TAS0728, A Covalent-binding, HER2-selective Kinase Inhibitor Shows Potent Antitumor Activity in Preclinical Models

This article is featured in Highlights of This Issue, p. 731

HER2 is a member of the ERBB family of receptor tyrosine kinases, which includes EGFR, HER2, HER3, and HER4 (1). Genetic abnormalities in HER2 have been reported in various cancers (2, 3), and it has been recognized as an oncogenic driver gene (4, 5). HER2 forms homo- or heterodimers with ERBB family kinases to induce activation of HER2 signal transduction associated with enhanced survival and cell growth signals in cancer cells. Targeting of HER2 has been shown to be an effective antitumor therapy in breast and gastric cancers (6–8). Although anti-HER2 antibodies, including trastuzumab, pertsuzumab, and the antibody–drug conjugate, T-DM1, are approved for use in patients with HER2-overexpressing breast cancers, there is still a need for effective therapies in patients who are refractory to HER2-targeting antibodies.

Lapatinib, a reversible HER2 and EGFR dual kinase inhibitor, is used in combination with capecitabine for the treatment of HER2-overexpressing breast cancer (9). Although HER2 is an oncogenic driver gene, the response rate of lapatinib as a monotherapy was less than 10% in chemotherapy-refractory HER2-positive breast cancer (10). Compared with other molecular targeting therapies such as a TRK inhibitor (11), ALK inhibitors (12), and EGFR inhibitors (13), the lapatinib's response rate was much lower. A preclinical study that reported the resilience of the HER2/HER3 pathway may provide insights into the clinical outcomes associated with lapatinib therapy. Indeed, the buffering activity of the HER2/HER3 pathway was shown to be related to the HER3 reactivation mechanism, and full inactivation of HER2 with high-dose lapatinib may overcome the negative feedback reactivation of HER3 (14). Consistent with this, extremely high doses of lapatinib as a single-agent treatment with 5-day intermittent dosing elicited dramatic clinical responses, although concomitant use of a CYP inhibitor was required to achieve and maintain high plasma concentrations (15). Therefore, robust inhibition of HER2 may result in dramatic responses in HER2-addicted tumors.

Various covalent-binding irreversible inhibitors of HER2 have been developed and have been shown to exhibit robust and sustained target engagement in preclinical models (16, 17). However, these reported HER2-inhibitory covalent binders are not selective for HER2 and instead act as pan-ERBB tyrosine kinase inhibitors (TKI) that block the activity of ERBB family kinases, including EGFR. Although EGFR is a well-established therapeutic target in cancer, inhibition of this protein in normal tissues can result in dose-limiting rashes and gastrointestinal (GI) issues, particularly diarrhea to the level of grade 3 or 4 toxicity (18). This was clearly reported in the LUX-BREAST 1/3 studies of afatinib (19, 20) and the ExteNET trial of neratinib (21). Therefore, the specificity for HER2 inhibition excluding EGFR may allow for more effective dosing to overcome the resiliency of the HER2/HER3 pathway in HER2-activated cancers and improve the clinical response rates of conventional HER2 TKIs. However, identification of HER2-selective covalent inhibitors is still challenging.

In this study, we screened for compounds with high potency and high selectivity for HER2. Here, we report TAS0728, a novel covalent-binding inhibitor of HER2 kinase with high specificity for HER2, but not EGFR. We characterized the inhibitory activities of this novel compound in HER2-overexpressing and -mutated cells and evaluated its antitumor activity in vivo. Assessment of TAS0728 in patients with cancer is ongoing in a clinical trial (NCT03410927).

TAS0728 was synthesized at Tsukuba Research Institute, Taiho Pharmaceutical Co., Ltd. by the procedure described in Supplementary Fig. S1. Lapatinib was purchased from LC Laboratories. Afatinib was purchased from Selleck Chemicals. Reagents were dissolved in dimethyl sulfoxide (DMSO) for in vitro assays. For in vivo experiments, TAS0728 was dissolved in 0.1 N HCl solution or 0.1 N HCl with 0.5% hydroxypropyl methylcellulose (HPMC). Lapatinib and afatinib were suspended in vehicle, as described in previous reports (16, 22).

