BBT-176, a Novel Fourth-Generation Tyrosine Kinase Inhibitor for Osimertinib-Resistant EGFR Mutations in Non–Small Cell Lung Cancer

First-, second-, and third-generation EGFR inhibitors have substantially improved the survival of patients with advanced, EGFR-mutant non–small cell lung cancer (NSCLC; refs. 1–10). For example, osimertinib, a third-generation, irreversible, and mutant-specific inhibitor, has improved progression-free survival (PFS) in patients with EGFR-mutant NSCLC, in both heavily pretreated and untreated populations (1, 11) and in adjuvant settings (12).

However, acquired resistance invariably emerges after treatment with osimertinib, leading to disease progression (13). The predominant resistance mechanism following osimertinib treatment is a tertiary point mutation at the C797 residue of EGFR, in which the cysteine within the ATP-binding site is substituted with serine and, therefore, prevents the formation of a covalent bond between the mutant EGFR and osimertinib (14, 15). This C797S mutation has been found to account for 10% to 26% of osimertinib-resistant cases in second-line and 7% in first-line settings (16, 17). Furthermore, the C797S mutation also confers cross-resistance to all other third-generation EGFR tyrosine kinase inhibitors (TKI), such as rociletinib and narzartinib due to their similar binding mode, greatly limiting treatment options for these patients (18). The current National Comprehensive Cancer Network (RRID:SCR_012959) guidelines recommend first-line treatment with osimertinib for patients bearing EGFR exon 19 deletion (19Del) or L858R substitution (19). However, after progression on osimertinib, there are no approved targeted therapy options available, and patients must turn to local therapies or systemic chemotherapies for treatment. Anecdotal cases of efficacy from combinations of third- and first-generation EGFR TKI (20) or brigatinib plus cetuximab have been reported (21) but do not represent generally accepted practice.

Currently, the combination of amivantamab, a bispecific antibody to EGFR and c-MET (mesenchymal–epithelial transition factor), and lazertinib, a third-generation EGFR TKI, is being investigated in multi-national phase I clinical trials (CHRYSALIS, NCT02609776; CHRYSALIS-2, NCT04077463), and promising early data have been presented (22). Other clinical trials exploring the benefit of immuno-oncology therapeutic strategies such as immune checkpoint inhibitors are ongoing (e.g., KEYNOTE 789, NCT03515837; CHECKMATE 722, NCT02864251), but data on their effectiveness and safety for treating NSCLC are not yet available.

For patients with NSCLC with disease progression on osimertinib, there is an urgent need for a next-generation EGFR TKI that is active against C797S-containing mutations. Preclinical evaluation of BLU-945, a fourth-generation EGFR TKI, has shown antitumor activity in an in vivo model of NSCLC (23) and is currently under clinical investigation either as monotherapy or in combination with osimertinib (NCT04862780).

BBT-176, a novel, orally available fourth-generation EGFR TKI was designed to selectively and noncovalently inhibit triple-mutant EGFR 19Del/T790M/C797S and L858R/T790M/C797S at nanomolar concentrations. Preclinical data from engineered Ba/F3 cells and patient- and cell-derived xenografts demonstrated the effectiveness of BBT-176 across single-, double-, and triple-mutant models. Here we report the preclinical findings of BBT-176 that prompted its evaluation in clinical trials as monotherapy in patients with EGFR-mutant NSCLC previously treated with at least one EGFR TKI (NCT04820023). We also introduce the first-in-human trial of BBT-176, and report two clinical cases of patients from this trial who showed clinical response by both circulating tumor DNA (ctDNA) analyses and conventional radiologic imaging.

The murine pro-B cell line Ba/F3 (RCB0805) and myelomonocytic, macrophage-like, BALB/C mouse leukemia cells (WEHI-3) were provided by the RIKEN Bio Resource Center (Tsukuba, Japan). Ba/F3 cells were maintained in RPMI1640 medium (Fujifilm Wako Pure Chemical Corporation), supplemented with 10% FBS (Gibco BRL), 1% penicillin-streptomycin (Fujifilm Wako Pure Chemical Corporation), and conditioned media from WEHI-3 (10%) as a source of IL3, and cultured at 37°C in a humid atmosphere with 5% CO₂. Cell lines A-431, A549, NCI-H1299, and NCI-H2073 were obtained from ATCC, and the LoVo cell line was obtained from European Collection of Authenticated Cell Cultures. BBT-176 was synthesized by Wuxi AppTec (Changzhou, China). First- to third-generation EGFR TKI [erlotinib (catalog No. S7786), afatinib (S1011), dacomitinib (S2727), and osimertinib (S7297)], brigatinib (S8229; a multi-kinase inhibitor active against mutant EGFR), and the anti-EGFR antibody cetuximab (A2000) were purchased from Selleck Chemicals; cetuximab was stored at 4°C. Each TKI was dissolved in dimethylsulfoxide (DMSO; Sigma-Aldrich) and preserved at −80°C.

