CH7233163 Overcomes Osimertinib-Resistant EGFR-Del19/T790M/C797S Mutation

Recent approaches targeting oncogenic drivers have revolutionized the treatment of lung cancer. Among them, the first success was in targeting EGFR (1). EGFR-activating mutations, such as an exon 19 deletion (Del19) and L858R substitution, have been reported in 10% to 50% of patients with non–small cell lung cancer (NSCLC; refs. 2, 3). These patients respond well to first/second-generation EGFR tyrosine kinase inhibitors (TKI), including gefitinib, erlotinib, and afatinib, which has made them the standard-of-care initial therapy for advanced EGFR-mutant NSCLC (4–6). However, despite initial marked and sometimes durable responses, patients invariably develop acquired resistance within 9 to 14 months of treatment. The most common resistance mechanism, detected in 50% of these patients, is a secondary single-nucleotide substitution in EGFR exon 20, resulting in a T790M mutation (7), which decreases drug potency by increasing the ATP affinity of mutated EGFR and hindering drug binding (8, 9).

Recently, osimertinib, a third-generation EGFR-TKI, potently and irreversibly inhibited the effects of EGFR-activating mutations with or without T790M, exhibiting low affinity for WT EGFR (10). Consistent with its aforementioned therapeutic profile, osimertinib displays remarkable efficacy in treating patients with both treatment-naïve EGFR mutations and EGFR T790M mutations. Given a progression-free survival (PFS) of 18.9 months, osimertinib is more effective than first-generation EGFR-TKIs as a first-line treatment (11); furthermore, with a PFS of 9.9 to 12.3 months, osimertinib is more effective than chemotherapy for patients with NSCLC harboring the EGFR-T790M mutation (12, 13). However, the C797S mutation is reportedly the most common mechanism underlying on-target osimertinib resistance in 10% to 30% of these patients, similar to those receiving first/second-generation EGFR-TKIs (14–20). C797 is the site of covalent binding for all known irreversible EGFR-TKIs, and because these agents are obligate covalent binders, they become 100- to 1,000-fold less effective at inhibiting cell proliferation and EGFR phosphorylation in the presence of the C797S mutation (15). Therefore, new-generation EGFR-TKIs are needed to overcome the osimertinib-resistant EGFR-triple mutations (Del19/T790M/C797S or L858R/T790M/C797S). Recently, mutant-selective allosteric inhibitors, EAI-001/045, and its derivative JBJ-04–125–02 (21, 22), were reported as potential therapeutic strategies to overcome the EGFR-L858R/T790M/C797S mutations. However, these compounds cannot inhibit Del19/T790M/C797S. Therefore, novel EGFR-TKIs potently effective against EGFR triple mutations, especially Del19/T790M/C797S, are required. In this study, we screened a massive chemical library to identify CH7233163, a novel EGFR-TKI capable of overcoming EGFR-Del19/T790M/C797S.

A431, NCI-H1975, and HCC827 cell lines were obtained from the ATCC), cultured in accordance with the supplier's instructions and confirmed as Mycoplasma-negative via previously reported culturing- or PCR-based methods. Cell assays were performed within 20 passages after thawing.

NIH3T3 cells (ATCC) were transduced with lentiviruses harboring genes encoding the EGFR-d746–750/T790M/C797S or EGFR-L858R/T790M/C797S mutant, generated from the pCDH-CMV-MCS-EF1-Puro vector (System Biosciences). Cells stably expressing these mutants were subsequently selected in medium supplemented with puromycin. CH7233163 and EAI-045 were synthesized by Chugai Pharmaceutical. Osimertinib was purchased from Combi-Blocks. The detailed protocol for the synthesis of CH7233163 is shown in Supplementary Figs. S1 and S2.

Recombinant EGFR proteins were purchased from SignalChem (Del19/T790M/C797S: E10–122UG, L858R/T790M/C797S: E10–122VG) or Carna Biosciences (WT: 08–115). Biochemical assays were carried out using the time-resolved fluorescence resonance energy transfer (TR-FRET) LANCE system (PerkinElmer). The details about assay condition was shown in Supplementary Table S1. The inhibitory activity against each kinase was evaluated as described previously (23).

