound mutations make the patient sensitive again to crizotinib or alectinib, but some compound mutations, such as the G1202R and L1196M compound mutation (hereinafter referred to as G1202R+L1196M), are resistant to all ALK-TKIs. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Okada K, et al., 2019, EBioMedicine Vol.41,pp.105-119 [Non-Patent Document 2] Shaw AT, et al., 2016, N Engl J Med, Vol.374, pp.54-61 [Non-Patent Document 3] Yoda S, et al. 2018, Cancer Discov Vol. 8,pp.714-729 [Non-Patent Document 4] Recondo G, et al. 2020, Clin Cancer ResVol. 26, pp. 242-255 [Non-Patent Document 5] Taniguchi H, et al. 2019, Nat Commun Vol. 10, 259 [Non-Patent Document 6] Doebele RC, et al., 2012, Clin Cancer ResVol.18, pp.1472-1482 [Non-Patent Document 7] Hrustanovic G, et al., 2015, Nat Med Vol.21, pp.1038-1047 [Non-Patent Document 8] Maynard A, et al., 2020, Cell Vol.182, pp.1232-1251e1222 [Overview of the project] [Problems that the invention aims to solve]

[0008] As mentioned above, while molecularly targeted drugs are highly effective, the development of resistance is a problem. The present invention aims to provide a pharmaceutical agent that is effective against ALK-positive tumors that have acquired resistance to any drug. In particular, it aims to search for a drug that is effective against the I1171N+F1174I and I1171N+L1198H duplicate mutations. Furthermore, it aims to broaden the range of treatment options by searching for molecules that can be targeted by the drug, i.e., gene mutations that are effective, based on the similarity of the structures to which the drug binds, and expanding the scope of application. In particular, the scope of application of gilteritinib, which has been shown to overcome the above-mentioned duplicate mutations, was investigated. [Means for solving the problem]

[0009] The present invention relates to the application of the following drugs. (1) A pharmaceutical composition for treating ALK fusion gene-positive tumors, characterized by containing gilteritinib as an active ingredient. (2) The pharmaceutical composition for treating ALK fusion gene-positive tumors according to (1), wherein the ALK fusion gene-positive tumor is a wild-type ALK or a tumor that has acquired resistance to first-generation, second-generation, or lorlatinib. (3) The pharmaceutical composition for treating ALK fusion gene-positive tumors according to (1) or (2), wherein the ALK fusion gene-positive tumor is a double mutation with I1171N, I1171S, V1180L, L1196M, C1156Y. (4) The pharmaceutical composition for treating ALK fusion gene-positive tumors according to any one of (1) to (3), wherein the ALK fusion gene-positive tumor is an ALK fusion gene having at least two or more double mutations including mutations of I1171N / S / T, F1174I / L, L1196M, L1198F / H, G1202R, D1203N, F1245V, L1256F, G1269A. (5) The pharmaceutical composition for treating ALK fusion gene-positive tumors according to any one of (1) to (3), wherein the ALK fusion gene-positive tumor has at least one or more ALK mutations of T1151K, C1159Y, I1171N, I1171T, I1171S, F1174I, F1174V, V1180L, L1196M, L1196Q, L1198F, D1203N, F1245V, L1256F, or G1269A. (6) The pharmaceutical composition for treating ALK fusion gene-positive tumors according to any one of (1) to (5), wherein the ALK fusion gene-positive tumor is non-small cell lung cancer. (7) The pharmaceutical composition for treating non-small cell lung cancer according to (6) for use as primary treatment or secondary treatment. (8) A composition for treating tumors caused by AXL activation, NTRK fusion gene, LTK fusion gene, and / or ROS1 fusion gene mutation, characterized by containing gilteritinib as an active ingredient. A method for testing whether gilteritinib is effective for patients with ALK fusion gene-positive tumors, which comprises testing whether the kinase domain is wild-type or has at least one ALK mutation of T1151K, C1159Y, I1171N, I1171T, I1171S, F1174I, F1174V, V1180L, L1196M, L1196Q, L1198F, D1203N, F1245V, L1256F, or G1269A, and if either is the case, determining that gilteritinib is effective. (10)A method for testing whether gilteritinib is effective for patients with ALK fusion gene-positive tumors, which comprises testing whether G1202R or D1203 mutation exists. If G1202R exists alone, it is determined that gilteritinib has no effect. In the case of a double mutant of D1203 mutation or G1202R mutation and other mutations, it is considered that the effect of gilteritinib may be slightly attenuated. (11)A treatment method characterized by examining the cause of the target disease, and if it is any of ALK fusion gene-positive, AXL activation including Gas6 expression induction, NTRK1 fusion gene, LTK fusion gene, and / or ROS1 fusion gene mutation, administering a pharmaceutical composition containing gilteritinib as an active ingredient. (12)The treatment method according to (11), characterized in that the target disease is ALK fusion gene-positive and is a tumor that is wild-type ALK, or has acquired resistance to first-generation, second-generation, or lorlatinib. (13)A treatment method for tumors that have developed resistance to ALK-TKI, which comprises examining the resistance acquisition mechanism. If the resistance acquisition mechanism is AXL activation, gilteritinib alone is administered. If the resistance acquisition mechanism is resistance via the KRAS bypass pathway, gilteritinib and a KRAS inhibitor are used. If the resistance acquisition mechanism is resistance via the BRAF bypass pathway, gilteritinib and a BRAF inhibitor and / or a MEK inhibitor are used. If the resistance acquisition mechanism is resistance via the EGFR bypass pathway, gilteritinib and an EGFR inhibitor are administered in combination.