Studies to determine the EGFR kinase half maximal inhibitory concentration values (IC₅₀) were performed by Carna Biosciences, Inc. using a human recombinant EGFR kinase protein and an off-chip mobility shift assay (MSA). Test and reference compounds were diluted to experimental concentrations as described in Supplementary Appendix S1A. For the in vitro EGFR kinase activity measurement, 20 μL of solution at experimental concentration was incubated in a polypropylene 384-well microplate for 1 hour at room temperature. After incubation, 70 μL of termination buffer (QuickScout Screening Assist MSA; Carna Biosciences) was added, the sample was applied to the LabChip system (Perkin Elmer), and the kinase reaction was evaluated.

The IC₅₀ values of BBT-176 against EGFR, EGFR T790M/L858R, INSR, TNK1, MAP4K2, and PKN1 kinases were measured by Carna Biosciences, Inc. using an off-chip MSA. The IC₅₀ values of BBT-176 against other 76 kinases were measured by Eurofins-Cerep SA using a radiometric assay.

Each EGFR mutation was introduced into the Ba/F3 cells by retroviral gene transfer as previously described (24, 25). A brief description is included in Supplementary Appendix S1B.

A total of 2 × 10³ cells were seeded in each well of 96-well plates. After 24 hours, EGFR TKI were added at the indicated concentrations. After a 72-hour incubation, 10 μL of the Cell Counting Kit-8 solution (Dojindo Laboratories) was added to each well, and the plates were incubated for an additional 3 hours. Absorbance at 450 nm was measured using a multiplate reader (Tecan). The percentage of viable cells was evaluated and compared with those of DMSO-treated controls or PBS-treated controls.

BBT-176-resistant clones were established by N-Ethyl-N-nitrosourea (ENU, Sigma-Aldrich) mutagenesis, as previously described (26). A brief description is included in Supplementary Appendix S1C.

Ba/F3 cells with EGFR mutations were treated with BBT-176 with or without cetuximab at the indicated concentrations for 6 hours. The cells were then washed twice with PBS and resuspended in lysis buffer. Lysates were quantified using a BCA protein assay (Bio-Rad) and were electrophoresed and transferred to polyvinylidene difluoride membranes. Immunoblotting was performed according to the antibody manufacturers’ instructions. Antibodies were purchased from Cell Signaling Technology and are listed in Supplementary Appendix 1D. Immunoblots were scanned using an Amersham Imager 680 (GE Healthcare).

For Ba/F3 EGFR 19Del/C797S and EGFR 19Del/T790M/C797S xenograft models, each mouse was inoculated subcutaneously in the right flank with cells (0.5 × 10⁶) in 0.1 mL of PBS supplemented with BD Matrigel (1:1) for tumor development. The animals were randomized, and treatment was started when the average tumor volume reached 150 to 180 mm³ for the efficacy study. Ba/F3 EGFR xenograft studies were performed by Wuxi AppTec (Shanghai, China).

For PDX models, fresh tumor tissues from mice bearing established primary cancer tissues were harvested and cut into small pieces (∼2–3 mm in diameter). Tumor fragments were inoculated subcutaneously at the upper right dorsal flank into corresponding female BALB/c nude mice or female nu/nu mice, ages 35–42 days, for tumor development. The randomization started when the mean tumor size reached approximately 150 to 200 mm³. Implanted mice were treated with vehicle or BBT-176 (60 and 90 mg/kg, N = 8), once daily by oral gavage for the indicated time period. PDX studies were performed by LIDE Biotech (Shanghai, China).