The following antibodies were purchased from Cell Signaling Technology Japan, K.K. and used for Western blotting: anti-phospho HER2 (Tyr1196; #6942), anti-HER2 (#2165), anti-phospho-HER3 (Tyr1289; #4791), anti-HER3 (#12708), anti-phospho-p44/42 MAPK; Thr202/Tyr204; #4370), anti-p44/42 MAPK (#4695), anti-phospho-AKT (Ser473; #4060), anti-AKT (#4685), anti-BIM (#2933), anti-cleaved PARP; #9541), anti-β-actin (#8457), anti-phospho-EGFR (Tyr1068; #2234), anti-EGFR (#2232), and anti-rabbit IgG (#7074).

The recombinant human HER2 cytoplasmic domain (Carna Biosiences; 1 μmol/L) was incubated with TAS0728 (50 μmol/L) at room temperature for 24 hours. After reduction and alkylation with dithiothreitol (DTT; Thermo Fisher Scientific K.K.) and iodoacetamide (Thermo Fisher Scientific K.K.), the protein was digested at 37°C with trypsin/Lys-C (Promega) in digestion buffer with 1 mol/L, ammonium bicarbonate solution (pH 8.0). The mass spectrum of the compound/kinase complex was detected with a quadrupole time-of-flight mass spectrometer (Xevo G2-S QTof; Waters Corporation) coupled with an ultra-high-performance liquid chromatography system (ACQUITY UPLC I-Class; Waters Corporation) in positive electrospray ionization mode. This system was equipped with an Aeris WIDEPORE 3.6μ XB-C18 150 × 2.1 mm column (Phenomenex) and was operated at a flow rate of 0.3 mL/minute and a column temperature of 50°C. The gradient of mobile phase A [water, 0.1% formic acid (v/v)] and B [acetonitrile, 0.1% formic acid (v/v)] was performed as follows: 2% B for 3 minutes, 2%–50% B for 37 minutes, 50%–90% B for 10 minutes. The data were acquired in MS^E mode with collision energy of 4 eV for low-energy scans and ramped from 15 to 40 eV for high-energy scans. Further data analysis was performed using BioPharmaLynx1.3.3 software (Waters Corporation).

The inhibitory activity of HER2 kinase was measured using kinase profiling, performed by Carna Biosciences. The 50% inhibitory concentration (IC₅₀ value) of TAS0728 was determined on the basis of the in vitro peptide substrate phosphorylation activity of HER2. Three independent experiments were conducted.

The inhibitory activity of TAS0728 for 386 or 374 kinases was tested using Kinase Panel Assay at Reaction Biology Corporation. The assays were conducted in duplicate mode at concentrations of 0.1, 1, and 10 μmol/L TAS0728. Reactions were carried out in the presence of 10 μmol/L ATP.

The irreversibility of the kinase inhibition of compounds was evaluated by dilution with ATP solution following preincubation of recombinant human HER2 with compounds. The recombinant human HER2 (Carna Biosciences) was preincubated with 5 μmol/L TAS0728, 5 μmol/L lapatinib, or 125 μmol/L staurosporine in assay buffer consisting of 13.5 mmol/L Tris-HCl, 0.009% Tween20, and 2 mmol/L DTT at 25°C for 30 minutes. The compounds/kinase complexes were then diluted 125-fold with assay buffer containing 400 μmol/L ATP, 10 mmol/L MnCl₂, and 0.5 μmol/L FL-peptide 22. The recovery of enzyme activity from a preformed enzyme–inhibitor complex was measured using LabChip EZ Reader Mobility Shift Assays (PerkinElmer).

SK-BR-3 cells were purchased from Dainippon Pharmaceutical Co., Ltd. A-431, AU565, BT-474, Calu-3, and NCI-N87 cells were purchased from ATCC. KPL-4 cells were provided by Dr. Junichi Kurebayashi at Kawasaki Medical School (Kurashiki, Matsushima, Japan; ref. 23). The series of MCF10A cell lines stably expressing HER2-mutant genes was provided by Dr. Motoki Takagi at Fukushima Medical University (Fukushima, Japan), which were established as described previously (24). The primary human epidermal keratinocytes (NHEK-NEO) cells were purchased from Lonza Walkersville, Inc.