Brief Description of the Drawings

[0010] [Figure 1A] This figure shows that gilteritinib suppresses the phosphorylation of ALK and ALK duplication mutations, as demonstrated by Western blotting. [Figure 1B] This figure shows the inhibitory effects of alectinib, lorlatinib, and gilteritinib on the phosphorylation of ALK and ALK duplication mutations, and the activation of downstream signaling pathways, as analyzed by Western blotting. [Figure 1C] This figure shows the inhibitory effects of alectinib, lorlatinib, and gilteritinib on the phosphorylation of ALK and ALK duplication mutations, and the activation of downstream signaling pathways, as analyzed by Western blotting. [Figure 2A] This figure shows that the inhibitory activity of gilteritinib on ALK kinase decreases in an ATP concentration-dependent manner, meaning that gilteritinib directly inhibits ALK by competing with ATP. [Figure 2B] This figure shows the results of analyzing the changes in peptide phosphorylation induced by gilteritinib treatment using phosphoproteome analysis. [Figure 3A] This figure shows the inhibitory effect (IC50) on cell proliferation of ALK gene mutation-positive non-small cell lung cancer cell lines and non-Hodgkin lymphoma cell lines by gilteritinib and ALK-TKI. [Figure 3B] Figure showing the analysis of ALK autophosphorylation in ALK gene mutation-positive cancer cell lines by Western blotting. [Figure 3C] A diagram showing that gilteritinib induces apoptosis. [Figure 4A] This figure shows the effects of gilteritinib treatment on the receptor tyrosine kinase activity of various non-small cell lung cancer cells and human normal fibroblasts. [Figure 4B] This figure shows the results of evaluating the effect of gilteritinib using a xenograft mouse model. [Figure 5A] This figure shows the results of analyzing the survival rate (IC50) of Ba / F3 cells expressing ALK mutants with a single mutation found in tumors resistant to first- and second-generation ALK-TKIs, after treatment with lorlatinib, alectinib, and gilteritinib. [Figure 5B] This figure shows the survival rates of various EML4-ALK mutants expressed in Ba / F3 cells, analyzed using gilteritinib and TKIs known to be effective against ALK mutants. IC50 is shown. [Figure 6] This figure shows the results of analyzing ALK autophosphorylation by Western blotting after treating Ba / F3 cells expressing a single ALK mutant found in tumors resistant to first- and second-generation ALK-TKIs with gilteritinib. [Figure 7A] This figure shows the results of Western blotting analysis of the suppression of downstream signaling pathway activation after treating MCC-003 cells expressing EML4-ALK I1171N with gilteritinib. [Figure 7B] This figure shows the results of analyzing apoptosis induction in MCC-003 cells treated with gilteritinib or lorlatinib. [Figure 8A] This figure shows the results of evaluating the effect of gilteritinib after transplanting MCC-003 into mice. [Figure 8B] This figure shows the in vivo effects of gilteritinib on MCC-003 cells transplanted into mice, analyzed by ALK autophosphorylation and activation of downstream signaling pathways. [Figure 9A] A schematic diagram illustrating the timing of ALK mutations detected by biopsy in JFCR-049, a patient with non-small cell lung cancer, in relation to ALK-TKI treatment. [Figure 9B] This figure shows the effect of gilteritinib on cell viability in Ba / F3 cells expressing the ALK variant detected in patient JFCR-049. [Figure 9C] This figure shows the survival rates (IC50) for alectinib, lorlatinib, and gilteritinib, analyzed using Ba / F3 cells expressing duplicate mutations. [Figure 9D] This figure shows the analysis of ALK autophosphorylation induced by gilteritinib using Ba / F3 cells expressing duplicate mutations. [Figure 10]This figure shows the effects of alectinib, lorlatinib, and gilteritinib analyzed using a xenograft model with JFCR-028-3 cells expressing EML4-ALK4 I1171N+F1174I. Tumor regression was observed when treatment was replaced with gilteritinib after treatment with alectinib or lorlatinib. [Figure 11A] This figure schematically illustrates the timing of ALK mutations detected by biopsy in non-small cell lung cancer patients JFCR-134 and JFCR-016, in conjunction with ALK-TKI treatment. [Figure 11B] This figure shows the effect of gilteritinib on cell viability in Ba / F3 cells expressing ALK mutants with the G1202R or D1203N mutation. [Figure 11C] This figure shows the inhibitory activity of the ALK-TKI, gilteritinib, against MR347 cells (EML4-ALK-D1203N+L1196M) established from ALK-TKI-resistant non-small cell lung cancer patients. [Figure 11D] This figure shows the IC50 values ​​of alectinib, lorlatinib, and gilteritinib in Ba / F3 cells expressing the duplicated mutations G1202R+L1196M, D1203N+F1245V, and D1203N+L1196M. [Figure 11E] This figure shows the autophosphorylation of ALK in Ba / F3 cells expressing the duplicated mutations G1202R+L1196M, D1203N+F1245V, and D1203N+L1196M after treatment with gilteritinib. [Figure 12A] This figure shows the results of Western blotting analysis of the activation of AXL and downstream signaling pathways by alectinib and gilteritinib in H3122 cells overexpressing AXL. [Figure 12B] This figure shows the IC50 values ​​of alectinib and gilteritinib in H3122 cells overexpressing AXL. [Figure 13A] This figure shows the effects of gilteritinib analyzed using an H3122 cell xenograft model overexpressing AXL. [Figure 13B] Figure showing the effect of alectinib in an H3122 cell xenograft model. [Figure 14A] This figure shows the effects of gilteritinib, trametinib, and AMG510 on the KRAS signaling pathway, analyzed by Western blotting using MCC-003 cells expressing KRAS G12C. [Figure 14B] A figure showing the IC50 of gilteritinib against MCC-003 cells expressing KRAS G12C. [Figure 14C] This figure shows the effects of gilteritinib and AMG510 on the KRAS signaling pathway, analyzed by Western blotting using JFCR-028-3 cells expressing KRAS G12C. [Figure 14D] A figure showing the IC50 of gilteritinib in JFCR-028-3 cells expressing KRAS G12C. [Figure 15A] This figure shows the effects of gilteritinib in a xenograft model using MCC-003 cells expressing KRAS G12C. [Figure 15B] This figure shows the effects of gilteritinib in a xenograft model using JFCR-028-3 cells expressing KRAS G12C. [Figure 16A] This figure shows the survival rates after treatment with gilteritinib, alectinib, and afatinib, using JFCR-098 cells established from patients who acquired ALK-TKI resistance via the EGFR pathway. [Figure 16B] This figure shows the results of Western blotting analysis of the suppression of EGFR and downstream signaling pathway activation by gilteritinib and alectinib. [Figure 17A] This figure shows the results of an analysis of the effect of ALK-TKI on survival rate using TPM3-NTRK1 fusion gene-positive colorectal cancer cells (KM12). [Figure 17B] This figure shows the results of an analysis of the effect of ALK-TKI on survival rate using Ba / F3 cells expressing the TPM3-NTRK1 fusion gene. [Figure 17C] This figure shows the effects of gilteritinib on NTRK1 and downstream signaling pathways, analyzed using KM12 cells. [Figure 17D] This figure shows the effects of entrectinib and gilteritinib on apoptosis using KM12 cells. [Figure 17E] A figure showing the effects of entrectinib and gilteritinib in a xenograft model transplanted with KM12 cells. [Figure 17F] This figure shows the effects of entrectinib and gilteritinib on NTRK1 and downstream signaling pathways in transplanted tumors, analyzed by Western blotting. [Figure 18A] This figure shows the effect of gilteritinib on survival rate using Ba / F3 cells expressing TPM3-NTRK1 with the G667C mutation. [Figure 18B] This figure shows the IC50 values ​​of gilteritinib and entrectinib for G595R and G667C mutations in the NTRK1 fusion gene. [Figure 18C] This figure shows the effects of gilteritinib, entrectinib, and lorlatinib on survival rates using Ba / F3 cells expressing TPM3-NTRK1 with the G595R mutation. [Figure 18D] This figure shows the inhibitory effect of gilteritinib on NTRK1 activity in G667C and G595R mutations of the NTRK fusion gene, analyzed by Western blotting. [Figure 19A] This figure shows the effects of various inhibitors on the cell viability of HCC78 cells. [Figure 19B] This figure shows the effects of various inhibitors on the cell viability of JFCR-168 cells. [Figure 19C] This figure shows the effects of crizotinib and gilteritinib in a xenograft model transplanted with JFCR-168 cells. [Figure 20A] This figure shows that low-dose gilteritinib is effective in a xenograft model using JFCR-028-3 cells. [Figure 20B] This figure shows that low-dose gilteritinib is effective in a xenograft model using H2228 cells. [Figure 20C]This figure shows that low-dose gilteritinib is effective in a xenograft model using MCC-003 cells.