Tumor volumes were measured twice weekly in two dimensions using a caliper, and the volume was expressed in mm³ using the formula: volume = 0.5 a × b² where a and b are the long and short diameters of the tumor, respectively. The tumor sizes were then used for the calculations of T/C (treatment/control) values. The tumor volume was used for calculation of tumor growth index (TGI) of each group according to the following formula: TGI (%) = [1 – (T_ti – T_t0)/(V_ci – V_c0)] × 100. The body weight of mice was measured daily for the dosing phase, and the relative change of body weight (RCBW) of each mouse according to the following formula: RCBW (%) = (BW_i – BW₀)/BW₀ × 100.

After the end of the patient-derived EGFR 19Del/T790M/C797S xenograft study, tumor tissue samples were collected from the mice and cut to about 30–100 mg and then were placed in a 2-mL microcentrifuge tube, adding the protein extraction buffer (Cell Signaling Technology) with protease inhibitors and grinding the tumors with Tissuelyser (Qiagen) at 50 Hz for 5 minutes. The same amount of protein (30 μg) was then obtained from each suspension and subjected to 10% SDS–PAGE after which the separated proteins were transferred to a nitrocellulose membrane. After blocking with buffer containing 2.5% skim milk, the membrane was incubated overnight with 1:1,000 primary antibodies at 4°C. Blots were then washed and incubated with horseradish peroxidase (HRP)-anti-rabbit or HRP-anti-mouse antibodies at 1:2,000 dilution.

All contracted study protocols were reviewed and approved by Bridge Biotherapeutics Inc. All contracted experiments were designed, supervised, and audited by employees of Bridge Biotherapeutics Inc. The protocol and any amendments or procedures involving the care and use of animals in this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Wuxi AppTec or LIDE Biotech prior to conduct. During the study, the care and use of animals were conducted in accordance with established national and international regulations for laboratory animal protection, namely the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Informed consent was obtained from all patients in accordance with established national and international standards prior to use of tissue samples.

The IC₅₀ values were determined by a nonlinear regression curve fit utilizing a variable slope model with normalized response in GraphPad Prism version 8 (GraphPad Software). Charts were created in Microsoft Excel (Microsoft; bar charts) and GraphPad Prism (all other charts). Where indicated, the significance of difference between indicated groups is calculated by Student t test. For the in vivo efficacy study, all data were described as mean ± SEM. One-way ANOVA was performed to compare the tumor volume of each group with vehicle group. All data were analyzed with GraphPad Prism 8, with statistical significance defined as P < 0.05 between groups.

A first-in-human phase I study is ongoing to evaluate the safety of BBT-176, including a dose-escalation phase to characterize dose-limiting toxicities (DLT) and determine the recommended phase II dose (NCT04820023); preliminary data have been presented elsewhere (27). Eligibility criteria included advanced-stage NSCLC with an activating EGFR mutation and disease progression on at least one prior EGFR inhibitor. A measurable lesion was required for exploratory evaluation of efficacy. Starting from 20 mg once daily, BBT-176 dose levels were escalated after evaluating dose–DLT relationships and toxicity probabilities during the first 21 days of dosing. All enrolled patients underwent circulating tumor DNA (ctDNA) analysis (Guardant360, Guardant Health) at screening and every 6 weeks throughout treatment, which was compared to imaging at the same time points. Changes in allelic frequency were calculated as per the proprietary algorithm of Guardant Health (28). Intrapatient dose-escalation to the next dose level was allowed. Plasma samples were collected at prespecified time points at cycle 2, day 1 following multiple doses (steady state) of BBT-176 in the dose-escalation cohorts, and the plasma concentrations were determined by a validated LC/MS-MS method with the LLOQ established at 1 ng/mL. The trial was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines. All applicable regulatory requirements were fulfilled, and the protocol was approved by an Ethics Committee at all participating sites. All participants provided written informed consent before undergoing any study procedures or sharing any data, imaging, and tissue for the study.

Data will be made available to other investigators upon reasonable request to the corresponding authors. Small-molecule crystallographic data for BBT-176 have been submitted to the Cambridge Crystallographic Data Centre (https://www.ccdc.cam.ac.uk/structures/Search?ccdc=2261855).

