Supplementary Table S2 shows the case reports of clinical activity and AUC0-24h of TAS-121.

Inhibitory activity and kinase selectivity of TAS6417 in biochemical assays

Body weight change resulting from once-daily oral administration of TAS6417

Methods for in vitro kinase assay, genome sequence, and immunoblotting analysis

Synthesis procedures and characterization data for the synthesis of TAS6417

Supplementary methods shows "Synthesis of TAS-121" and the metohds that is relevant to the experiments shown in Supplementary datas.

Nucleic acid (NA) biomarkers play critical roles in drug development. However, the global regulatory guidelines for assessing quantification methods specific to NA biomarkers are limited. The validation of analytical methods is crucial for the use of biomarkers in clinical and post-marketing evaluations of drug efficacy and adverse reactions. Given that quantitative polymerase chain reaction (qPCR) and reverse transcription qPCR (RT-qPCR) methods are the gold standards for the quantification of NA biomarkers, the Biomarker Analytical Method Validation Study Group in Japan has discussed considerations and made recommendations for the development and validation of qPCR- and RT-qPCR-based analytical methods for endogenous NA biomarkers as drug development tools. This white paper aims to contribute to the global harmonization of NA biomarker assay validation.

Supplementary Fig. S4 shows dose-dependency of Cmax (A) and AUC0-24h (B) in nude mice.

Establishment of a cell line tool with an EGFR exon 20 insertion mutation

Supplementary Fig. S3 shows cytotoxicity of EGFR-TKIs for normal human epidermal keratinocytes.

Supplementary Fig. S5 shows anti-tumor effect of TAS-121 in HCC827 xenograft mouse models.

Supplementary Fig. S1 shows chemical structure and binding mode of TAS-121.

Supplementary Fig. S2 shows inhibitory activity of TAS-121 for ERBB family kinases.

Assignment of MSE fragmentation data for protease-digested peptide with TAS6417

Biomarkers are an important drug developmental tool. Assessment of quantitative analytical methods of biomarkers is not included in any regulatory documents in Japan. Use of biomarkers in clinical evaluations and supporting the post-marketing evaluation of drug efficacy and/or adverse reactions requires assessment and full validation of analytical methods for these biomarkers. The Biomarker Analytical Method Validation Study Group is a research group in Japan comprising industry and regulatory experts. Group members discussed and prepared this 'points to consider document' covering measurements of endogenous metabolites/peptides/proteins by ligand binding assays and chromatographic methods with or without mass spectrometry. We hope this document contributes to the global harmonization of biomarker assay validation.

TAC‐302 stimulates neurite outgrowth activity and is expected to restore urinary function in patients with lower urinary tract dysfunction. We conducted 2 phase 1, randomized, placebo‐controlled studies to confirm the safety and pharmacokinetics (PK) of TAC‐302 in healthy adult Japanese male volunteers. In the first‐in‐human single‐dose study (n = 60), TAC‐302 was administered at doses from 100 to 1200 mg after an overnight fast. The effects of a meal on the PK of TAC‐302 400 mg were also examined. A multiple‐dose study (n = 36) evaluated the effects of meal fat content on the PK of single doses of TAC‐302 (100, 200, or 400 mg) and multiple doses of TAC‐302 administered for 5 days (100, 200, and 400 mg twice daily). TAC‐302 showed linear PK up to doses of 1200 mg in the fasting state, and across the dose range of 100–400 mg in the fed state. No accumulation of TAC‐302 was observed. Food, particularly with high fat content, increased TAC‐302 plasma concentrations. No differences were observed in the adverse event incidence between the TAC‐302 and placebo groups in either study. TAC‐302 showed a wide safety margin.

