TAS6417, A Novel EGFR Inhibitor Targeting Exon 20 ... · low-energy scans and ramped from 15 to 40 eV for high-energy scans, and further data analysis was performed using BioPhar-maLynx1.3.3

Small Molecule Therapeutics

TAS6417, A Novel EGFR Inhibitor Targeting Exon20 Insertion MutationsShinichi Hasako1,2, Miki Terasaka1, Naomi Abe1, Takao Uno1, Hirokazu Ohsawa1,Akihiro Hashimoto1, Ryoto Fujita1, Kenji Tanaka1, Takashige Okayama1,Renu Wadhwa3, Kazutaka Miyadera1, Yoshimi Aoyagi1, Kazuhiko Yonekura2, andKenichi Matsuo1

Abstract

Activatingmutations in the EGFR gene are important targetsin cancer therapy because they are key drivers of non–small celllung cancer (NSCLC). Although almost all common EGFRmutations, such as exon 19 deletions and the L858R pointmutation in exon 21, are sensitive to EGFR-tyrosine kinaseinhibitor (TKI) therapies, NSCLC driven by EGFR exon 20insertion mutations is associated with poor clinical outcomesdue to dose-limiting toxicity, demonstrating the need for anovel therapy. TAS6417 is a novel EGFR inhibitor that targetsEGFR exon 20 insertion mutations while sparing wild-type(WT) EGFR. In cell viability assays using Ba/F3 cells engineeredto express humanEGFR, TAS6417 inhibited EGFRwith various

exon20 insertionmutationsmorepotently than it inhibited theWT. Western blot analysis revealed that TAS6417 inhibitedEGFR phosphorylation and downstream molecules in NSCLCcell lines expressing EGFR exon 20 insertions, resulting incaspase activation. These characteristics led to marked tumorregression in vivo in both a genetically engineeredmodel and ina patient-derived xenograft model. Furthermore, TAS6417 pro-vided a survival benefit with good tolerability in a lung ortho-topic implantation mouse model. These findings support theclinical evaluation of TAS6417 as an efficacious drug candidatefor patients with NSCLC harboring EGFR exon 20 insertionmutations. Mol Cancer Ther; 17(8); 1648–58. �2018 AACR.

IntroductionSomatic mutation of the EGFR is a major oncogenic driver

(1) and is present in approximately 30% to 50% and 10% to20% of non–small cell lung cancer (NSCLC) in Asians and inAmericans and Western Europeans, respectively (2–5). Acti-vating mutations in the EGFR kinase domain induce ligand-independent constitutive activation and subsequent down-stream molecule phosphorylation, leading to cancer cellgrowth and survival (6, 7). Because of its important role incancer, mutant EGFR is an important therapeutic target forlung cancer. Among a wide variety of somatic mutations inEGFR, exon 19 deletion mutations and L858R substitutionmutation in exon 21 are most common, accounting for over80% of mutations (8, 9). These common mutations are sen-sitive to ATP-mimetic EGFR tyrosine kinase inhibitors (TKI) inpreclinical models (10–12), and patients with NSCLC harbor-ing these drug-sensitive mutations show significant responses

to EGFR-TKI therapies, including gefitinib, erlotinib, and afa-tinib, compared with standard chemotherapies (13–18).

The next largest proportion of EGFR mutations is a family ofexon 20 insertions, which accounts for roughly 4% to 13% of allEGFR mutations in patients with NSCLC (9, 19–22); these muta-tions consist of in-frame insertions of 3 to 21 base pairs predom-inantlywithin the range of codons 762 to 774. These insertions, aswell as exon 19 deletions and L858R mutation, have character-istics of oncogenic driver mutations, which induce constitutiveactivation and cell transformation inpreclinical studies.However,in contrast to exon 19 deletions and L858R mutation, exon 20insertions do not sensitize the kinase domain to EGFR-TKIs,behaving as intrinsic resistant mutations (6, 23–25). Further-more, patients with NSCLC harboring these mutations exhibitpoor clinical responses to monotherapy with afatinib (26), gefi-tinib (25, 27), and erlotinib (21, 25, 28) because plasma con-centrations of these drugs in clinical settings are kept low by dose-limiting toxicity caused by wild-type (WT) EGFR inhibition (22,29, 30), indicating the importance of development of an EGFRinhibitor targeting exon 20 insertions.

Therefore, clinical trials for EGFR-TKIs designed to overcomeexon 20 insertion–mediated resistance in patients are now inprogress. AP32788, a TKI designed to inhibit exon 20 insertions inthe EGFR or HER2 genes, is currently in a phase I/II clinical trial(NCT02716116) to determine a recommended phase II dose inthe dose escalation cohort and the overall response rate in theexpansion cohorts. In addition, interventional studies againstEGFR exon 20 insertions have been initiated for poziotinib(NCT03066206 and NCT03318939), which is a pan-ErbB inhib-itor, and are planned for osimertinib (NCT03191149), which hasreceived approval for patients with NSCLC harboring T790Macquired resistance mutations in the exon 20 region.

1Discovery and Preclinical Research Division, Taiho Pharmaceutical Co., Ltd.,Tsukuba, Ibaraki, Japan. 2Early Development Strategy & Planning, Taiho Phar-maceutical Co., Ltd., Kandanishiki-cho, Tokyo, Japan. 3DBT-AIST InternationalLaboratory for AdvancedBiomedicine (DAILAB), National Institute of AdvancedIndustrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Shinichi Hasako, Taiho Pharmaceutical Co., Ltd., 3Okubo, Tsukuba, Ibaraki 300-2611, Japan. Phone: 812-9865-4527; Fax: 812-9865-2170; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-17-1206

�2018 American Association for Cancer Research.