We developed a novel series of reversible small-molecule inhibitors and identified BBT-176 as a potent inhibitor of C797S-related mutant forms, including EGFR 19Del/T790M/C797S, 19Del/C797S, and L858R/C797S. BBT-176 was designed as a reversible ATP-competitive inhibitor to overcome the drug resistance and lack of potency of osimertinib against mutant EGFR. BBT-176 has a distinct binding mode into triple mutant (19Del/T790M/C797S or L858R/T790M/C797S) EGFR compared to osimertinib. The structure and binding interactions of BBT-176 are visualized in Fig. 1. The pyrimidine moiety of the BBT-176 core structure forms two conventional hydrogen bonds with Met793 of the hinge region, the piperidine moiety forms salt bridges with Glu904 and Asp800, and multiple hydrophobic interactions of aromatic rings occur. The differences in spatial position of Lys745, Asp855, and Gly857 are observed in the active and inactive kinase domain (29). In the inactive state of EGFR, the lysine forms a salt bridge with Asp855 of the DFG motif and with Glu762 of the αC-helix. In contrast, in the active state of EGFR, Asp855 and Glu762 move, resulting in a missing interaction with Lys745. The additional interaction between sulfone group of BBT-176 and Lys745 could lead to stronger potency against triple-mutant EGFR and thus selectivity versus wild-type.

At K_m ATP concentration, BBT-176 exhibited single-digit nanomolar potencies in biochemical assay with EGFR 19Del/T790M/C797S (IC₅₀ = 1.79 nmol/L), 19Del/C797S (IC₅₀ = 4.36 nmol/L) and L858R/C797S (IC₅₀ = 5.35 nmol/L), whereas IC₅₀ values for osimertinib for these mutants were approximately 100-fold higher (Fig. 2A–C). To further characterize BBT-176, we tested whether BBT-176 is an ATP-competitive inhibitor. Using different ATP concentrations in the biochemical assay (Fig. 2D–F), the inhibitory activity of BBT-176 against EGFR 19Del/T790M/C797S, EGFR L858R/T790M/C797S, and L858R/C797S was significantly reduced at 1 mmol/L ATP concentration, suggesting that BBT-176 is an ATP-competitive inhibitor.

To evaluate off-target effects of BBT-176, it was tested against 82 kinases; their IC₅₀ values are presented in Supplementary Table S1. In addition to EGFR T790M/L858R, BBT-176 was active against ALK, JAK2, and others.

Next, we evaluated the activity of BBT-176 along with the first- and second-generation EGFR TKI against Ba/F3 cells carrying EGFR mutations (19Del or L858R), five exon 20 insertion mutations, EGFR TKI-mediated resistance mutations (EGFR T790M, EGFR C797S, EGFR T790M/C797S), and patient-derived lung cancer cell lines and determined the IC₅₀ of each drug by cell growth inhibition assays. BBT-176 showed strong activities against EGFR 19Del-based mutations (19Del/C797S and 19Del/T790M/C797S in Fig. 3A, 19Del and 19Del/T790M in Supplementary Fig. S1), with IC₅₀ values lower than 50 nmol/L (Fig. 3B). Among the drugs evaluated, only BBT-176 had an IC₅₀ value below 50 nmol/L for all of these mutations. The activity of BBT-176 against EGFR L858R–based mutations (L858R/C797S and L858R/T790M/C797S in Fig. 3A, L858R and L858R/T790M in Supplementary Fig. S1) was less potent than its activity against the 19Del-based mutations, with all IC₅₀ values higher than 100 nmol/L (Fig. 3B). BBT-176 showed low potency against five EGFR exon 20 insertion mutation models with IC₅₀ values higher than 200 nmol/L (Supplementary Fig. S2). The IC₅₀ of BBT-176 against wild-type EGFR was 10- and threefold higher than those against EGFR 19Del and L858R mutants, respectively. In addition, BBT-176 showed weaker antiproliferation activities (IC₅₀ range, 300 nmol/L–1 μmol/L) compared with osimertinib in various cell lines harboring wild-type EGFR (Fig. 3C and D).

To analyze potential mutations in EGFR that would reduce the activity of BBT-176, we performed ENU mutagenesis screening. The principle of this mutagen-based resistant mutation screening is as follows: Ba/F3 cells, which depend only on a specific driver mutation for their survival and proliferation, are exposed to ENU, a potent mutagen that induces a variety of point mutations (26, 30). By adding a growth-inhibitory agent such as an EGFR-TKI, only cells with mutations in the binding site of that drug will survive. Because (i) the activity of BBT-176 against Ba/F3 cells carrying EGFR L858R–based mutations was lower than that those against EGFR 19Del–based mutations and (ii) the growth-inhibitory activity against IL3-dependent parental Ba/F3 cells was also observed at about 1,000 nmol/L of BBT-176 (Fig. 3B), the effective range for blocking EGFR L858R–mediated signals is narrow. Therefore, we decided to focus on analyzing the resistance mechanism for EGFR 19Del–based mutations (19Del, 19Del/C797S, and 19Del/T790M/C797S) in this study.