Cells were incubated in medium containing serial dilutions of the compound in a 96-well culture plate (Corning) or PrimeSurface96U plates (Sumitomo Bakelite) at 37°C for 4 or 7 days. The number of living cells was then determined using CellTiter-Glo Luminescent Cell Viability Assay (Promega). Luminescence was measured using EnVision Xcite (PerkinElmer). Antiproliferative activity and IC₅₀ values were determined in a manner similar to the TR-FRET-based EGFR biochemical assays.

Western blotting was performed as described previously (24). The following primary antibodies were used: anti-phospho-EGFR (Tyr1068; #2234), anti-EGFR (#4267), anti-phospho-ERK1/2 (#4370), anti-ERK1/2 (#4695), anti-phospho-AKT (#4060), anti-AKT (#9272; Cell Signaling Technology), and anti-α-tubulin (AbD Serotec). Signals were detected using Chemi-Lumi One Super (Nacalai Tesque), followed by LAS-4010 (GE Healthcare). Images were edited using ImageQuant TL (GE Healthcare).

The kinase domain of human EGFR (residues 695–1022) containing L858R/T790M/C797S/E865A/E866A/K867A mutation was expressed in Sf9 cells with a His-tag and GST-tag, which was eliminated by TEV protease cleavage during purification; thereafter, the kinase domain was purified using Ni affinity and GST affinity chromatography. Then, Lambda Protein Phosphatase and TEV protease treatment were performed and subsequently, additional Ni affinity, size-exclusion, and GST-affinity chromatography were conducted. The purified protein was concentrated and stored at −80°C until use.

Crystals were obtained at 21°C from sitting drops using reservoir solutions 0.92–1.0 mol/L succinic acid, 0.1 mol/L HEPES (pH 7.0), 1% w/v polyethylene glycol monomethyl ether 2,000 through vapor diffusion. Crystals were soaked in the solution containing 1% w/v polyethylene glycol monomethyl ether 2,000, 50 mmol/L HEPES pH 7.1, 815 mmol/L succinic acid, 4% DMSO, and 0.4 mmol/L CH7233163 for 3 hours at 21°C, or 0.4 mmol/L osimertinib overnight at 21°C and subsequently flash-cooled in liquid nitrogen after addition of 20% ethylene glycol. Diffraction data of L858R/T790M/C797S-CH7233163 were obtained at 100 K at beamline BL45XU in SPring-8, using the PILATUS3 6M detector (DECTRIS Ltd.) with the Zoo system (25). Diffraction data of L858R/T790M/C797S–osimertinib was obtained at 95K at beamline BL17A in Photon Factory using an EIGER X16M (DECTRIS Ltd.). The dataset was processed with autoPROC, which utilized XDS, POINTLESS, AIMLESS, CCP4, and STARANISO (26–31). The initial structures were determined through molecular replacement with dimple in CCP4 suite with the structure of PDB ID 5EM7 as a search model. A chemical restraint dictionary was generated using ACEDRG and Grade (32, 33). The crystal contains one monomer of L858R/T790M/C797S–compound complex in the asymmetric unit. The models were manually regenerated using Coot, refined with REFMAC5, Phenix, and Buster to a final resolution of 2.32 Å for CH7233163 complex and 2.05 Å for osimertinib, and evaluated with MolProbity (34–38). Crystallographic data and refinement statistics are shown in Supplementary Table S2. The structures are deposited in the Protein Data Bank with PDB ID: 6LUB, and 6LUD for CH7233163, and osimertinib, respectively. All graphical presentations were prepared using PyMOL (39).