[0011] As detailed below, gilteritinib is effective against tumors that have acquired ALK-TKI resistance due to mutations in the kinase domain of ALK. Furthermore, even if the acquisition of resistance to ALK-TKI is via a bypass pathway, gilteritinib alone can be effective if it is due to AXL activation, and if it is via the KRAS, BRAF, or EGFR pathway, it can be effective in combination with drugs that suppress those respective pathways.

[0012] If a patient develops resistance to existing ALK-TKIs due to treatment, whether the mutation is one that would be effective against gilteritinib can be determined by detecting the ALK gene mutation using known testing methods, such as PCR-based mutation detection, sequencing, or the use of antibodies that specifically detect mutations. Furthermore, even if no mutation is detected in the ALK gene and a bypass pathway is suspected, mutations or activation of KRAS, BRAF, and EGFR can be detected using known methods such as PCR-based mutation detection, sequencing, or antibodies.

[0013] 1. Searching for drugs that overcome ALK-TKI resistance mutations. The inventors have found and reported that some EML4-ALK duplication mutations that have acquired lorlatinib resistance regain sensitivity to existing ALK-TKIs (Non-Patent Literature 1). However, none of the approved ALK-TKIs are effective against the EML4-ALK I1171N+F1174I and I1171N+L1198H duplication mutations. Therefore, in order to search for drugs that can overcome these mutations, EML4-ALK I1171N+F1174I or EML4-ALK I1171N+L1198H were introduced into Ba / F3 cells (obtained from RIKEN BRC), and the cell proliferation inhibitory effect was examined using an inhibitor library mainly consisting of available kinase inhibitors in the expressed cells. The mutant gene-introduced cells used below were created by incorporating the mutated gene into a lentiviral vector and introducing it into cells. Furthermore, the EML4-ALK fusion gene and its variants used below were constructed based on the fusion gene disclosed in Non-Patent Document 1, unless otherwise specified.

[0014] Table 1 shows the survival rates of cells treated with DMSO, with the values ​​set to 100 after adding various drugs at a 50 nM concentration and measuring them using the CellTiter-Glo assay (Promega) 72 hours later. The results showed that gilteritinib, a drug used to treat acute myeloid leukemia with FLT3 gene mutations, exhibited growth inhibitory effects against EML4-ALK wild-type, I1171N+F1174I, and I1171N+L1198H duplication mutations. The sources of the drugs used are as follows. Crizotinib, brigatinib, lorlatinib: Shanghai Biochempartner; Gilteritinib: Shanghai Biochempartner or Biovision; Alectinib, ceritinib: ActiveBiochem; Entrectinib: Medchem Express; Afatinib: ChemieTek; Trametinib: AdooQ Bioscience Brigatinib was dissolved in ethanol, while the other drugs were dissolved in DMSO before use.

[0015] [Table 1]

[0016] In addition to crizotinib, ceritinib, alectinib, brigatinib, and lorlatinib, which are approved as ALK inhibitors, entrectinib also showed growth inhibitory effects against wild-type ALK fusion genes, but none of the drugs showed strong efficacy against duplication mutations. In contrast, gilteritinib showed strong cell proliferation inhibitory effects against I1171N+F1174I and I1171N+L1198H duplication mutations.

[0017] 2. Analysis of the mechanism of action of gilteritinib Since gilteritinib is a multi-kinase inhibitor, we analyzed whether its inhibitory effect on cell proliferation against duplication mutations is due to a direct action on its target, ALK duplication mutations. Ba / F3 cells expressing EML4-ALK wild-type, I1171N+F1174I, and I1171N+L1198H duplication mutations were treated with varying concentrations of gilteritinib for 3 hours, and ALK autophosphorylation was analyzed by Western blotting (Figure 1(A)). It was revealed that gilteritinib inhibits autophosphorylation of both ALK wild-type and duplication mutants. This suggests that it directly targets ALK.

[0018] Next, we analyzed whether gilteritinib suppresses ALK autophosphorylation using the EML4-ALK-positive non-small cell lung cancer cell line H3122 (obtained from MGH Cancer Center). H3122 cells and H3122 cells overexpressing the I1171N+F1174I duplication mutation were treated with alectinib, lorlatinib, and gilteritinib, and ALK phosphorylation and phosphorylation of AKT, ERK, and S6, which are located in the downstream signaling pathway of ALK, were analyzed by Western blotting (Figure 1(B), (C)). In the parental H3122 cell line, alectinib and lorlatinib also inhibited the phosphorylation of ALK and molecules in the downstream signaling pathway, but only gilteritinib had the effect of inhibiting the phosphorylation of ALK and molecules in the downstream signaling pathway at low concentrations in cells overexpressing I1171N+F1174I. Furthermore, the degree of inhibition of ALK phosphorylation and downstream signaling phosphorylation correlated with gilteritinib concentration, suggesting that gilteritinib directly inhibits ALK.

[0019] Furthermore, to confirm that gilteritinib directly inhibits ALK, ALK kinase activity was measured using an in vitro kinase assay at varying ATP concentrations (Figure 2(A)). ALK kinase activity was measured using the ADP-Glo ​​assay kit (Promega) in the presence of ATP from 1 μM to 1 mM. ALK kinase activity was inhibited in an ATP concentration-dependent manner, and IC50 was observed. 50 This increases in a ATP concentration-dependent manner. This indicates that gilteritinib inhibits ALK kinase activity by competing with ATP. Therefore, it was shown that gilteritinib directly inhibits ALK.