Cell lines were cultured in McCoy 5A medium supplemented with 10% heat-inactivated FBS (SK-BR-3 cells), DMEM with 10% heat-inactivated FBS (BT-474 and A-431 cells), RPMI1640 medium supplemented with 10% heat-inactivated FBS (AU565, NCI-N87 cells, and NCI-N87-luc), DMEM supplemented with 5% heat-inactivated FBS (KPL-4 cells), or Eagle minimum essential medium supplemented with 10% heat-inactivated FBS (Calu-3 cells). NHEK-Neo cells were cultured using a KGM-Gold Bullet Kit (Lonza Walkersville, Inc.). MCF10A cells were cultured in DMEM/Ham F-12 medium supplemented with 10 μg/mL insulin, 500 ng/mL hydrocortisone, 5 μmol/L forskolin, and 5 % heat-inactivated horse serum. Cell lines were authenticated by short tandem repeat profiling, tested negative for Mycoplasma, and used within 7 passages between experiments.

Cells were seeded in 96-well plates and then incubated overnight. Medium containing a compound or DMSO control was added, and cells were then incubated for 72 hours. The cell population densities were then measured using CellTiter-Glo 2.0 Reagent (Promega Corporation).

Cells were seeded in 6-well plates and cultured overnight. Compounds were added to the culture medium at concentrations as indicated for 3 hours followed by harvesting for Western blot analysis.

For studies using MCF10A cell line, cells were seeded in 12-well plates and incubated overnight. Compounds were added to the culture medium and incubated for 3 hours. The cells were harvested to perform Western blot analysis.

In the pharmacodynamics time-course study, SK-BR-3 cells were seeded in 100-mm dishes and cultured overnight. Compounds were added and incubated for 3 or 48 hours. The cells were harvested to perform Western blot analysis.

Cells were lysed in cell protein extraction buffer containing Cell Extraction Buffer (Life Technologies Corporation), cOmplete Mini Protease Inhibitor Cocktail (Roche Diagnostics K.K.), and PhosSTOP Phosphatase Inhibitor Cocktail (Roche Diagnostics). Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes using a Trans-Blots Turbo RTA Midi PVDF Transfer Kit (Bio-Rad Laboratories, Inc.). Membranes were blocked with Blocking One-P Blocking Reagent (Nacalai Tesque, Inc.) and probed with appropriate primary antibodies. The membranes were then incubated with anti-rabbit IgG antibody. The proteins were detected using luminol-based enhanced chemiluminescence with SuperSignal West Dura (Thermo Fisher Scientific K.K.) or SuperSignal West Femto (Thermo Fisher Scientific K.K.). Luminescent images were captured with ImageQuant LAS 4010 or Amersham Imager 600 QC (GE Healthcare Japan).

All animal experiments were conducted with approval of the Institutional Animal Care and Use Committee of Taiho Pharmaceutical Co., Ltd. and carried out according to the guidelines for animal experiments of Taiho Pharmaceutical Co., Ltd. Nude mice (BALB/cAJcl-nu/nu) were purchased from CLEA Japan, Inc. For pharmacokinetics, TAS0728 was administered orally to male nude mice. Plasma samples were collected according to the sampling schedule (predose, 0.25, 0.5, 1, 2, 4, 6, and 24 hours) after dosing. Plasma samples were analyzed using liquid chromatography with tandem mass spectrometry (LC/MS-MS).

For in vivo pharmacodynamics evaluation, suspensions of NCI-N87 cells were implanted subcutaneously into the side flanks of 6-week-old male nude mice. Mice were randomized into the control and treatment groups (n = 3). Tumors were collected from animals, snap frozen in liquid nitrogen, and stored in a deep freezer for pharmacodynamics analysis. Tumor tissues were lysed in lysis buffer containing Sample Diluent, Concentrate 2 (R&D Systems, Inc.), cOmplete Mini Protease Inhibitor Cocktail (Roche Diagnostics), and PhosSTOP Phosphatase Inhibitor Cocktail (Roche Diagnostics), and processed for Western blot analysis as described above.