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In this study, we report the preclinical characterization ofTAS6417, a novel EGFR-TKI with a unique scaffold fitting intothe ATP-binding site of the EGFR hinge region. The structurediffers from that of all previously known small-molecule EGFR-TKIs, and our results demonstrate the selective potent inhibitoryeffect of TAS6417 on EGFR exon 20 insertions compared withWTEGFR, leading to an efficacious anticancer effect in several in vivomodels.

Materials and MethodsMaterials

TAS6417was synthesized at TaihoPharmaceuticalCo., Ltd. Thestructure was confirmed by ESI-MS and NMR, and its purityexceeded 99% as measured by HPLC. Afatinib was purchasedfrom Selleck Chemicals, and erlotinib from LC Laboratories.

Mass spectrometry analysisThe human recombinant EGFR D770_N771insNPG was

purchased from Carna Biosciences. The recombinant enzymeat 1 mmol/L was incubated with 50 mmol/L TAS6417 at 37�C for2 hours. After reduction and alkylation with DTT (ThermoFisher Scientific) and iodoacetamide (Thermo Fisher Scientific),the protein was digested with trypsin/Lys-C (Promega) fol-lowed by Asp-N (Thermo Fisher Scientific). The peptide–com-pound complex was detected with an LC/MS system consistingof an Acquity UPLC I-Class and Xevo G2-S QT of mass spec-trometer equipped with an ESI ion source (Waters Corpora-tion) using an Aeris WIDEPORE 3.6m XB-C18 150 � 2.1 mmcolumn (Phenomenex) at a flow rate of 0.3 mL/minute and acolumn temperature of 50�C. The gradient of mobile phase A[water, 0.1% formic acid (v/v)] and B [acetonitrile, 0.1% formicacid (v/v)] was performed as follows: 2% B for 3 minutes, 2% to50% B for 37 minutes, 50% to 90% B for 10 minutes. The datawere acquired in MSE mode with collision energy of 4 eV forlow-energy scans and ramped from 15 to 40 eV for high-energyscans, and further data analysis was performed using BioPhar-maLynx1.3.3 software (Waters Corporation).

Cell cultureTheNCI-H1975,HCC827,NCI-H23,NCI-H460, andNIH/3T3

cell lines were obtained from ATCC, and the Ba/F3 and PC-9 celllines were from RIKEN BioResource Center. The LXF 2478L cellline was provided by Charles River Discovery Research Services,GmbH, who generated the original cell line from its proprietarypatient-derived xenograft (PDX) model. The NHEK-Neo cell linewas obtained from Lonza. Ba/F3 cells were cultured in RPMI1640supplementedwith 10%FBS (MPBiomedicals, LLC) and1ng/mLmIL-3 (Cell Signaling Technology), NIH/3T3 cells were culturedin D-MEM containing 10% NBCS (Thermo Fisher Scientific),NHEK-Neo cells were cultured in Keratinocyte Basal Medium(KBM) containing KGM-Gold SingleQuots (Lonza), and otherhuman lung cancer cells were cultured in RPMI1640 containing10% FBS (MP Biomedicals, LLC); all cells were cultured at 37�Cwith 5% CO2. All cell lines were authenticated by short tandemrepeat profiling before purchase or use in this study.

Genetically engineered cell linescDNA constructs ofWT andmutant EGFRwere transfected into

Ba/F3 andNIH/3T3 cells usingNucleofector (Lonza), followed byclone selection using puromycin. The mutant EGFRs tested in

this study were A763_Y764insFQEA, V769_D770insASV,D770_N771insSVD, D770_N771insG, H773_V774insNPH,H773_V774insPH, delE746_A750 (exon 19 deletion), anddelE746_A750 þ T790M (exon 19 deletion þ T790M). ForNCI-H1975 EGFR D770_N771insSVD cells, NCI-H1975 cellsexpressing D770_N771insSVD mutant EGFR were established asdescribed above, and then endogenous L858R/T790M mutantEGFR was knocked out by XTN TALEN-mediated mutagenesis(Transposagen Biopharmaceuticals). NCI-H1975 EGFRD770_N771insSVD_Luc cells were established by transfectionof luciferase expression vector into NCI-H1975 EGFRD770_N771insSVD cells, followed by clone selection usinghygromycin B. After establishment, all cell lines were authenti-cated by short tandem repeat profiling, and sequencing analysiswas performed to confirm the integration of WT and mutantEGFR. As for NCI-H1975 EGFR D770_N771insSVD cells, thepresence of knockoutmutation bymutagenesis was also validatedwith both sequencing and immunoblotting analysis as describedin Supplementary Methods.

Cell proliferation assayCell proliferation was assessed using the CellTiter-Glo lumi-

nescent cell viability assay (Promega). For a panel of Ba/F3 celllines expressing human EGFRs, the cells were plated at anoptimal density onto 96-well plates, followed by 72 hours ofexposure to the compound in mIL-3-free conditions. Ba/F3parent cells and Ba/F3 EGFR WT cells were stimulated with 1ng/mL mIL-3 and 50 ng/mL EGF, respectively, during com-pound treatment. For a panel of human lung cancer cell linesand NHEK-Neo cells, the cells were plated as described aboveand incubated for 24 hours for attachment, followed by com-pound treatment for 72 hours. After compound exposure, avolume of CellTiter-Glo reagent equal to the volume of medi-um in each well was added, followed by measurement ofluminescence in each well using an EnSpire Multimode PlateReader (Perkin Elmer). IC50 and GI50 values were determinedusing the SAS software package in EXSUS (CAC Croit).