[0020] Furthermore, phosphoproteome analysis was performed to analyze the quantitative changes in peptide phosphorylation induced by gilteritinib treatment. Cell lysates of ALK-positive lung cancer cells treated with gilteritinib or DMSO were processed using a standard method, and phosphoproteome analysis was performed (Figure 2(B)). Arrows indicate phosphorylated ALK peptides. Gilteritinib was shown to significantly suppress ALK phosphorylation. Although data are not shown here, gilteritinib treatment also significantly suppressed the phosphorylation of ALK adapter proteins such as IRS1 / 2, SOS2, and SH2B2. These results indicate that gilteritinib directly inhibits ALK by competing with ATP.

[0021] 3. Effect of gilteritinib on inhibiting proliferation We investigated whether gilteritinib has a growth inhibitory effect on non-small cell lung cancer with ALK rearrangement. Cell viability assays were performed using EML4-ALK fusion gene-positive non-small cell lung cancer cells (H2228 (obtained from ATCC) and H3122 (obtained from MGH Cancer Center)) and primary cultured cells derived from ALK fusion gene-positive lung cancer patients (JFCR-018-1 and JFCR-028-3). Cells with "JFCR" in their name are cells established by the Japanese Foundation for Cancer Research. ICs for gilteritinib and ALK-TKI in each cell line were also performed. 50 This is shown in Figure 3(A). In all cell types, gilteritinib showed a growth inhibitory effect at a low concentration comparable to that of lorlatinib. Furthermore, gilteritinib also showed a growth inhibitory effect on KARPAS299 cells, a human non-Hodgkin lymphoma cell line that is positive for the NPM-ALK fusion gene. In other words, it is shown to have therapeutic effects not only on non-small cell lung cancer but also on tumors that are positive for the ALK gene mutation.

[0022] These non-small cell lung cancer cell lines were treated with gilteritinib, alectinib, and lorlatinib, and autophosphorylation was analyzed by Western blotting. In all cell lines, gilteritinib inhibited ALK autophosphorylation (Figure 3(B)). Although not shown here, gilteritinib also suppressed the activation of downstream signaling pathways such as AKT, ERK, S6, and STAT3.

[0023] Furthermore, the effect of gilteritinib on apoptosis induction was analyzed by flow cytometry (Figure 3(C)). H3122 cells were treated with DMSO, lorlatinib, and gilteritinib at concentrations of 100 nM each. After 72 hours, the cells were stained with annexin V and propidium iodide, and the percentage of cells in which apoptosis was induced was measured. Both gilteritinib and lorlatinib induced apoptosis in approximately 50% of the cells.

[0024] Several driver genes other than ALK exist for non-small cell lung cancer. Therefore, we investigated the effects of gilteritinib on non-small cell lung cancer caused by causative genes other than ALK gene mutations. Cells with EGFR-activating mutations (HCC827, obtained from ATCC), PC9 (obtained from RIKEN BRC), KRAS mutation-positive cells (A549, obtained from RIKEN BRC), H460 (obtained from ATCC), patient-derived cells with BRAF mutations (JFCR-256-3), and human normal lung fibroblasts (TIG-3, obtained from RIKEN BRC) were treated with varying concentrations of gilteritinib, and the inhibitory effects on EGFR or downstream signaling pathway activity, as well as the induction of apoptosis, were analyzed by Western blotting. In JFCR-028-3 cells, which are ALK gene mutation-positive cells, gilteritinib showed inhibition of ALK autophosphorylation, inhibition of phosphorylation of proteins in the ALK-downstream signaling pathway, and induction of apoptosis. However, gilteritinib showed little to no inhibitory effect on cells positive for any of the EGFR, KRAS, or BRAF gene mutations (Figure 4(A)).

[0025] Since gilteritinib showed growth inhibitory effects in vitro against ALK gene mutation-positive cancers, we then investigated whether it would be effective in vivo. H3122 or JFCR-028-3 was transplanted subcutaneously into BALB / c nu / nu mice, and tumor volumes reached 150 mm². 3 Once the target level was reached, gilteritinib was administered orally at a dose of 30 mg / kg to the solvent only as a control group, and in addition to the JFCR-028-3 transplant group, alectinib was administered orally at a dose of 30 mg / kg once daily for 5 days a week, and the effects were analyzed (n=6 in each group). Tumor volume was measured three times a week. Gilteritinib also showed a significant tumor growth inhibitory effect in in vivo studies (Figure 4(B)).

[0026] Patients resistant to alectinib have been reported to have gene mutations in the ALK kinase domain involving amino acid substitutions such as I1171T / N / S, V1180L, G1202R, and L1196M. ALK variants with single mutations identified in alectinib-resistant tumors were expressed in Ba / F3 cells, and cell viability after 72 hours of culture with gilteritinib, alectinib, and lorlatinib was calculated using the CellTiter-Glo assay. IC 50 We calculated this (Figure 5(A)).

[0027] Except for the EML4-ALK I1171T (4.17nM), EML4-ALK I1171N (6.13nM), EML4-ALK I1171S (2.86nM), EML4-ALK V1180L (1.45nM), EML4-ALK L1196M (20.4nM), and EML4-ALK G1202R (168nM), gilteritinib showed IC50 values ​​of 30nM or less for all mutations except the G1202R mutation. 50 This indicated that gilteritinib has a high inhibitory effect on cell proliferation against these mutations.

[0028] In addition to these alectinib-resistant mutations, we also analyzed the cell proliferation inhibitory effects of mutations reported to be crizotinib and ceritinib-resistant. As a result, IC in each mutant 50The EML4-ALK mutations observed were EML4-ALK C1156Y (0.66nM), EML4-ALK F1174V (3.41nM), EML4-ALK F1245V (1.41nM), EML4-ALK G1269A (1.39nM), EML4-ALK T1151K (1.24nM), EML4-ALK F1174I (4.72nM), EML4-ALK L1196Q (25.5nM), and EML4-ALK D1203N (53.0nM). Except for the D1203N mutation, gilteritinib showed a strong inhibitory effect on proliferation. Furthermore, gilteritinib also showed an IC (intracytoplasmic sperm injection) effect against EML4-ALK L1256F, a lorlatinib-resistant mutation recently detected by the inventor. 50 It showed an inhibitory effect on cell proliferation even at a low concentration of 0.34 nM.