All in vivo experiments were performed in accordance with the protocol approved by the Chugai Institutional Animal Care and Use Committee. All animal experiments were performed in accordance with the “Guidelines for the Accommodation and Care of Laboratory Animals” at Chugai Pharmaceutical Co. Ltd. All animals were housed in a pathogen-free environment under controlled conditions (temperature: 20°C–26°C, humidity: 40%–70%, and light/dark cycle: 12/12 hours). Chlorinated water and irradiated food were provided ad libitum. The animals were allowed to acclimatize and recover from shipping-related stress for 1 week prior to the study. Mouse health was monitored daily.

Cells were suspended in 200 mL HBSS or PBS and injected subcutaneously into the right flank of the nude mice (CAnN.Cg-Foxn1<nu>/CrlCrlj nu/nu, Charles River Laboratories). Tumor size was measured using a gauge twice per week, and the tumor volume (TV) was calculated using the following formula: TV = ab²/2, where a is the length of the tumor, and b is the width. Once the tumors approached a volume of approximately 150 to 250 mm³, animals were randomly segregated into groups (n = 4 in each group), and treatment was initiated. Compounds were orally administered once a day. Resected tumors were lysed for Western blotting.

To obtain a new EGFR-TKI capable of overcoming EGFR-Del19/T790M/C797S mutation, we performed high-throughput inhibitor screening against the EGFR-Del19/T790M/C797S enzyme using a chemical library with over 1 million compounds. As a result, we obtained several hits with previously unreported chemical scaffolds. Among the derivatives, we found a lead compound that can strongly inhibit EGFR-Del19/T790M/C797S, which we then intensively modified and improved its potency. Finally, we identified CH7233163 (Fig. 1A) as a potent inhibitor of EGFR-Del19/T790M/C797S. CH7233163 exhibited subnanomolar potency in a biochemical assay with EGFR-Del19/T790M/C797S (IC₅₀ = 0.28 nmol/L, Fig. 1B). In contrast, EAI-045, an allosteric EGFR inhibitor, didn't show inhibitory activity in this assay (IC₅₀ >1,000 nmol/L), which is consistent with a previous report (22). Next, we investigated whether the inhibitory activity of CH7233163 shown in the biochemical assay reflected the biological activities of cells. For this purpose, we established NIH3T3 cells expressing the EGFR-Del19/T790M/C797S mutation (Del19/T790M/C797S_NIH3T3). CH7233163 potently inhibited the proliferation of Del19/T790M/C797S_NIH3T3 cells with an IC₅₀ of 20 nmol/L (Fig. 1C). Furthermore, we examined the ability of CH7233163 to inhibit cellular EGFR phosphorylation via Western blotting. CH7233163 potently and dose dependently blocked the EGFR phosphorylation in the Del19/T790M/C797S_NIH3T3 cells (Fig. 1D). Moreover, we assessed the time dependency of the inhibitory activity of CH7233163 against cellular EGFR and downstream AKT and ERK1/2 phosphorylation. Cellular EGFR-Del19/T790M/C797S phosphorylation was decreased by CH7233163 treatment from 0.5 hour, and this phenomenon was retained for 24 hours (Fig. 1E). Similarly, the downstream AKT and ERK1/2 phosphorylation was persistently decreased upon CH7233163 treatment in Del19/T790M/C797S_NIH3T3 cells, suggesting that CH7233163 can inhibit Del19/T790M/C797S signaling.

In addition to these in vitro pharmacologic effects, CH7233163 exhibited a moderate half-life of 6 hours and a high area under the curve of 3,390 h/ng/mL (AUClast) upon orally administering 10 mg/kg to mice (Supplementary Table S3). The mean plasma levels reached 43 nmol/L at 24 hours postdose, and this plasma concentration substantially exceeds the in vitro IC₅₀ value shown in Fig 1C for Del19/T790M/C797S_NIH3T3 cells. Accordingly, we next performed a pharmacodynamic study using Del19/T790M/C797S_NIH3T3 xenografted tumors in mice. As shown in Fig. 2A, CH7233163 clearly inhibited EGFR phosphorylation 6 hours after oral administration, and this activity was retained 24 hours after oral administration; however, it was partly reduced, suggesting the importance of daily dosing. On the basis of these findings, we administered CH7233163 daily for an in vivo efficacy study using a Del19/T790M/C797S_NIH3T3 xenograft mouse model. Consequently, tumor growth was significantly reduced at every dose, and potent tumor regression was observed at 100 mg/kg (Fig. 2B). No differences in body weight or gross signs of toxicity were observed between control- and CH7233163-treated mice at any dose. Thus, CH7233163 has potent therapeutic efficacy against tumors with EGFR-Del19/T790M/C797S in vivo. These results suggest that CH7233163 is a potent EGFR-Del19/T790M/C797S inhibitor.