Nude mice (BALB/cAJcl-nu/nu) were purchased from CLEA Japan, Inc. Severe combined immunodeficient (SCID) mice (CB17/Icr-Prkdcscid/CrlCrlj) were purchased from Charles River, Japan, Inc. Suspensions of cells were implanted subcutaneously into the side flanks of 6-week-old nude mice. The tumor volume (TV, mm³) was calculated as the length (mm) × width (mm)²/2. During the treatment period, TV and body weight (BW) were measured twice per week. When the TV_final was smaller than the TV_initial in the treatment group, tumor regression was judged to have occurred. As an indicator of changes in BW during the dosing period, the BW change (BWC; %) was calculated as 100 − ([BW on each measurement day] − [BW_initial])/(BW_initial), where BW_initial is the body weight on the allocation day. Dunnett tests or Aspin–Welch t test were used as a statistical method to compare the tumor volume data in the drug-treated and control groups.

To establish a peritoneal dissemination model with HER2-addicted cancer cells, NCI-N87-luc cells stably expressing the luciferase gene were established by transfection with the luciferase expression vector. The cells were cloned using promycine and authenticated by short tandem repeat profiling. The expression of luciferase activity was confirmed using a Dual-Glo Luciferase Assay System (Promega Corporation). Cell suspensions (4 × 10⁷ cells/mL) in PBS were inoculated intraperitoneally into SCID mice, as described previously (25). Engraftment of tumors in mice was confirmed using bioluminescence values that were evaluated with an IVIS Lumina II Imaging System (Perkin Elmer Inc.) and randomized into each treatment group. Compounds were administered once daily. The increased life span was calculated using the median survival time (MST) in each treatment group. The mice were euthanized by isoflurane inhalation upon becoming moribund. The survival times of the drug treatment and control groups were compared using a log-rank test. Differences with P values <0.05 were considered statistically significant.

TAS0728 is a pyrazolopyrimidine carboxamide analogue with an acrylamide group as a covalent binding moiety to target the cysteine residue in the kinase domain of HER2. The chemical structure is shown in Fig. 1A. The IC₅₀ value (mean ± SD) of TAS0728 against recombinant human HER2, as determined by in vitro kinase assays, was 36 ± 3 nmol/L.

Mass spectrometry analysis showed that TAS0728 covalently modified the cysteine residue at position 805 of recombinant human HER2 protein, suggesting that TAS0728 covalently bound to the recombinant human HER2 cytoplasmic domain (Fig. 1B).

To further characterize the covalent binding of TAS0728 to HER2, time-course of inhibitory activity of TAS0728 was evaluated in a high-concentration ATP reaction mixture after an enzyme–inhibitor complex was formed. The reversible HER2 inhibitor lapatinib was used as a reference (26). The lapatinib/HER2 complex showed the recovery of kinase activity after dilution with ATP, suggesting the dissociation of lapatinib from HER2 protein. In contrast, TAS0728 was not affected for 200 minutes after adding high concentration of ATP, indicating that the inhibitory activity of TAS0728 was not sensitive to ATP concentration once TAS0728 bound to HER2 (Fig. 1C).

Collectively, these findings suggested that TAS0728 formed a covalent bond with HER2 and irreversibly inhibited the kinase activity of HER2.

To investigate selectivity of TAS0728, enzyme assays of a panel of 386 or 374 kinases were conducted. TAS0728 at 0.1 μmol/L showed an inhibition rate of greater than 80% (≥ 80% inhibition of activity) for three of the 386 kinases tested (Fig. 2A and D). In a panel of 374 kinases, 12 and 19 kinases were inhibited by more than 80% at 1 and 10 μmol/L, respectively (Fig. 2B–D). IC₅₀ values of TAS0728 against HER2 and EGFR recombinant, which were used in the kinase panels, were 13 nmol/L and 65 nmol/L, respectively. IC₅₀ values of TAS0728 for HER2, EGFR, and other off-target kinases were listed in Supplementary Table S1. Moreover, the LeadProfilingScreen containing 68 nonkinase molecules (Eurofins Panlabs) revealed that TAS0728 at 10 μmol/L did not bind to the 68 targets. The list of nonkinase targets tested is shown in Supplementary Table S2.