[0029] Furthermore, the effects of TKIs known to be effective against ALK mutants, and the effects of gilteritinib on ALK mutants were analyzed. Similar to the above, in a cell model in which various mutants were expressed in Ba / F3 cells, ALK-TKIs, or crizotinib, alectinib, ceritinib, brigatinib, lorlatinib, entrectinib, and gilteritinib, which are known to be effective against ALK mutants, were added. Cell viability after 72 hours of culture was calculated using the CellTiter-Glo assay, and IC was obtained. 50 We sought the following (Figure 5(B)). As a result, it became clear that gilteritinib, compared to approved ALK-TKIs, is effective at low concentrations against various ALK variants, not just the I1171N+F1174I and I1171N+L1198H variants.

[0030] Many ALK mutations have been identified to date. Gilteritinib is thought to be effective against these combinations as well. For example, the I1171N / T / S mutation is thought to have overlapping mutations with the following mutations: T1151ins, F1174I / L, L1196M, L1198F / H, G1202R, D1203N, F1245V, L1256F, and G1269A. Furthermore, the V1180L mutation is expected to have overlapping mutations with T1151ins, F1174I / L, L1196M, L1198F / H, G1202R, D1203N, F1245V, L1256F, and G1269A. The G1202R mutation is expected to have overlapping mutations with F1174I / L, L1196M, L1198F / H, and G1269A. The L1196M mutation is expected to have overlapping mutations with F1174I / L, L1198F / H, G1202R, D1203N, F1245V, L1256F, and G1269A. Gilteritinib is expected to be effective against these overlapping mutations as well. In this context, I1171N / T / S indicates a mutant in which the I at position 1171 is mutated to either N, T, or S. The same applies to other mutants.

[0031] Furthermore, autophosphorylation of these single-mutation ALK gene mutants induced by gilteritinib treatment was analyzed by Western blotting (Figure 6). Corresponding to the growth-inhibiting effect of gilteritinib, autophosphorylation was completely suppressed in all mutants except G1202R and D1203N upon treatment with 50 nM gilteritinib.

[0032] In addition to cell models using Ba / F3, the inhibitory effect of gilteritinib was confirmed using MCC-003 cells (established by the Cancer Research Society from patient samples from Miyagi Cancer Center) that were derived from patients who had become resistant to alectinib and possessed the EML4-ALK-I1171N mutation. MCC-003 cells were treated with alectinib, lorlatinib, and gilteritinib for 6 hours, and the phosphorylation of ALK and downstream signaling pathways was analyzed by Western blotting (Figure 7(A)). In MCC-003 cells as well, gilteritinib suppressed ALK autophosphorylation and the activation of downstream signaling pathways.

[0033] Furthermore, it was confirmed that gilteritinib induced apoptosis in MCC-003 cells (Figure 7(B)). MCC-003 cells were treated with DMSO, lorlatinib, and gilteritinib at a concentration of 100 nM each as a solvent, stained with annexin V and propidium iodide 72 hours later, and the proportion of cells in which apoptosis was induced was measured. In both the cells treated with gilteritinib and lorlatinib, apoptosis was induced in about 20% of the cells.

[0034] MCC-003 cells were transplanted into BALB / c nu / nu mice, and the effect of gilteritinib was analyzed using a xenograft model. MCC-003 cells were transplanted subcutaneously into mice, and after the average tumor volume reached 150 mm 3 ³, only the solvent, 30 mg / kg of alectinib, or gilteritinib was orally administered once a day, 5 days a week, as a control. The experiment was conducted with 8 mice in each group, and the tumor volume was measured three times a week (Figure 8(A)). A significant regression of the tumor was observed in the gilteritinib-administered group, whereas no significant regression of the tumor was observed in the alectinib-administered group. No decrease in body weight was observed in all groups until day 15.

[0035] In the MCC-003 cell tumor transplanted into mice, it was analyzed by Western blotting whether the autophosphorylation of ALK and the activation of the downstream signaling system were suppressed by the administration of gilteritinib (Figure 8(B)). Suppression of the autophosphorylation of ALK and the activation of the downstream signaling system was observed by the administration of gilteritinib, whereas no significant suppression was observed by the administration of alectinib.

[0036] Lorlatinib is effective against most single gene mutations that cause resistance to first- and second-generation ALK-TKIs. However, the inventors have previously reported that lorlatinib resistance can be acquired due to duplicate mutations in the ALK kinase domain (Non-Patent Literature 2, 3). Furthermore, the inventors have identified a case in which the ALK I1171S+G1269A duplicate mutation resulted in lorlatinib resistance (Figure 9(A)). Patient JFCR-049 received chemotherapy (4 cycles of cisplatin / pemetrexed / bevacizumab) followed by treatment with crizotinib, alectinib, and lorlatinib. The resistance mutation was detected in liver metastases after lorlatinib treatment. The I1171S+G1269A duplicate mutation is sensitive to the second-generation ALK-TKIs ceritinib and brigatinib, and the patient's tumors regressed with treatment with ceritinib.

[0037] As shown above, since gilteritinib was effective against mutations that had acquired resistance to first-generation and second-generation ALK-TKIs, we evaluated whether it was effective against duplication mutations that had acquired resistance to lorlatinib. We introduced the EML4-ALK I1171S+G1269A duplication mutant into Ba / F3 cells and expressed it, and analyzed the effect of gilteritinib on cell survival (Figure 9(B)). Ba / F3 cells were expressed with EML4-ALK wild-type, EML4-ALK I1171S single-gene mutant, and EML4-ALK I1171S+G1269A duplication mutant, and gilteritinib was added to the culture medium. After 72 hours, cell survival was evaluated by the CellTiter-Glo assay. IC of single-gene mutant (I1171S) 50 The IC was 2.86 nM, representing a duplicate mutation (I1171S+G1269A). 50 The cell proliferation inhibitory effect of gilteritinib was observed in both single-gene mutations and duplicated-gene mutations, with a mutation size of 23.5 nM.

[0038] Furthermore, we expressed duplicate mutations with I1171N in Ba / F3 cells and analyzed the effects of alectinib, lorlatinib, and gilteritinib on cell proliferation. Gilteritinib showed IC50 or less in all cases, even with the I1171N duplicate mutation. 50(I1171N+F1174I, 23.5nM; I1171N+F1174L, 3.15nM; I1171N+L1196M, 14.0nM; I1171N+L1198F, 1.64nM; I1171N+L1198H, 6.95nM; I1171N+L1256F, 0.41nM; I1171N+G1269A, 11.4nM) were observed, demonstrating a high inhibitory effect on cell proliferation (Figure 9(C), Figure 5(B)).