Caspase induction analysisCaspase induction was assessed using the Caspase-Glo 3/7

assay (Promega). Cells were plated at optimal density onto 96-well plates and incubated for 24 hours for attachment, followedby compound treatment for 24 hours. After compound exposure,a volume of Caspase-Glo 3/7 reagent equal to the volume ofmedium in each well was added, followed by measurement ofluminescence in each well as described above. The measurementwas correctedwith respect to cell viability determined as describedabove, and relative caspase-3/7 activation against DMSO controlwas determined.

Immunoblotting analysisFor cultured cell lines, cells were seeded at optimal density onto

culture plates or dishes with medium including 10% serum.Following culture for 24 hours, the cells were exposed to thecompound and harvested at the indicated time points. NIH/3T3EGFR WT was stimulated with 50 ng/mL EGF during compoundexposure. Xenograft tumors and mouse skin tissues were excisedat each time point after a single dosing of test compound. Thecollected samples were lysed and homogenized with RIPA buffersupplemented with a protease and phosphatase inhibitor cocktail(Thermo Fisher Scientific) on ice. Following centrifugation, the

TAS6417 as a Novel EGFR-TKI Specific to Exon 20 Insertions

www.aacrjournals.org Mol Cancer Ther; 17(8) August 2018 1649




supernatants were collected, and protein concentrations weredeterminedusing the Pierce BCAProteinAssayKit (ThermoFisherScientific).

For quantification of pEGFR, proteins at optimal concentra-tions were dispensed into Wes 12 to 230 kDa prefilled plates(ProteinSimple) with primary antibody, anti-rabbit secondaryantibody, and streptavidin horseradish peroxidase (HRP) and itssubstrate, followed by separation with capillary electrophoresisand chemiluminescence detection using Simple Western (Pro-teinSimple). Peak areas (in the range of 162–198 kDa) were usedto determine IC50 values using the SAS software package in EXSUS(CAC Croit).

For Western blotting analysis, proteins were separated bySDS-PAGE using 4% to 15% Criterion TGX precast gels (Bio-Rad Laboratories) and transferred to PVDF membranes using aTrans-Blot Turbo Transfer System (Bio-Rad). After probing withthe primary antibodies, the membranes were incubated withHRP-conjugated secondary antibody. The antigen–antibodycomplexes were visualized using ECL prime (GE Healthcare),followed by detection with the Amersham Imager 600 QC (GEHealthcare).

Primary and secondary antibodies were obtained from CellSignaling Technologies and GE Healthcare, respectively.

In vivo efficacy experimentsAll animal protocols were reviewed and approved according to

regional Institutional Animal Care and Use Committees. For theLXF 2478 and LU0387 models, animal experiments were per-formed by Charles River Discovery Research Services, GmbH, andCrown Bioscience Inc., respectively. Male BALB/cAJcl-nu/nu miceand female F344/NJcl-rnu/rnu rats were obtained from CLEAJapan, NMRI nu/nu mice from Charles River Laboratories, andBALB/c nude mice from Harlan Laboratories; animals rangedfrom 4 to 6 weeks of age.

In the subcutaneous implantation model, LXF 2478 andLU0387, human lung cancer PDXs, were transplanted intoNMRI nu/nu mice and BALB/c nude mice, respectively. Thegenetically engineered cell lines were suspended in PBS andinoculated into BALB/cAJcl-nu/nu mice for NCI-H1975 EGFRD770_N771insSVD (0.8� 107 cells/100 mL) and NIH/3T3 EGFRH773_V774insNPH(0.5�107 cells/100mL), and into F344/NJcl-rnu/rnu rats forNCI-H1975 EGFRD770_N771insSVD (1.6� 107

cells/200mL). After reachingoptimal tumor volume, animalswererandomized into groups (six to eight animals per group) andorally administered the compound once daily for 10, 14, or 28days. In both PDXmodels, the treatment period was followed byan observation period of 14 days. Antitumor activity was evalu-ated using tumor volume calculated according to the followingformula: [tumor volume] ¼ [major axis] � [minor axis]2 � 0.5.Statistical analysis was performed using repeated measuresANOVA followed by Dunnett test for tumor volume (P < 0.05).

In the orthotropic implantation model, NCI-H1975 EGFRD770_N771insSVD_Luc cells were suspended in 50% PBS and50% Matrigel (BD Biosciences) and directly inoculated into thelungs of BALB/cAJcl-nu/nu nudemice (2.4� 106 cells/20 mL). Sixdays after inoculation, a luciferin solution was administered viathe tail vein, and then photon values were measured by the IVISImaging System (Perkin Elmer) to confirm engraftment, followedby grouping using the values. Starting on the following day,animals were orally administered the compound once daily.Survival benefit was evaluated according to median survival time

(MST), and increase in life span (ILS) was calculated using thefollowing formula: ILS (%)¼ ([MSTof the treated group]/[MST ofcontrol] – 1) � 100. Statistical analysis was performed using theWilcoxon test for MST (P < 0.05).

TAS6417 was formulated in 0.1 N HCl with or without 0.5%hypromellose (HPMC) solution. Afatinib was formulated in a0.5% methylcellulose (MC), 0.4% Tween80 suspension.

Pharmacokinetic analysisThe plasma concentrations of TAS6417 were determined in

BALB/cAJcl-nu/nu mice and F344/NJcl-rnu/rnu rats followingmultiple oral administrations for 14 days at effective doses (threeanimals per group). The plasma samples were collected beforeadministration on Day 14, as well as at 0.5-, 1-, 2-, 4-, 8-, and 24-hour post-administration for mice and 0.25-, 0.5-, 1-, 2-, 4-, 8-,and 24-hour post-administration for rats. The plasma concentra-tions were measured with the LC/MS-MS.