To investigate the effects of TAS0728 on the phosphorylation of HER2 in cells and the specificity excluding EGFR, HER2-overexpressing SK-BR-3 cells and EGFR-overexpressing A-431 cells were treated with TAS0728 for 3 hours. At concentrations ranging from 30 to 1,000 nmol/L, TAS0728 strongly inhibited the phosphorylation of HER2 in SK-BR-3 cells. On the other hand, phosphorylation of EGFR was only weakly inhibited even at a concentration of 1,000 nmol/L in A-431 cells (Fig. 2C). In contrast, afatinib inhibited the phosphorylation of HER2 in SK-BR-3 cells at concentrations ranging from 100 to 300 nmol/L and inhibited the phosphorylation of EGFR in A-431 cells at concentrations ranging from 10 to 300 nmol/L. These results suggested that TAS0728 had higher selectivity for HER2 than EGFR.

In addition to amplification or overexpression of HER2, mutations in HER2, an alternative activation mechanism, have been reported in several cancers (3). To investigate the effects of TAS0728 on the phosphorylation of mutated HER2 in cells, MCF10A cells stably expressing wild-type HER2 protein or various HER2 mutants were treated with TAS0728. Phosphorylation of wild-type or mutant HER2 was examined in control and treated cells. The inhibitory effects of TAS0728 on the phosphorylation of various HER2 mutants (G309A, S310F, R678Q, L755_T759 deletion, D769H, V777L, V842I, and R869C) was equivalent to that of wild-type HER2 (Fig. 2D). A slightly higher concentration was required to inhibit the phosphorylation of HER2 L755S and ex20insYVMA, although this concentration was still lower than that required for inhibition of wild-type EGFR. These results suggested that TAS0728 had inhibitory effects on the phosphorylation of various HER2 mutants and on overexpressed wild-type HER2 at a concentration lower than that for inhibition of wild-type EGFR.

To examine the cell line–selective cytotoxicity of TAS0728 for HER2-amplified and EGFR-activated cell lines, the drug concentration of 50% growth inhibition (GI₅₀) values were determined for seven cancer cell lines, including HER2 gene–amplified cells, wild-type EGFR gene-amplified cells, and primary normal human epidermal keratinocytes showing EGF-dependent cell growth (Table 1). TAS0728 potently inhibited the in vitro proliferation of five HER2-amplified cell lines, and the GI₅₀ values were less than 10 nmol/L. For KPL-4 cells, a HER2-amplified, PIK3CA-mutated breast cancer cell line, the GI₅₀ value of TAS0728 was relatively higher than those of the other five HER2-amplified cell lines. The GI₅₀ values of TAS0728 for EGFR-amplified A-431 cells and EGFR-expressing normal keratinocytes (NHEK) were 450 ± 220 nmol/L and 620 ± 310 nmol/L, respectively, which was more than 10-fold higher than those of HER2-amplified cell lines. In contrast, the GI₅₀ values of afatinib were almost equivalent for both HER2-amplified cell lines and EGFR-activated cell lines, except in KPL-4 cells. These findings suggested that TAS0728 had potent inhibitory activities against cancer cell lines with HER2 amplification, but showed reduced activity in cells addicted to EGFR signals.

In a previous study, the feedback reactivation of HER2/HER3 signaling was observed during lapatinib exposure in HER2-positive breast cancer cells (14). To compare the time-dependent pharmacodynamic activities of TAS0728 and lapatinib in SK-BR-3 cells, the cells were treated with TAS0728 or lapatinib for 3 and 48 hours at concentrations ranging from 30 to 300 nmol/L. Western blotting analysis was performed to investigate the inhibitory effects of compounds on the phosphorylation of HER2, HER3, AKT, and ERK. In addition, the expression of BIM (a key effector of apoptosis; ref. 27) and cleaved PARP (a marker of apoptosis; ref. 28) was examined. Although lapatinib initially inhibited HER2 signaling after 3 hours of exposure, reactivation of HER2 and downstream molecules, including HER3, AKT, and ERK, was observed at 48 hours (Fig. 3). In contrast, TAS0728 showed sustained inhibition of HER2, HER3, AKT, and ERK phosphorylation at concentrations ranging from 100 to 300 nmol/L during the 48-hour incubation. BIM and cleaved PARP were markedly increased after treatment of SK-BR-3 cells with TAS0728 for 48 hours at concentrations ranging from 30 to 300 nmol/L, although these changes were not observed in the lapatinib group. These results suggested that TAS0728 had superior sustained inhibitory activity toward HER2 and downstream factors compared with lapatinib and that these effects were associated with apoptosis induction of cancer cells.