[0039] Furthermore, we analyzed the effect of gilteritinib on autophosphorylation in mutations resistant to these first and second-generation ALK-TKIs. I1171S single-gene mutations and duplicate mutations were expressed in Ba / F3 cells, and the cells were harvested 3 hours after gilteritinib treatment and analyzed by Western blotting (Figure 9(D)). In all the analyzed mutants, ALK autophosphorylation was inhibited by gilteritinib.

[0040] Next, JFCR-028-3 cells expressing the lorlatinib-resistant duplication mutation ALK I1171N+F1174I were transplanted into mice, and the effect of gilteritinib was evaluated in an in vivo model. JFCR-028-3 cells expressing EML4-ALK I1171N+F1174I were transplanted into BALB / c nu / nu mice, and the tumor volume was 200 mm². 3 Once tumor volume was reached, either the solvent alone, alectinib (30 mg / kg), lorlatinib (5 mg / kg), or gilteritinib (30 mg / kg) was administered orally once daily for 5 days a week (n=6 in each group). On day 41, the alectinib and lorlatinib-treated groups were switched to gilteritinib treatment (30 mg / kg, once daily, 5 days a week) and the experiment continued. Tumor volume was measured three times a week (Figure 10).

[0041] In the alectinib and lorlatinib treatment groups, tumor regrowth was observed within a short period, but in the gilteritinib treatment group, complete tumor regression was observed for more than 50 days. Furthermore, in the alectinib and lorlatinib treatment groups, rapid tumor regression was observed when gilteritinib was administered after tumor regrowth had occurred. These results suggest that gilteritinib is likely to be effective against tumors that have become resistant to currently approved ALK-TKIs.

[0042] Mutations that have led to resistance to ALK-TKIs, including lorlatinib, include the ALK L1196M+G1202R and D1203N+F1245V duplication mutations. In JFCR-134, the L1196M+G1202R mutation was detected after treatment with crizotinib and lorlatinib, and in JFCR-016, the D1203N+F1245V duplication mutation was detected after treatment with crizotinib and alectinib (Figure 11(A)). Furthermore, since there have been reports that the L1196M+D1203N duplication mutation has led to resistance to all clinically used ALK-TKIs (Non-Patent Literature 4), we investigated whether gilteritinib has a cell proliferation inhibitory effect against these three duplication mutations.

[0043] We expressed EML4-ALK wild-type cells, the three duplicate mutations that have acquired resistance to existing ALK-TKIs, and single mutations of G1202R and D1203N in Ba / F3 cells and examined the effect of gilteritinib on cell survival (Figure 11(B)). As a result, relatively high concentrations of gilteritinib were required to inhibit the three duplicate mutations other than ALK wild-type, or the single mutations of G1202R and D1203N.

[0044] MR347 cells, established from ALK-TKI-resistant non-small cell lung cancer patients, possess the EML4-ALK-D1203N+L1196M duplicate mutation. These cells were used to investigate the effects of ALK-TKIs and gilteritinib on cell viability (Figure 11(C)). Neither drug showed a significant inhibitory effect on cell viability.

[0045] Double mutations D1202R+L1196M, D1203N+F1245V, and D1203N+L1196M were expressed in Ba / F3 cells, and IC for alectinib, lorlatinib, and gilteritinib was performed. 50 The following was calculated (Figure 11(D)): IC for gilteritinib against cell viability. 50 The mutations were D1202R+L1196M, 117nM; D1203N+F1245V, 64nM; and D1203N+L1196M, 109nM. Unlike the previously shown duplication mutations including I1171N / S, gilteritinib did not show a significant inhibitory effect on cell proliferation at low concentrations against these gene mutations.

[0046] Furthermore, we investigated whether gilteritinib suppresses ALK autophosphorylation in these duplicate mutations (Figure 11(E)). As a result, gilteritinib hardly suppressed ALK autophosphorylation. As shown above, gilteritinib is effective against a very large number of resistance mutations compared to existing ALK-TKIs, but it is not sufficiently effective against some of the mutations mentioned above.

[0047] 4. Effect of gilteritinib on bypass mechanisms involved in ALK-TKI resistance Drug resistance in ALK fusion gene-positive cancers can be broadly classified into ALK-independent drug resistance, i.e., activation of pathways that bypass ALK, such as EGFR, cMET, KRAS, BRAF, and AXL, and ALK-dependent mechanisms resulting from the occurrence of a second mutation in ALK, as described above. As discussed so far, gilteritinib has been shown to be effective against a considerable number of mutations in the ALK-dependent pathway. However, resistance acquisition through bypass pathways is also a problem, so we investigated whether gilteritinib can suppress pathways that bypass ALK and acquire resistance. Although not shown here, gilteritinib is not affected by AXL activation induced by Gas6 expression and is effective against resistant cells.

[0048] In EGFR-positive lung cancer, decreased responsiveness to EGFR-TKIs has been reported to correlate with AXL activation (Non-Patent Literature 5). Therefore, we analyzed whether gilteritinib suppresses the activation of AXL and its downstream signaling pathways by Western blotting. AXL was overexpressed in the EML4-ALK-positive non-small cell lung cancer cell line H3122, and treated with alectinib and gilteritinib. Western blotting was used to analyze AXL and its downstream signaling pathways (Figure 12(A)). Gilteritinib suppressed the activation of AXL and its downstream MAPK and PI3-AKT signaling pathways, but the addition of alectinib did not inhibit AXL or its downstream signaling pathways.

[0049] IC for survival rates of alectinib and gilteritinib 50 Upon investigation, it was found that overexpression of AXL leads to increased IC of alectinib 50 Although a significant increase was observed, gilteritinib remained largely unaffected by AXL overexpression, showing values ​​below 5 nM.

[0050] H3122 cells overexpressing AXL were transplanted subcutaneously into BALB / c nu / nu mice, resulting in an average tumor volume of 150 mm². 3After reaching a certain stage, patients were divided into control groups: those receiving only the solvent, those receiving alectinib (30 mg / kg), and those receiving gilteritinib (30 mg / kg). Forced oral administration was administered once daily, five days a week. On day 25, the alectinib-treated group was randomly divided into two groups: one continuing alectinib and the other switching to gilteritinib (n=6 in each group). While the alectinib group initially showed tumor growth inhibition, tumor growth became apparent after about three weeks. In contrast, tumor growth was suppressed in the groups receiving gilteritinib, or those switched from alectinib to gilteritinib (Figure 13(A)). The fact that gilteritinib also showed tumor growth inhibition in the group switched from alectinib to gilteritinib indicates that it has a growth inhibitory effect on tumors that have become resistant to alectinib. The effect of alectinib on the parent strain H3122 was confirmed using a xenograft model in the same manner as described above, and it was found that the proliferation of H3122 was completely suppressed by alectinib (Figure 13(B)).