ResultsTAS6417 is a novel inhibitor of EGFR exon 20 insertion

TAS6417,with6-methyl-8,9-dihydropyrimido[5,4-b]indolizineas its core structure, is a novel small-molecule inhibitordesigned to be fit to the ATP binding site of the EGFR hingeregion and to exhibit inhibitory activity against exon 20 inser-tion mutations. The chemical structure is shown in Fig. 1A andits synthetic procedure is described in Supplementary Fig. S1.MS analysis demonstrated that TAS6417 covalently modifiedthe cysteine residue at position 797 of recombinant EGFR,harboring an in-frame insertion mutation in the exon 20 region(Fig. 1B; Supplementary Table S1).

TAS6417 inhibited the in vitro phosphorylation activity ofEGFR and its mutants including an exon 20 insertion mutation(D770_N771insNPG), with IC50 values ranging from1.1� 0.1 to8.0 � 1.1 nmol/L (Supplementary Table S2A). TAS6417 wasfurther evaluated according to its selectivity for EGFR over otherkinases as described in Supplementary Methods. Of the 255kinases tested, TAS6417 inhibited 25 kinases other than EGFRwith IC50 values less than 1,000 nmol/L, which was 100 timeshigher than its IC50 value against WT EGFR. It inhibited only sixkinases containing TXK, BMX, HER4, TEC, BTK, and JAK3, withIC50 values less than 10 times that against WT EGFR, as shown inSupplementary Table S2B.

The cellular potency of TAS6417 against EGFR exon 20 inser-tion mutations was evaluated using genetically engineered NIH/3T3 cells. After 6 hours of exposure, TAS6417 showed potentinhibition of EGFR phosphorylation in a panel of NIH/3T3 cellsstably expressing exon 20 insertion mutant human EGFRs, withan IC50 value of <4.48 nmol/L for A762_Y763insFQEA, 78.2 �27.4 nmol/L for V769_D770insASV, 23.4 � 2.5 nmol/L forD770_N771insSVD, 45.7 � 19.8 nmol/L for D770_N771insG,66.7� 19.0 nmol/L forH773_V774insNPH, and 117� 31 nmol/L for H773_V774insPH (Table 1A). In contrast, the IC50 value ofTAS6417 for WT EGFR was 368 � 181 nmol/L. Furthermore,TAS6417 inhibited proliferation of Ba/F3 cells driven by variousEGFR exon 20 insertionmutations, with IC50 values ranging from5.05�1.33nmol/L to150�53nmol/L (Fig. 1C).Consistentwithan intracellular target inhibition assay in NIH/3T3 cells, TAS6417showed selectivity against WT EGFR (IC50 ¼ 676� 304 nmol/L).In a cell proliferation assay in a Ba/F3 cell panel, the selectivity forexon 20 insertions over the WT, represented as the WT/mut ratio,

Hasako et al.

Mol Cancer Ther; 17(8) August 2018 Molecular Cancer Therapeutics1650




was 134-fold for A763_Y764insFQEA, 6.37-fold forV769_D770insASV, 17.4-fold for D770_N771insSVD, 17.2-foldfor D770_N771insG, 4.51-fold for H773_V774insNPH, and4.55-fold for H773_V774insPH (Fig. 1D). In contrast, afatinib

and erlotinib showed no selectivity against exon 20 insertions,with less than one-fold WT/mut ratios except for that ofA763_Y764insFQEA. Although A763_Y764insFQEA is an inser-tion mutation at the exon 20 region in the EGFR gene, it has been

Figure 1.

TAS6417, an EGFR-TKI targeting exon 20 insertion mutations. A, Chemical structure of TAS6417, which contains 6-methyl-8,9-dihydropyrimido[5,4-b]indolizine asits core unit.B,MSE spectrum confirmation for protease-digested peptide [LLGICLTSTVQLITQLMPFGCLL] with TAS6417. TAS6417-treated EGFRD770_N771insNPGwas digestedwith protease and analyzed by LC-MSE peptidemapping. Identification of TAS6417 attached peptidewas confirmed using theMSE fragmentation data.Fragment ions containing C797 (red) were shifted corresponding to the molecular weight of TAS6417, suggesting that TAS6417 covalently bound to EGFRD770_N771insNPG.C, Inhibitory activity of TAS6417, afatinib, and erlotinib on cell proliferation in a panel of Ba/F3 cells engineered to express humanEGFRs. The cellswere cultured with the compounds for 72 hours in mouse IL3-free conditions. Ba/F3 EGFR WT cells were stimulated with 50 ng/mL EGF during compoundtreatment. IC50 valueswere determinedwith theCellTiter-Glo assay.D,Exon 20 insertionmutation selectivity of TAS6417, afatinib, and erlotinib. Using the IC50 valuesshown in C, the WT/mut ratio was determined.






reported as an EGFR-TKI-sensitive mutation, and patients withNSCLC harboring this mutation have shown clinical responses toboth afatinib and erlotinib (9, 25). These data demonstrated thepotency of TAS6417 against cells driven by EGFR exon 20 inser-tions withmutant selectivity, the spectrum of which was differentfrom that of other EGFR-TKIs.