Abstract TAS-121 is a novel orally active selective covalent inhibitor of the mutant EGFR. We performed preclinical characterization of TAS-121 and compared its efficacy and selectivity for common EGFR mutations (Ex19del and L858R), first- and second- generation EGFR-tyrosine kinase inhibitor (EGFR-TKI) resistance mutation (T790M), and uncommon mutations (G719X and L861Q) with those of other EGFR-TKIs. We also commenced investigation of the clinical benefits of TAS-121. The IC50 for intracellular EGFR phosphorylation was determined by using Jump-In GripTite HEK293 cells transiently transfected with EGFR expression vectors. Mouse xenograft models were used to evaluate the antitumor activity of TAS-121. TAS-121 potently inhibited common activating and resistance EGFR mutations to the same extent as another third-generation EGFR-TKI (osimertinib). In addition, TAS-121 showed equivalent inhibitory activity against some uncommon mutations such as G719X and L861Q. Furthermore, TAS-121 demonstrated greater selectivity for mutant EGFRs versus the wild-type EGFR compared with other EGFR-TKIs. Moreover, TAS-121 displayed antitumor activity in SW48 (EGFR G719S) and NCI-H1975 (EGFR L858R/T790M) xenograft models, and achieved an objective response in patients with NSCLC with EGFR mutations including G719A mutation. In conclusion, TAS-121 is a novel third-generation EGFR-TKI and demonstrates antitumor activities in patients with NSCLC expressing either common or uncommon EGFR mutations.

Title: TAS-121, TAS-121 shows effect on EGFR G719X mutation. Abstract TAS-121 is a novel orally active selective covalent inhibitor of the mutant epidermal growth factor receptor (EGFR). We performed preclinical characterization of TAS-121 and compared its efficacy and selectivity for common EGFR mutations (Ex19del and L858R), first- and second- generation EGFR-tyrosine kinase inhibitor (EGFR-TKI) resistance mutation (T790M), and uncommon mutations (G719X and L861Q) with those of other EGFR-TKIs. We also commenced investigation of the clinical benefits of TAS-121. The 50% inhibitory for intracellular EGFR phosphorylation was determined by using Jump-In GripTite HEK293 cells transiently transfected with EGFR expression vectors. Mouse xenograft models were used to evaluate the antitumor activity of TAS-121. TAS-121 potently inhibited common activating and resistance EGFR mutations to the same extent as another third-generation EGFR-TKI (osimertinib). In addition, TAS-121 showed equivalent inhibitory activity against some uncommon mutations such as G719X and L861Q. Furthermore, TAS-121 demonstrated greater selectivity for mutant EGFRs versus the wild-type EGFR compared with other EGFR-TKIs. Moreover, TAS-121 displayed antitumor activity in SW48 (EGFR G719S) and NCI-H1975 (EGFR L858R/T790M) xenograft models, and achieved an objective response in NSCLC patients with EGFR mutations including G719A mutation. In conclusion, TAS-121 is a novel third-generation EGFR-TKI and demonstrates anti-tumor activities in patients with NSCLC expressing either common or uncommon EGFR mutations.

Abstract Activating mutations in the EGFR gene are important targets in cancer therapy because they are key drivers of non–small cell lung cancer (NSCLC). Although almost all common EGFR mutations, such as exon 19 deletions and the L858R point mutation in exon 21, are sensitive to EGFR-tyrosine kinase inhibitor (TKI) therapies, NSCLC driven by EGFR exon 20 insertion mutations is associated with poor clinical outcomes due to dose-limiting toxicity, demonstrating the need for a novel therapy. TAS6417 is a novel EGFR inhibitor that targets EGFR exon 20 insertion mutations while sparing wild-type (WT) EGFR. In cell viability assays using Ba/F3 cells engineered to express human EGFR, TAS6417 inhibited EGFR with various exon 20 insertion mutations more potently than it inhibited the WT. Western blot analysis revealed that TAS6417 inhibited EGFR phosphorylation and downstream molecules in NSCLC cell lines expressing EGFR exon 20 insertions, resulting in caspase activation. These characteristics led to marked tumor regression in vivo in both a genetically engineered model and in a patient-derived xenograft model. Furthermore, TAS6417 provided a survival benefit with good tolerability in a lung orthotopic implantation mouse model. These findings support the clinical evaluation of TAS6417 as an efficacious drug candidate for patients with NSCLC harboring EGFR exon 20 insertion mutations. Mol Cancer Ther; 17(8); 1648–58. ©2018 AACR.