[0051] Next, we investigated the KRAS signaling pathway, which is known as another bypass pathway. Lineage switching and activation of the MAPK signaling pathway have been reported to be important mechanisms of resistance to ALK-TKIs (Non-Patent Literature 6, 7). Furthermore, it has been reported that even in EML4-ALK-positive tumors, there are KRAS G12C and KRAS G13D mutations in cases of recurrence after treatment with multiple ALK-TKIs (Non-Patent Literature 8). Therefore, we focused on KRAS G12C and investigated whether gilteritinib is effective against this mutation.

[0052] We analyzed the inhibition of downstream KRAS signaling pathway activity using MCC-003 cells expressing KRAS G12C by Western blotting. We analyzed whether the activation of the KRAS signaling pathway was inhibited by adding gilteritinib, trametinib (which inhibits the MAPK signaling pathway by inhibiting MEK), and AMG510 (obtained from Medchem Express), a KRAS G12C-specific inhibitor, either individually or in combination (Figure 14(A)). Gilteritinib could not completely suppress the KRAS signaling pathway. Furthermore, IC50 was used to assess survival rates. 50 When calculated, it showed a high value (Figure 14(B)).

[0053] Similar analyses were performed using Western blotting with JFCR-028-3 cells expressing KRAS G12C. When KRAS G12C was introduced into JFCR-028-3 cells, some suppression of KRAS downstream signaling pathway activation was observed even with gilteritinib alone (Figure 14(C)). Furthermore, regarding survival rate, gilteritinib alone showed high IC50. 50 This was shown (Figure 14(D)).

[0054] Based on these results, it was considered necessary to use a combination of a KRAS G12C-specific inhibitor and gilteritinib to overcome KRAS G12C-mediated resistance. Therefore, a combination study of AMG510 and gilteritinib was conducted in an in vivo model. MCC-003 cells expressing KRAS G12C were transplanted subcutaneously into BALB / c nu / nu mice, with an average tumor volume of 175 mm². 3After reaching a certain stage, participants were divided into control groups: one receiving only the solvent, the other receiving AMG510 (100 mg / kg), the other gilteritinib (30 mg / kg), and the other receiving a combination of both AMG510 and gilteritinib. Forced oral administration was performed once daily, five days a week (n=6 in each group). Tumor volume was measured five times a week (Figure 15(A)). The same experiment was also conducted using JFCR-028-3 cells expressing KRAS G12C (Figure 15(B)). In both cell types, neither AMG510 nor gilteritinib alone showed complete inhibition of tumor growth; only when both were administered in combination did they show an inhibitory effect on tumor growth. While this study investigates the effect of gilteritinib on KRAS mutations, it is thought that it may also be effective against the BRAF-mediated bypass pathway. BRAF is a molecule located downstream of KRAS, and its activating mutations are not only recognized as driver oncogenes in melanoma, lung cancer, and colorectal cancer, but are also occasionally found in acquired resistance cases of ALK and EGFR-positive lung cancer. Therefore, in cases where ALK inhibitor resistance has developed due to BRAF mutations, combining gilteritinib with a BRAF inhibitor or a drug that inhibits MEK, a signaling molecule located downstream of BRAF, can lead to overcoming resistance.

[0055] Next, we analyzed the EGFR signaling pathway, which is known as another bypass pathway. JFCR-098 cells were established from patients who acquired ALK-TKI resistance via the EGFR pathway. JFCR-098 cells were treated with alectinib, gilteritinib, and in combination with the EGFR inhibitor afatinib, and their effects on cell viability were analyzed (Figure 16(A)). Significant cell proliferation inhibition was observed with alectinib, or with gilteritinib in combination with an EGFR inhibitor. In particular, the combination of gilteritinib and afatinib strongly suppressed cell proliferation. Furthermore, activation of downstream signaling pathways by Western blotting was also suppressed by the combination of afatinib and gilteritinib (Figure 16(B)).

[0056] 5. Effects of gilteritinib on ROS1 and NTRK fusion genes Various driver gene mutations have been identified in non-small cell lung cancer, one of which is due to rearrangement of the ROS1 or NTRK gene. Three NTRK genes are known: NTRK1, NTRK2, and NTRK3, all of which are fusion genes involved in the pathogenesis of cancer. When simply referred to as an NTRK fusion gene, it indicates that it is a fusion gene of one of NTRK1, NTRK2, or NTRK3. ROS1 rearrangement accounts for approximately 1% of non-small cell lung cancers, and NTRK rearrangement accounts for approximately 0.1%. Since the tyrosine kinase domains of ROS1 and NTRK have structural similarities to the kinase domain of ALK, it is known that several ALK-TKIs have inhibitory effects on the kinase activity of ROS1 and NTRK. Therefore, the effect of gilteritinib was investigated using a cell model with Ba / F3 cells.

[0057] The effect of gilteritinib on survival was analyzed in the TPM3-NTRK1 fusion gene-positive colorectal cancer cell line KM12 and in Ba / F3 cells into which the TPM3-NTRK1 fusion gene had been introduced (Figure 17(A), (B)). IC of KM12 cells treated with gilteritinib. 50 IC of Ba / F3 with TPM3-NTRK1 fusion gene expression below 30 nM 50 Both cell lines showed high sensitivity to gilteritinib, with a gene strength of 13.3 nM. Entrectinib is a kinase inhibitor used as an inhibitor of NTRK and ROS1 fusion genes.

[0058] Downstream signaling pathways were analyzed using KM12 cells by Western blotting (Figure 17(C)). Corresponding with the survival rate results, gilteritinib suppressed the activation of NTRK1 and downstream signaling pathways. Furthermore, the effect of gilteritinib on apoptosis was analyzed (Figure 17(D)). Similar to entrectinib, gilteritinib induced apoptosis in approximately 50% of cells.

[0059] The effect of gilteritinib was analyzed using a xenograft model with KM12 cells (Figure 17(E)). KM12 cells were transplanted subcutaneously into BALB / c nu / nu mice with an average tumor volume of 100 mm². 3 After reaching a certain stage, patients were divided into control groups receiving either the solvent only, entrectinib (30 mg / kg), or gilteritinib (30 mg / kg), and were administered orally once daily for 10 days (n=6 in each group). Tumor volume was measured three times a week. Gilteritinib, like entrectinib, suppressed tumor growth. Furthermore, gilteritinib also suppressed the activation of NTRK1 and downstream signaling pathways in the tumors (Figure 17(F)).