To further characterize the profiles of TAS0728, pharmacokinetics and pharmacodynamics were evaluated in mice. In the pharmacokinetics study in male nude mice, the plasma concentration–time profiles on day 14 were determined after daily oral administration of TAS0728 at doses of 7.5, 15, 30, and 60 mg/kg/day for 14 days. The plasma concentration reached C_max less than 0.5 hours after dosing. For the dose range tested (7.5–60 mg/kg/day), plasma concentration of TAS0728 at each time point increased in a dose-dependent manner. TAS0728 was rapidly eliminated within 24 hours, and the biological half-life of TAS0728 at 60 mg/kg in mice was less than 1 hour. At 6 hours after administration, most TAS0728 was eliminated from plasma (Fig. 4A).

Pharmacodynamic analysis at 60 mg/kg/day of TAS0728 revealed that phosphorylation of HER2, HER3, AKT, and ERK was decreased compared with that in the control group at 0.5, 1, 4, and 8 hours after administration. The inhibition was continued up to 8 hours after administration and recovered to around 75% at 12 hours (Fig. 4B). These analyses revealed that sustained target inhibition was observed after elimination of TAS0728 in the plasma of mice.

We next determined the in vivo efficacy of TAS0728 in a NCI-N87 HER2–amplified human gastric cancer mouse model. The animals were orally administered TAS0728 at different doses; all doses were well tolerated in all mice because no significant loss in body weight was observed (Supplementary Fig. S2). Furthermore, dose-dependent antitumor effect was observed. Significant tumor regression was observed in mice treated with 60 mg/kg/day (Fig. 5A). Afatinib (20 mg/kg/day) also showed antitumor effects in this model, as described previously (16).

We next evaluated the in vivo efficacy of TAS0728 in another model bearing HER2-amplified breast tumors. In this model, administration of TAS0728 at 30 or 60 mg/kg/day resulted in tumor regression. In contrast, tumor shrinkage was not observed in the lapatinib-treated group (Fig. 5B).

To evaluate the effects of TAS0728 on survival and the tolerability of long-term daily dosing, we established an NCI-N87 peritoneal dissemination model and then evaluated and compared the efficacy and tolerability of once-daily oral dosing of TAS0728, afatinib, and lapatinib. The dosing was started on day 7 after implantation. The MSTs were 60 days in the untreated control group and 77 days in lapatinib-treated mice. In contrast, no mice died in the afatinib- or TAS0728-treated groups during the 120-day experimental period (Fig. 5C). In this model, any evident toxicity, including diarrhea and body weight loss, was not observed by the long-term dosing of TAS0728.

Collectively, these data demonstrated not only the potent antitumor effect of TAS0728 but also the good tolerability of long-term administration.

In this study, we aimed to identify a next-generation HER2 inhibitor with both high activity and high selectivity for HER2. Accordingly, we identified TAS0728 as a highly selective covalent inhibitor of HER2 kinase. TAS0728 is an amino-pyrazolopyrimidine class compound, which has a distinct chemical structure compared with reported HER2-inhibitory compounds, such as lapatinib (quinazoline class), afatinib (quinazoline class), and neratinib (cyanoquinoline class; ref. 29). We demonstrate the pharmacologic profile of TAS0728 in in vitro and in vivo preclinical models.