TAS6417 inhibits in vitro cell growth and EGFR signaling inhuman cancer cell lines driven by EGFR exon 20 insertions

The effect of TAS6417 on tumor cell proliferation was inves-tigated using a panel of seven human lung cancer cell linesexpressing different EGFRs. The panel consisted of LXF 2478Lcells established from a PDX expressing an EGFR exon 20 inser-tion mutation (V769_D770insASV), NCI-H1975 EGFRD770_N771insSVD cells in which the endogenous alleles withL858RandT790Msubstitutionswere knockedout and exogenousEGFR with an exon 20 insertion mutation (D770_N771insSVD)was transfected (Supplementary Fig. S2), HCC827 and PC-9 cellsexpressing an exon 19 deletion mutation (delE746_A750) of theEGFR gene, NCI-H1975 cells with substitution mutations inexons 20 and 21 (T790M with L858R), and NCI-H23 and NCI-H460 cells expressing a KRAS mutation without EGFR mutation.TAS6417 suppressed the growth of cells expressing exon 20insertion mutations of the EGFR gene, with a GI50 value of86.5 � 28.5 nmol/L for LXF 2478L cells and 45.4 � 2.6 nmol/L for NCI-H1975 EGFR D770_N771insSVD cells (Table 1B).TAS6417 also potently inhibited proliferation in other cell linesharboring activating mutations or acquired resistance mutations,with mean GI50 values of 1.92 � 0.21 nmol/L to 7.12 � 0.60nmol/L. In contrast, the effect of TAS6417 on cell proliferation innormal human epidermal keratinocytes (NHEK-Neo), of whichWT EGFR is implicated in the growth and survival (31, 32), wasmoderate. Also, TAS6417 had no effect on EGFR-independentproliferation in NCI-H23 or NCI-H460 cells.

Furthermore, EGFR signal inhibition and apoptosis inductionby TAS6417 were confirmed by assessing the expression of phos-

pho-proteins and caspase activation markers (Fig. 2A and B).Culture with cells for 6 hours resulted in a concentration-depen-dent decrease of phospho-EGFR and its downstream molecules,including ATK andERK, in both LXF 2478L cells andH1975 EGFRD770_N771insSVD cells, indicating inhibition of EGFR signaltransduction. Accordingly, TAS6417 led to increased levels of Bim,which is a pro-apoptosis protein whose expression is regulated byAKT and ERK signaling (33), and cleaved PARP, which is cleavedby caspases and is a marker of cells undergoing apoptosis (34), inboth cell lines after treatment for 24 hours. Consistent withWestern blotting analysis, chemiluminescence analysis revealedthat TAS6417 caused a concentration-dependent increase in rel-ative caspase-3/7 activity in both cell lines compared with that inthe control 24 hours after treatment, implying induction ofapoptosis (Fig. 2C and D). In summary, these findings suggestthat TAS6417 inhibits EGFR signal transduction, leading to cellgrowth inhibition and apoptosis induction in NSCLC cells drivenby EGFR exon 20 insertion mutations.

TAS6417 causes persistent tumor regression in vivo in EGFRexon 20 insertion-driven tumor models

To investigate in vivo antitumor efficacy of TAS6417 fortumors driven by EGFR exon 20 insertion mutations, weconducted experiments against the three major mutations,namely V769_D770insASV, D770_N771insSVD, andH773_V774insNPH. TAS6417 was administered orally oncedaily for 14 days to mice implanted with NCI-H1975 EGFRD770_N771insSVD xenografts, resulting in dose-dependenttumor growth inhibition (Fig. 3A). At final evaluation on Day15, all groups treated with TAS6417 showed statistically sig-nificant inhibition of tumor growth compared with that of thecontrol group. Although TAS6417 administered at 50 and 100mg/kg/day showed marked tumor growth inhibition withtreatment/control (T/C) ratios of 51% and 19%, respectively,200 mg/kg/day TAS6417 achieved tumor regression with a T/Cof 5%. The tumor regression occurred quickly after TAS6417

Table 1. Cellular potency of TAS6417

A. Inhibitory activity of TAS6417 against pEGFR in NIH/3T3 cell lines expressing human EGFRIC50 value, nmol/L

EGFR status Mean � SD

WT (þEGF) 368 � 181A763_Y764insFQEA <4.48V769_D770insASV 78.2 � 27.4D770_N771insSVD 23.4 � 2.5D770_N771insG 45.7 � 19.8H773_V774insNPH 66.7 � 19.0H773_V774insPH 117 � 31Ex19del 1.35 � 0.17Ex19delþT790M 0.868 � 0.152

B. Inhibitory activity of TAS6417 against cell proliferation in lung cancer cell lines and primary keratinocytesGI50, nmol/L

TAS6417 Afatinib ErlotinibCell line Mean � SD Mean � SD Mean � SDLXF 2478L 86.5 � 28.5 71.7 � 21.5 1040 � 365NCI-H1975 EGFR D770_N771insSVD 45.4 � 2.6 221 � 45 2340 � 313HCC827 1.92 � 0.21 1.61 � 0.13 10.0 � 2.3PC-9 2.96 � 1.56 3.36 � 1.79 66.3 � 22.6NCI-H1975 7.12 � 0.60 182 � 30 >3,000NCI-H23 >3,000 1710 � 151 >3,000NCI-H460 >3,000 >3,000 >3,000NHEK-Neo 729 � 373 33.2 � 16.7 557 � 219

Hasako et al.





treatment and persisted during the treatment period. Regardingtolerability, mean body weight loss over 5% was not observedin any groups (Supplementary Fig. S3A). In this model, afatinibadministered at 20 mg/kg showed minimal, but statisticallysignificant (P < 0.049), tumor growth inhibition with a T/C of69%. In rats bearing the same xenograft tumors, similar efficacywas obtained from lower doses compared with those in themouse model (Fig. 3B). Oral administration of TAS6417 at over10 mg/kg once daily for 14 days led to statistically significantinhibition of tumor growth (T/C of 20%), and 20 mg/kg/dayled to complete inhibition of tumor growth with a T/C of 7%.At 40 mg/kg, the sustained tumor regression (T/C of 3% at Day15) was observed soon after TAS6417 administration withoutany effect on body weight gain. Pharmacokinetic analysisrevealed that the plasma concentration of TAS6417, adminis-tered at 50 and 100 mg/kg to mice, decreased rapidly in the first4 hours (Fig. 3F). In contrast, although the effective doses in therat model exhibited lower maximum plasma concentration(Cmax) values than those in the mouse model, the plasmaconcentrations tended to be more persistent, lasting up to 8hours.