[0060] Furthermore, we also examined mutations in the NTRK gene fusion. NTRK1 G667C and G595R were the first mutations reported to cause entrectinib resistance. Although several drugs, such as ponatinib, have been reported to be effective against the NTRK1 G667C mutation, none of them have been approved. We analyzed whether gilteritinib is effective against this mutation. Ba / F3 cells expressed TPM3-NTRK1-G667C were treated with gilteritinib, entrectinib, and lorlatinib, and cell viability was analyzed (Figure 18(A)). The G667C mutation was more sensitive to gilteritinib than the wild type, and IC 50 The saturation level was 12.7 nM (Figure 18(B), (C)). However, neither gilteritinib nor entrectinib had any effect on survival rate in the G595R mutant (Figure 18(B)). Furthermore, analysis of the effect on NTRK1 phosphorylation revealed that gilteritinib suppressed NTRK1 phosphorylation in the G667C mutant (Figure 18(D)).

[0061] Next, we analyzed the effects on ROS1 fusion genes. HCC78 cells are cancer cells possessing the SLC34A2-ROS1 fusion gene, and JFCR-168 cells are cancer cells possessing the CD74-ROS1 fusion gene. In both cell types, gilteritinib showed an inhibitory effect on cell proliferation (Figure 19(A), (B)). Furthermore, we analyzed the effect of gilteritinib in a xenograft model using JFCR-168 cells (Figure 19(C)). JFCR-168 cells were transplanted subcutaneously into SCID-Beige mice with an average tumor volume of 150 mm². 3 After reaching a certain stage, patients were divided into control groups receiving only the solvent, crizotinib (100 mg / kg), or gilteritinib (30 mg / kg), and forced oral administration was performed once daily for 5 days a week (n=3 in each group). Tumor volume was measured 3 times a week. Significant tumor regression was observed with both gilteritinib and crizotinib.

[0062] In the experiments using the xenograft model described above, the effect was analyzed after administering 30 mg / kg of gilteritinib, but we also investigated whether a lower dose of gilteritinib would be effective. JFCR-028-3 cells, H2228 cells, and MCC-003 cells were transplanted subcutaneously into BALB / C nu / nu mice, with an average tumor diameter of 150 mm. 3 After reaching the target level, the effects were analyzed using either the solvent alone or gilteritinib administered orally once daily as controls (Figure 20). The kinase domain of the ALK gene was wild-type in JFCR-028-3 cells and H2228 cells, while alectinib-resistant MCC-003 cells had the I1171N mutation.

[0063] In a xenograft model using JFCR-028-3 cells, tumor cell proliferation was not observed even when gilteritinib was administered at 3 mg / kg, showing a significant difference compared to the control group administered DMSO. Furthermore, tumor regression was observed in the 6 mg / kg dose group, and the tumor had completely regressed by day 9 of gilteritinib administration (Figure 20(A)). In a xenograft model using H2228 cells, tumor regression was observed at all concentrations of gilteritinib administered at 6 mg / kg, 10 mg / kg, and 30 mg / kg (Figure 20(B)). In a xenograft model using MCC-003 cells resistant to alectinib, tumor regression was observed when gilteritinib was administered at 10 mg / kg (Figure 20(C)).

[0064] As described above, gilteritinib has been shown to be effective against tumors that have developed resistance to first-generation and second-generation ALK-TKIs. In particular, it is effective against duplicate mutations for which there were previously no effective drugs. Furthermore, gilteritinib monotherapy can overcome resistance to ALK-TKI resistant cancers mediated by NTRK, ROS1 fusion genes, and AXL, and combination therapy can overcome resistance to ALK-TKI resistance mechanisms mediated by bypass pathways such as KRAS, BRAF, and EGFR.

Claims

1. A pharmaceutical composition for the treatment of ALK fusion gene-positive tumors, comprising gilteritinib as an active ingredient, The aforementioned ALK fusion gene-positive tumor, A pharmaceutical composition characterized by a tumor having overlapping mutations with I1171N, I1171S, V1180L, L1196M, and C1156Y.

2. A pharmaceutical composition for the treatment of ALK fusion gene-positive tumors, comprising gilteritinib as an active ingredient, The aforementioned ALK fusion gene-positive tumor, A pharmaceutical composition characterized by a tumor having at least two or more overlapping mutations, including the mutations I1171N / S / T, F1174I / L, L1196M, L1198F / H, G1202R, D1203N, F1245V, L1256F, and G1269A.

3. A pharmaceutical composition for the treatment of ALK fusion gene-positive tumors, comprising gilteritinib as an active ingredient, The aforementioned ALK fusion gene-positive tumor, A pharmaceutical composition characterized by a tumor having at least one mutation of T1151K, C1156Y, I1171N, I1171T, I1171S, F1174I, F1174V, V1180L, L1196M, L1196Q, L1198F, D1203N, F1245V, L1256F, or G1269A.

4. A pharmaceutical composition for treating ALK fusion gene-positive tumors according to any one of claims 1 to 3, characterized in that the ALK fusion gene-positive tumor is non-small cell lung cancer.

5. A pharmaceutical composition for the treatment of non-small cell lung cancer according to claim 4, for use as a primary or secondary treatment.

6. A tumor treatment composition for tumors caused by an NTRK1 fusion gene, an NTRK1 fusion gene having a G667C mutation, or a ROS1 fusion gene, characterized by containing gilteritinib as an active ingredient.

7. A method to support testing whether gilteritinib is effective in ALK fusion gene-positive tumor patients, The kinase domain is detected to be wild-type or to have at least one ALK mutation from T1151K, C1156Y, I1171N, I1171T, I1171S, F1174I, F1174V, V1180L, L1196M, L1196Q, L1198F, D1203N, F1245V, L1256F, or G1269A. A diagnostic support method to determine if gilteritinib is effective if either of the following conditions is met.

8. A method to support testing whether gilteritinib is effective in ALK fusion gene-positive tumor patients, Detect whether the G1202R or D1203 mutation is present. If G1202R is present alone, it is determined that gilteritinib will not be effective. A diagnostic support method that acknowledges that the effectiveness of gilteritinib may be slightly reduced in cases of duplicate mutations involving the D1203 mutation or the G1202R mutation and other mutations.

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