TAS0728 was found to be a covalent-binding inhibitor of HER2 kinase. TAS0728 covalently bound to Cys in HER2 and inhibited kinase activity. Unlike lapatinib, the inhibitory activity of TAS0728 for HER2 kinase was not affected by the presence of high concentration of ATP after binding to HER2 kinase, which is a particular characteristic of covalent-binding TKIs. To achieve durable inhibition of target kinase activity in cancer cells, irreversible inhibition would be an advantage of TKIs. A previous report suggested that the HER2/HER3 signal in HER2-amplified cancer cells is resilient; thus, higher doses of lapatinib, which are required to block HER2/HER3 signaling, may result in dramatic antitumor responses in HER2-positive cancer cells (14, 15). In our study, although lapatinib initially inhibited the phosphorylation of HER2 and the downstream pathway, reactivation of these molecules was observed after 48 hours of continuous exposure, consistent with previous reports. In contrast, TAS0728 induced durable inhibition of the phosphorylation of HER2 and downstream molecules after 48 hours of treatment. Consistent with these findings, increased BIM protein and cleaved PARP were observed in the TAS0728-treated group, suggesting that apoptosis was induced in these cancer cells. Moreover, in vivo, TAS0728 induced tumor shrinkage in xenograft models, accompanied by increased BIM expression and cleaved PARP levels in the tumor tissues. In contrast, lapatinib did not induce tumor regression in mice. The different pharmacologic activities of TAS0728 and lapatinib are presumably due to differences in the binding modes of the two compounds. Thus, TAS0728 has the potential to exceed the clinical activity of lapatinib as a single agent.

TAS0728 showed high selectivity for HER2 and reduced activity for EGFR. Compared with afatinib, TAS0728 had higher specificity for HER2 than for EGFR, as demonstrated in the panel enzymatic assay, cellular pharmacodynamics assay, and cytotoxicity assay. To date, various covalent inhibitors have been developed for the treatment of HER2-overexpressing cancers. However, a common dose-limiting toxicity among EGFR-inhibitory compounds is grade 3 diarrhea, which is most often caused by inhibition of wild-type EGFR (18). The concept of avoiding EGFR-related toxicity may be supported by clinical data with osimertinib, which shows high specificity for EGFR T790M over wild-type EGFR; when using osimertinib, mild rash and diarrhea were reported in only a minority of patients (30).

In mouse models, administration of TAS0728 did not induce diarrhea during the treatment at efficacious doses. However, it is still unclear whether TAS0728-dependent EGFR-related toxicity is reduced in humans compared with that in other TKIs, because the vulnerability to those adverse effects and pharmacokinetic profile may differ between humans and mice. There is a limit of the study using mouse models to expect EGFR-related toxicity of TAS0728 in human, and further evaluation in the clinical setting is needed to confirm the hypothesis. Currently, a phase I study of TAS0728 in patients with solid tumors is ongoing, and data describing pharmacokinetic profiles and adverse events will be reported elsewhere.

In conclusion, TAS0728 was found to be an orally available, highly selective covalent inhibitor of HER2. The high specificity for HER2 may prevent EGFR-related toxicity and to achieve more effective dosing in patients. TAS0728 may be a promising therapeutic option for the treatment of cancers harboring HER2 gene abnormalities and would be expected to have an improved therapeutic window compared with current HER2 inhibitors.

No potential conflicts of interest were disclosed.

Conception and design: H. Irie

Development of methodology: H. Irie, K. Ito, A. Hashimoto, K. Funabashi

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): H. Irie, K. Ito, Y. Fujioka, K. Oguchi, A. Fujioka, A. Hashimoto, H. Ohsawa, K. Funabashi, H. Araki, Y. Kawai, T. Shimamura

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): H. Irie, K. Ito, Y. Fujioka, K. Oguchi, A. Fujioka, A. Hashimoto, H. Ohsawa, K. Tanaka, K. Funabashi, H. Araki, S. Ohkubo, K. Matsuo

Writing, review, and/or revision of the manuscript: H. Irie, K. Tanaka, R. Wadhwa, S. Ohkubo, K. Matsuo

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): K. Tanaka

Study supervision: H. Irie, S. Ohkubo, K. Matsuo

Other (Chemical design and synthesis): Y. Kawai, T. Shimamura

The authors thank Drs. Teruhiro Utsugi, Yoshikazu Iwasawa, and Kazuhiko Yonekura for their insightful discussion. The authors also thank all the departments at the Discovery and Preclinical Research Division of Taiho for the support they provided for this work. All research activities were founded by Taiho Pharmaceutical Co., Ltd.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.