To further understand the characteristics of CH7233163, we then checked whether CH7233163 is an ATP-competitive inhibitor. For this purpose, we utilized different ATP concentrations in a Del19/T790M/C797S biochemical assay. As shown in Fig. 3A, the inhibitory activity of CH7233163 against Del19/T790M/C797S was reduced with the higher ATP concentration, suggesting that CH7233163 is an ATP competitive inhibitor. Generally, it is difficult for simple ATP-competitive inhibitors like ATP analogues to selectively inhibit EGFR-T790M mutations (Del19/T790M and L858R/T790M) over EGFR WT, because the binding affinity of ATP to EGFR-T790M and EGFR WT is almost the same (9). However, selectivity to EGFR WT is important for EGFR-TKIs, because WT EGFR inhibition causes side-effects such as rashes and/or diarrhea, and these WT EGFR-derived toxicities cause dose-limiting effects (40, 41). Therefore, we tested whether CH7233163 can selectively inhibit the activities of Del19/T790M/C797S mutant over EGFR WT. In the biochemical assay, CH7233163 preferentially inhibited the effect of Del19/T790M/C797S as compared with that of WT EGFR (Fig. 3B). This mutant selectivity against EGFR-WT is similar to that of osimertinib (Fig. 3B, right). In addition, antiproliferation activity of CH7233163 against A431 cells (EGFR-WT) was much weaker (IC₅₀ of 1200 nmol/L) compared with Del19/T790M/C797S_NIH3T3 cells (IC₅₀ of 20 nmol/L; Fig. 3C). Furthermore, CH7233163 did not reduce EGFR phosphorylation in A431 cells (EGFR WT) even at 1,000 nmol/L concentration (Fig. 3D). These results suggest that CH7233163 is an ATP-competitive inhibitor that selectively inhibits EGFR-Del19/T790M/C797S mutation over EGFR WT.