NIH/3T3, a mouse fibroblast cell line, does not engraft nor-mally in nude mice, but tumorigenicity can be rendered bymutant EGFR expression (6). In vivo antitumor efficacy ofTAS6417 againstH773_V774insNPH, an exon 20 insertionmuta-tion, was evaluated in nude mice implanted with NIH/3T3 cellsgenetically engineered to express humanEGFRwith themutation.In this model, TAS6417was administered orally once daily for 10days. Consistently, with data from the NCI-H1975 EGFRD770_N771insSVD xenograft model, TAS6417 administered at50 mg/kg/day or more led to statistically significant inhibition oftumor growth in a dose-dependent manner at final evaluation onDay 11 (Fig. 3C). Tumor regression was achieved at 200 mg/kg/day, corresponding to a T/C ratio of 2%; T/C ratios of 25%and6%were observed at the lower doses of 50 and 100 mg/kg/day,respectively. Furthermore, the in vivo activity of TAS6417 againstEGFR H773_V774insNPH was assessed in mice with LU0387human lung cancer xenografts, a PDX model (Fig. 3D). In thismodel, 50 mg/kg/day for 28 days induced statistically significanttumor growth inhibition with a T/C of 16%. Striking and persis-tent tumor regression was observed at 100mg/kg/day, with a T/Cof 2% at Day 28. Although afatinib dosed at 20 mg/kg also

Figure 2.

Inhibition of EGFR signaling by TAS6417 in NSCLC cell lines with EGFR exon 20 insertion mutations. A and B, Effect of TAS6417 on EGFR signaling in LXF 2478L cells(A) and NCI-H1975 EGFR D770_N771insSVD cells (B). The cells were cultured with TAS6417 or afatinib and harvested 6 and 24 hours after treatment.Phosphorylation of EGFR-Tyr1068, AKT-Ser473, and ERK1/2-Thr202/Tyr204 and expression of BIM and cleaved PARP were determined using immunoblottinganalysis. C and D, Caspase3/7 activation by TAS6417 in LXF 2478L cells (C) and NCI-H1975 EGFR D770_N771insSVD cells (D). The cells were treated withTAS6417 or afatinib for 24 hours. Caspase-3/7 activation relative to that of the DMSO control was determined using the Caspase-Glo 3/7 and CellTiter-Gloassays. � , P < 0.05; �� , P < 0.01; �� , P < 0.001.






inhibited tumor growth, tumor volume still gradually increased,corresponding to a T/C of 35% at Day 28. After completion oftreatment and a 2-week dose-free observation period, the tumorsof all tested groups grew over time, resulting in T/C ratios of 31%for 50mg/kg/day TAS6417, 7% for 100mg/kg/day TAS6417, and61% for 20mg/kg/day afatinib. Althoughmean body weight lossover 5% was not observed for TAS6417 treatment groups, thebodyweight of the afatinib treatment groupwas decreased acutely

by over 10% at Day 4, after which it recovered over time duringthe treatment period (Supplementary Fig. S3D).

In the LXF 2478 human lung cancer PDX model with EGFRV769_D770insASV, one of the most common exon 20 insertionmutations, daily oral administration of TAS6417 resultedin partial to complete remission, and the T/C ratio at Day 28for the 100 and 200 mg/kg/day groups was 3% and 0.1%,respectively (Fig. 3E). The tumors remained at their reduced sizes

Figure 3.

In vivo antitumor efficacy of once-daily oral administration of TAS6417.A, Inhibition of tumor growth inmice bearing NCI-H1975 EGFRD770_N771insSVD xenograftsover 14-day treatment with TAS6417 and afatinib. B, Inhibition of tumor growth in rats bearing NCI-H1975 EGFR D770_N771inSVD xenografts over 14-daytreatmentwith TAS6417.C, Inhibition of tumor growth inmice bearingNIH/3T3 EGFRH773_V774insNPH allografts over 10-day treatmentwith TAS6417 and afatinib.D, Inhibition of tumor growth in mice bearing lung cancer PDX with EGFR H773_N774insNPH (LU0387) over 28-day treatment with TAS6417 and afatinib,followed by 14-day observation period. E, Inhibition of tumor growth in mice bearing lung cancer PDX with EGFR V769_D770insASV over 28-day treatment withTAS6417 and afatinib, followed by 14-day observation period. F, Plasma concentration profiles over time on Day 14 in nude mice and nude rats. Controlgroup was administered the vehicle for TAS6417. Data are presented as mean tumor volume � SEM or mean plasma concentration � SD in each group.