To further analyze the kinase selectivity of CH7233163, we applied KINOMEscan assay for CH7233163 at 100 nmol/L against 468 kinases (42). The assay concentration (100 nmol/L) is set to over 100-fold higher than that of IC₅₀ value against EGFR-Del19/T790M/C797S mutant in biochemical assay (Fig. 1B). As a result, CH7233163 displayed excellent selectivity across the human kinome with an S-score [10] = 0.015 (Fig. 4). Only five nonmutant and three mutant kinases other than EGFR were inhibited at the 10% cutoff: BUB1 (% inhibition: 100), CLK1 (% inhibition: 99), MELK (% inhibition: 98), CLK4 (% inhibition: 95), MAP4K4 (% inhibition: 92), FLT3-D835V (% inhibition: 94), FLT3-ITD (% inhibition: 91), and FLT3-ITD/D835V (% inhibition: 95). This high selectivity across the human kinome is consistent with the lack of gross signs of toxicity in the mouse efficacy study, as shown in Fig. 2B. In the case of EGFR, 10 mutants were inhibited by CH7233163 at the 10% cutoff: 5 types of Del19 mutations (E746-A750del, L747-E749del/A750P, L747-S752del/P753S, L747-T751del/Sins, and S752-I759del), 2 types of L858R mutations (L858R, and L858R/T790M), T790M, G719S and L861Q. This inhibitory profile suggested that CH7233163 can inhibit not only EGFR-Del19/T790M/C797S, but also EGFR-L858R/T790M/C797S triple-mutation and other various EGFR mutations. To confirm this possibility, we firstly performed several assays of EGFR-L858R/T790M/C797S triple mutation. CH7233163 potently inhibited the phosphorylation activity of L858R/T790M/C797S in a biochemical assay with an IC₅₀ value of 0.25 nmol/L (Fig. 5A), which was almost same for Del19/T790M/C797S. In addition, CH7233163 showed potent antiproliferation activity against L858R/T790M/C797S–expressing NIH3T3 (L858R/T790M/C797S_NIH3T3) cells (IC₅₀ = 45 nmol/L; Fig. 5B). Furthermore, CH7233163 blocked the phosphorylation of EGFR-L858R/T790M/C797S dose dependently in the cells (Fig. 5C). Next, we checked whether CH7233163 inhibits EGFR-single-activating mutations (Del19 and L858R) and EGFR-double mutations (Del19/T790M and L858R/T790M), as suggested by the KINOMEscan assay result (Fig. 4). As shown in Supplementary Table S4, CH7233163 potently and selectively inhibited both EGFR-single-activating mutations (Del19 and L858R) and EGFR-double mutations (Del19/T790M and L858R/T790M) over EGFR-WT in biochemical assays (IC₅₀ values: 0.17–0.41 nmol/L). Moreover, CH7233163 showed potent and mutant selective antiproliferation activities against the tumor cells harboring EGFR-single-activating mutations (Del19 and L858R) and EGFR-double mutations (Del19/T790M and L858R/T790M) (Supplementary Table S5). To further confirm these properties, we evaluated its in vivo efficacy against EGFR-single-activating mutation and EGFR-double mutations. Therein, we used human lung cancer cell lines NCI-H1975 and HCC827 harboring the EGFR double mutation (L858R/T790M) and single activating mutation (Del19), respectively. Consistent with the in vitro antiproliferation spectrum, CH7233163 potently inhibited tumor growth in both tumor xenograft models and its potency was similar to that of osimertinib (Fig. 5D and E). These data suggest that CH7233163 selectively inhibits not only EGFR-Del19/T790M/C797S, but also various EGFR mutants, such as EGFR-L858R/T790M/C797S, EGFR double mutation (Del19/T790M and L858R/T790M) and single activating mutation (Del19 and L858R).