Hasako et al.





during the 2-week, treatment-free observation period. Althoughafatinib delayed tumor growth with a T/C ratio of 23% at Day 28,the tumors showed significant regrowth during the observationperiod. In this model, the maximum mean body weight loss was3% for the control group, 4% for the TAS6417 100 mg/kg/daygroup, 11% for the TAS6417 200 mg/kg/day group, and 12% forthe afatinib 20mg/kg/day group (Supplementary Fig. S3E). Solelyin the afatinib treatment group, a low grade of dry, flaky, orreddened skin was observed around 3 weeks after initiation ofadministration. These findings demonstrate that TAS6417achieved persistent tumor regression in EGFR exon 20 inser-tion-driven tumors in both genetically engineered xenograft andPDX models, and that TAS6417 was more efficacious and moretolerable than afatinib in an animal model.

TAS6417 inhibits mutant EGFR in tumors but not WT EGFR inskin tissues

To determine whether the in vivo efficacy was associated witheffective inhibition of EGFR signaling, phosphorylation of EGFRand its downstream effectors was assessed in tumors by Westernblotting after a single dose of TAS6417. In mice bearing xenografttumors expressing EGFR D770_N771insSVD, a single dose ofTAS6417 at 50 mg/kg or more caused intensive reduction ofphospho-EGFR (pEGFR) at 1 hour (Fig. 4A). Consistent with thisfinding, phosphorylation of molecules downstream of EGFR,including phospho-AKT (pAKT) and phospho-ERK (pERK), wasalso decreased. In contrast, the effect of TAS6417 on pEGFR inmouse skin tissues expressing WT EGFR was partial even at thehighest dose of 200 mg/kg, as shown in Fig. 4B. Furthermore, atime-course analysis of EGFR signaling inhibition in NCI-H1975

EGFR D770_N771insSVD tumors was conducted following asingle dose of TAS6417 at 100 mg/kg. The inhibition of pEGFRwas observed 1 hour after dosing andwasmaintained partially for6 hours. In parallel, pATK and pERK were partially inhibited for 6hours to levels comparable with the control at 24 hours, as shownin Fig. 4C. Similar results were obtained in rats with NCI-H1975EGFR D770_N771insSVD xenografts, as shown in Fig. 4D.TAS6417 administered at 20 mg/kg, which achieved completesuppression of tumor growth, induced a significant decrease inpEGFR, leading to reduction of pAKT and pERK at 1 hour. Theinhibitory effect was still noted at 6 hours, and phosphorylationof EGFR, ATK, and ERK recovered by 24 hours. These pharma-codynamic changes correlatedwell with plasma concentrations ineach model. These results suggest that TAS6417 achieved morepotent inhibition of mutant EGFR in tumors than of WT EGFR inskin tissues. The data also indicate that the ability of TAS6417 tosustain the inhibition of EGFR signaling may contribute to itspotent antitumor efficacy in the mouse and rat xenograft models.

TAS6417 prolonged survival of animals bearing lung cancerTAS6417 was further evaluated for its survival benefit in mice

with intrapulmonary NCI-H1975 EGFR D770_N771insSVDxenografts. As a result of daily oral administration of TAS6417,dose-dependent prolonged survival was observed (Fig. 5A).TAS6417 administered at 100 and 200 mg/kg/day providedstatistically significant prolongation ofmedian survival comparedto that of the control group, with ILS of 59% and 102%, respec-tively. The afatinib group exhibited no statistically significant ILS.Although the body weight of mice in the control group tended toincrease by approximately 30days after implantation, it decreased

Figure 4.

EGFR inhibition by TAS6417 in the H1975 EGFR D770_N771insSVD xenograft model. A and B, In vivo inhibitory activity of TAS6417 on EGFR in xenograft tumors andskin tissues. Mice bearing NCI-H1975 EGFR D770_N771insSVD xenografts were administered TAS6417 and afatinib. One and 2 hours after administration ofTAS6417 and afatinib, respectively, xenograft tumors and skin tissues were collected. Phosphorylation of EGFR-Tyr1068 in xenograft tumors (A) and in skin tissues(B) was detected by immunoblotting. C and D, Duration of inhibitory activity of TAS6417 against EGFR signaling in mice (C) and rats (D). NCI-H1975 EGFRD770_N771insSVD xenograftswere collected at each time point. Phosphorylation of EGFR-Tyr1068, AKT-Ser473, and ERK1/2-Thr202/Tyr204was determined usingimmunoblotting analysis.






over time during the median survival period until animal death.In contrast, the TAS6417-treated group exhibited an increase inthe body weight beyond 30 days. After this period, decreases inbody weight associated with animal death were observed, just asin the control group (Fig. 5B). These data demonstrate not onlythe survival benefit of TAS6417 in mice with intrapulmonaryxenograft tumors driven by EGFR exon 20 insertion mutations,but also the good tolerability of long-term daily administration.

DiscussionIn this article, we describe the characteristics of TAS6417, a

novel EGFR inhibitor with potential for the treatment of cancerharboring EGFR exon20 insertionmutations. TAS6417 is a kinaseinhibitor that covalentlymodifies C797 in the ATP-binding site ofEGFR, showing inhibitory activity against WT and mutant EGFRincluding an exon20 insertionmutation in biochemical assays. Ina panel of 255 kinases, TAS6417 at 1,000 nmol/L exhibited >50%inhibition against 25 kinases other thanEGFRandonly six kinases(TXK, BMX, BTK, HER4, TEC, JAK3) whose ATP-binding pocketsharbor a cysteine at a structural position homologous to C797 inEGFR (35), suggesting high selectivity of TAS6417 against EGFRover other kinases. These biochemical data related to kinaseselectivity are supported by the results of the cell proliferationassay. Although cells driven by mutant EGFR were sensitive toTAS6417, NCI-H23 cells and NCI-H460 cells, which exhibitEGFR-independent cell growth did not respond to TAS6417.Furthermore, cell-based assays of intracellular EGFR inhibitiondemonstrated the selectivity of TAS6417 against mutant EGFRs,including exon 20 insertion mutations over WT EGFR. Among awide variety of exon 20 insertion mutations in EGFR, the mostcommonmutations are V769_D770insASV, D770_N771insSVD,and H773_V774insNPH, which account for approximately 50%

of all mutations. TAS6417 inhibited these major mutations andalso other insertion mutations, including A763_Y764insFQEA,D770_N771insG, and H773_V774insPH, while sparing WTEGFR, suggesting that TAS6417 has potent and selective inhibi-tory activity against a diverse range of insertion mutations in theexon 20 region of the EGFR gene over WT EGFR.