To further understand the molecular mechanism by which CH7233163 potently and selectively inhibits EGFR triple-mutations, we determined the three-dimensional structure of a CH7233163 and triple-mutated EGFR complex. To do this, we utilized the L858R/T790M/C797S, not Del19/T790M/C797S, because we cannot purify the EGFR-Del19/T790M/C797S protein to a crystal grade. EGFR-L858R/T790M/C797S (residues 695–1022 with E865A/E866A/K867A mutations) was expressed in insect cells, and the crystal structure of the CH7233163 and EGFR-L858R/T790M/C797S complex was determined. As shown in Fig. 6A and B, CH7233163 binds to the ATP-binding pocket of EGFR-L858R/T790M/C797S, confirming that CH7233163 is an ATP-competitive inhibitor, as suggested in Fig. 3A. Figure 6B showed that CH7233163 interacted with multiple amino acids within the ATP-binding pocket through hydrogen bonds and CH/π interactions, but none of these interactions suggest covalent binding. Interestingly, CH7233163 does not interact with C797S residue, which is the unique amino acid in EGFR-triple mutations. We then determined the structures of complexes of osimertinib with EGFR-L858R/T790M/C797S to compare the binding mode. CH7233163 binds more deeply within the ATP-binding pocket than the osimertinib and interacts with not only the P-loop and hinge regions but also the gatekeeper residue T790M directly, with which osimertinib does not interact (Fig. 6C). These results suggest that multiple noncovalent interactions, including with T790M, may contribute to the potency of CH7233163 against triple-mutant EGFR. In addition, T790M is absent from EGFR-WT, and so the interaction between CH7233163 and T790M would further contribute to mutant-selective inhibition over EGFR-WT. Furthermore, we compared the structures between the EGFR–CH7233163 complex and EGFR–EAI-001 complex (PDB ID: 6LUB and 5D41, respectively), because the allosteric EGFR inhibitor EAI-001 reported to bind to the αC-helix-out conformation of EGFR (22). Figure 6D revealed that the two structures have dramatically different αC-helix conformations, suggesting that CH7233163 binds to EGFR-L858R/T790M/C797S with an αC-helix-in conformation. We also compared the structure of EGFR–CH7233163 complex (PDB ID: 6LUB) with reported two αC-helix-in conformations of EGFR (PDB ID: 2ITZ and 5XDK). As shown in Supplementary Fig. S3, αC-helix structure is very similar among three structures. This result also supports that CH7233163 binds to EGFR-L858R/T790M/C797S with an αC-helix-in conformation. This mode of CH7233163 binding to EGFR in an αC-helix-in conformation would be very important for inhibiting the Del19/T790M/C797S mutation. This is because EGFR-Del19 mutations may not form αC-helix-out conformation (43), and reported allosteric inhibitors which bind to the αC-helix-out conformation, cannot inhibit EGFR-Del19/T790M/C797S. Although no one succeed to gain crystal structures of EGFR-Del19 mutants, recent in silico molecular dynamics (MD) simulations revealed that Del19 mutations inferred that the reduced length and flexibility of N-terminal portion of aC-helix locks the helix αC-in state and prohibits transition to the inactive conformation. The EGFR structure (PDB IB: 1M17) is reported as a similar αC-helix-in structure to that of Del19 by MD simulation (44), and so we additionally superimposed our crystal structure (PDB ID: 6LUB) with this crystal structure (PDB ID: 1M17). As shown in revised Supplementary Fig. S4, αC-helix-in structure is similar between two structures. These findings suggest that CH7233163 is a noncovalent ATP-competitive inhibitor for EGFR-Del19/T790M/C797S that utilizes multiple interactions with the EGFR's αC-helix-in conformation.

Osimertinib resistance resulting from EGFR triple-mutations (Del19/T790M/C797S and L858R/T790M/C797S) has been reported. Because no therapies have been developed to attenuate the effects the Del19/T790M/C797S mutation thus far, there is a pressing need for novel approaches to overcome this therapeutic resistance. Herein, we generated a novel EGFR-TKI, CH7233163, which potently and selectively inhibits EGFR-Del19/T790M/C797S mutation over EGFR WT. Certain allosteric inhibitors potentially attenuating the osimertinib-resistant EGFR-L858R/T790M/C797S mutation have been reported, such as EAI-001/045, JBJ-04–125–02 (21, 22); however, these compounds are unable to inhibit the EGFR-Del19/T790M/C797S mutation (Fig. 1B and C; refs. 21, 22). Therefore, the identification of CH7233163, which potently inhibits Del19/T790M/C797S, is of therapeutic significance. In addition, EGFR-Del19 mutations probably does not form the αC-helix-out conformation due to the shortening the loop leading into the αC-helix (43). Hence, it is reasonable to conclude that CH7233163 binding to EGFR with αC-helix-in conformation (Fig. 6D) is the mechanism underlying the potent inhibitory activities of this compound against Del19/T790M/C797S (Fig. 1B, 1C, and 2B). Furthermore, EAI-001/045 as monotherapy failed to display antitumor effects even against L858R/T790M/C797S both in vitro and in vivo (Fig. 5B; ref. 22). The molecular mechanism behind this phenomenon is similarly associated with EAI-001/045 binding to EGFR's αC-helix-out conformation. The αC-helix-out is an inactive conformation of EGFR, and interestingly, this inactive form of EGFR can induce the EGFR signaling without kinase activity through asymmetric dimerization (22, 45). Therefore, combinatorial treatment with anti-EGFR antibody to block asymmetric dimerization, is required for sufficient antitumor activities. However, EGFR-WT inhibition causes side-effects such as rashes and/or diarrhea, and these WT EGFR-derived toxicities cause dose-limiting effects in various anti-EGFR therapies (40, 41). Unlike EGFR-TKIs, anti-EGFR antibodies are not selective toward EGFR mutants and WT EGFR; therefore, the therapeutic window for combinatorial treatment with anti-EGFR antibody and EGFR-TKI would be narrower than that of monotherapy with mutant selective EGFR-TKI. Thus, the compound profile of CH7233163, which selectively inhibit EGFR-Del19/T790M/C797S over EGFR WT and potent antitumor activity upon monotherapy, would benefit osimertinib-resistant patients. Recently, new ATP-competitive EGFR TKIs, BI-4020 and its derivatives, were reported as mutant-selective EGFR-Del19/T790M/C797S inhibitors (46). However, these compounds are reported as 10-fold weaker potency against L858R/T790M/C797S in biochemical assays. In contrast, CH7233163 potently inhibited not only EGFR-Del19/T790M/C797S but also various EGFR mutations, including L858R/T790M/C797S triple mutations, double mutations and single-activating-mutations (Fig. 5; Supplementary Table S4 and S5). This profile would help attenuate osimertinib resistance, potentially because drug-resistant EGFR-mutated NSCLC contains various EGFR mutations based on the intratumor heterogeneity. For example, coexisting T790M and T790–wild-type clones or coexisting Del19 and L858R clones have been previously reported in single patient after treatment with first/second-generation EGFR-TKIs (47, 48). Therefore, compared with the specific inhibitor of EGFR-triple mutations, CH7233163′s ability to potently and selectively inhibit various EGFR mutations (Del19/T790M/C797S, L858R/T790M/C797S, Del19/T790M, L858R/T790M, Del19 and L858R), is potentially necessary to overcome osimertinib resistance, considering the intratumoral heterogeneity of human tumors. In addition to overcoming osimertinib resistance, CH7233163 potentially prevent on-target EGFR resistance in the initial therapy setting. In case of osimertinib, this compound can inhibit both EGFR activating-mutations and first-generation resistant EGFR-T790M mutations (10). The FLAURA clinical trial designed to test the efficacy of osimertinib as first-line treatment showed the superior efficacy than that of first-generation EGFR TKIs, and osimertinib prevented T790M-mediated on-target resistance (11, 49, 50). Therefore, CH7233163′s ability to potently inhibit various EGFR mutations (Del19/T790M/C797S, L858R/T790M/C797S, Del19/T790M, L858R/T790M, Del19 and L858R) potentially reduce on-target resistance and induce superior clinical efficacy. On the basis of this evidence, CH7233163 could greatly benefit osimertinib-resistant patients, especially those with the EGFR-Del19/T790M/C797S mutation. Although further studies are required to assess the safety of animal tests for clinical trials, we expect that CH7233163 may have the potential for clinical evaluation.

All of the authors are employees of Chugai Pharmaceutical Co., Ltd.

K. Kashima: Conceptualization, data curation, formal analysis, validation, writing-original draft, project administration. H. Kawauchi: Data curation, formal analysis, validation. H. Tanimura: Data curation, formal analysis, validation. Y. Tachibana: Data curation, formal analysis, validation. T. Chiba: Conceptualization, data curation, formal analysis, validation. T. Torizawa: Resources, validation. H. Sakamoto: Resources, validation, project administration, writing-review and editing.

The authors thank Ayano Nakamura and Maiko Izawa for their technical support; Toshihiro Aoki and Masami Kochi for compound generation; Kotaro Ogawa for pharmacokinetic analyses; and Sho Akai, Takaaki Fukami, Nobuhiro Oikawa, Tadakatsu Takahashi, Takuo Tsukuda, Kiyoaki Sakata, Toshihiko Fujii, Toshiyuki Mio, Yoshiki Kawabe, Hitoshi Iikura, and Junichi Nezu for the helpful discussions. This study was funded by Chugai Pharmaceutical Co., Ltd.

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