Patients with NSCLC harboring commonmutations, includingexon 19 deletions and L858R, show striking responses to EGFR-TKIs, including gefitinib, erlotinib, and afatinib, leading to pro-longed progression-free survival compared with that of standardchemotherapy (13–18). However, the clinical response of NSCLCdriven by EGFR exon20 insertionmutations to these EGFR-TKIs ismuch lower (25–28). Given that current EGFR-TKIs are notselective against exon 20 insertion mutations and that clinicaldoses are thus limited by toxicity related to WT EGFR inhibition,poor clinical outcomesmay be the result of an insufficient plasmaconcentration for inhibition (22, 29, 30). Therefore, the devel-opment of an EGFR inhibitor selective against mutations intumors rather than WT EGFR in normal tissues is extremelyimportant for the treatment of NSCLC driven by EGFR exon 20insertion mutations. This study demonstrates that TAS6417 exhi-bits selective inhibition of such mutations over WT EGFR in vitroand in vivo. In the cell proliferation assay, TAS6417 exhibitedmorepotent inhibitory activity in human cancer cell lines harboringmutant EGFRs than inprimary keratinocytes. Consistentwith this,TAS6417 achieved remarkable and durable inhibition of mutantEGFR and its downstream effectors in tumors, while sparing WTEGFR in skin tissues. This mutation-selective characteristic led tosignificant in vivo antitumor activity in mouse and rat models.Notably, once-daily oral administration of TAS6417 at 100mg/kgachieved persistent tumor regression with good tolerability in aPDXmodel of EGFR exon 20 insertions, including V769_D770in-sASV and H773_V774insNPH. In contrast, afatinib induced

Figure 5.

Survival benefit of once-daily oraladministration of TAS6417 in theorthotropic model. A, Kaplan–Meiersurvival curve of BALB/cAJcl-nu/nunude mice directly inoculatedwith NCI-H1975 EGFRD770_N771insSVD_Luc cells intothe lungs. Six days after implantation,mice were grouped according tophoton values measured by an IVISImaging System followed by once-daily administration of TAS6417 andafatinib. B, Body weight change ofeach mouse in the control andTAS6417 200mg/kg/day groups afterallocation. Control group wasadministered the vehicle for TAS6417.

Hasako et al.





tumor growth inhibition but not tumor regression, and sometoxic signatures, including body weight loss over 10% and skinsymptoms, were observed. In addition to the tumor growthinhibition experiments, a lung orthotropic implantation modelin mice demonstrated the survival benefits of TAS6417, withtolerability in long-term daily administration.

Although EGFR exon 20 insertion mutations are uncommon,occurring in only 2% to 3% of NSCLC cases, there is an unmetmedical need for patients. A few clinical trials of EGFR-TKIs forEGFR exon 20 insertionmutation-positive NSCLC are in progress,including trials for AP32788 (NCT02716116), osimertinib(NCT03191149), and poziotinib (NCT03066206 andNCT03318939). As described in this article, TAS6417 has aunique core unit differing from other reported EGFR-TKIs anda unique mutation selectivity profile, achieving remarkable anti-tumor efficacy and prolonged survival in preclinical models.These findings support clinical evaluation of TAS6417 as anefficacious drug candidate for patients with NSCLC driven byEGFR exon 20 insertion mutations.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: S. Hasako, Y. AoyagiDevelopment of methodology: S. Hasako, M. Terasaka, A. Hashimoto,R. Fujita, T. Okayama, K. Miyadera

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Hasako, M. Terasaka, H. Ohsawa, A. Hashimoto,R. Fujita, K. Tanaka, T. OkayamaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Hasako, M. Terasaka, N. Abe, A. Hashimoto, R.Fujita, K. Tanaka, T. Okayama, R. WadhwaWriting, review, and/or revision of the manuscript: S. Hasako, R. Wadhwa, K.Miyadera, K. Yonekura, K. MatsuoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S. Hasako, K. MiyaderaStudy supervision: S. Hasako, K. Yonekura, K. MatsuoOther (chemical design and synthesis): T. Uno

AcknowledgmentsThe authors thank Drs. Teruhiro Utsugi and Yoshikazu Iwasawa for their

insightful discussion. The authors also thank the overall departments at theDiscovery and Preclinical Research Division of Taiho for the support theyprovided for this work. All research activities were founded by Taiho Pharma-ceutical Co., Ltd.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received December 4, 2017; revised March 15, 2018; accepted May 4, 2018;published first May 10, 2018.

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2018;17:1648-1658. Published OnlineFirst May 10, 2018.Mol Cancer Ther Shinichi Hasako, Miki Terasaka, Naomi Abe, et al. MutationsTAS6417, A Novel EGFR Inhibitor Targeting Exon 20 Insertion

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TAS6417, A Novel EGFR Inhibitor Targeting Exon 20 ... · low-energy scans and ramped from 15 to 40 eV for high-energy scans, and further data analysis was performed using BioPhar-maLynx1.3.3