By subjecting Ba/F3 cells to ENU mutagenesis, we successfully generated multiple resistant clones that exhibited increasing BBT-176 concentrations shown in Fig. 4A. The full-length EGFR sequence was analyzed from RNA extracted from resistant clones, but none showed additional mutations in the EGFR gene. This result is contrary to the results of ENU mutagenesis studies with other TKI, where most resistant clones showed additional mutations in the tyrosine kinase domain (25, 31). For each mutation, two resistant clones obtained at the highest concentrations of BBT-176 were isolated, and their sensitivity to BBT-176 was measured (Fig. 4B and C). The IC₅₀ of BBT-176 against each resistant clone increased compared with those against each parental cell. However, the increase was mild in each case (within two- to fivefold)_. Furthermore, osimertinib was still active against EGFR, 19Del–derived resistant clones (Supplementary Fig. S3). These results indicate that the resistance observed did not arise from a secondary mutation in EGFR, and the resistant cells were still dependent on EGFR signaling for survival. Western blot analysis showed the enhanced levels of phosphorylated EGFR (p-EGFR) in the BBT-176-resistant Ba/F3 cells with the 19Del/T790M/C797S mutation (Fig. 4D).

Previous preclinical and clinical case studies have shown that the anti-EGFR antibody cetuximab enhanced the activity of first- or second-generation EGFR TKI (32, 33). Therefore, we evaluated the activity of BBT-176 in combination with cetuximab. We first evaluated the activity of cetuximab as a monotherapy against Ba/F3 cells carrying various mutations. Cetuximab showed growth-inhibitory activity against EGFR 19Del, L858R, 19Del/C797S, L858R/C797S, and L858R/T790M/C797S cells, with IC₅₀ values lower than 1 μg/mL, but not against EGFR 19Del/T790M, and L858R/T790M cells, which had IC₅₀ values higher than 100 μg/mL (Supplementary Fig. S1). Unlike a previous report (34), cetuximab as monotherapy showed potent activity against Ba/F3 cells carrying the EGFR 19Del mutation in our study. Subsequently, we evaluated the activity of BBT-176 in the presence of cetuximab (10 μg/mL). Enhanced activity of BBT-176 was observed, especially against 19Del/T790M/C797S with an IC₅₀ value lower than 1 nmol/L, and the IC₅₀ values of BBT-176 combined with cetuximab were 50-fold lower compared with BBT-176 alone (Fig. 5A). Western blot analysis for Ba/F3 cells with EGFR 19Del/T790M/C797S showed the potentiated inhibition of downstream signaling in the combination treatment (Supplementary Fig. S4). The combination effect of osimertinib and cetuximab against EGFR 19Del/T790M/C797S was also evaluated, but the decrease in IC₅₀ values was only about half (Fig. 5A). BBT-176 combined with cetuximab synergistically suppressed the growth of EGFR 19Del/T790M/C797S–expressing cells, whereas no synergistic benefit was obtained when combined with osimertinib (Fig. 5B). Furthermore, we tested the combination of BBT-176 and cetuximab against the resistant clones obtained by the ENU mutagenesis. Cetuximab alone was able to overcome Ba/F3 cells carrying EGFR 19Del or 19Del/C797S-derived BBT-176–resistant clones as well as in parental cells (Supplementary Fig. S1 and Supplementary Fig. S5). In BBT-176–resistant Ba/F3 cells expressing EGFR 19Del/T790M/C797S, the enhanced activity of BBT-176 combined with cetuximab was observed, with IC₅₀ lower than 5 nmol/L and a synergistic suppression of growth (Fig. 5C and D).

To determine whether BBT-176 is effective in animal models, we tested BBT-176 in mice bearing Ba/F3 EGFR 19Del/C797S or Ba/F3 EGFR 19Del/T790M/C797S xenografts. As shown in Fig. 6A, BBT-176 suppressed tumor growth in a dose-dependent manner with complete inhibition at 90 mg/kg in the Ba/F3 EGFR 19Del/C797S xenograft. In combination with osimertinib, BBT-176 at 90 mg/kg also resulted in complete tumor growth inhibition in Ba/F3 EGFR 19Del/T790M/C797S xenografts (Fig. 6A). In the vehicle-treated groups, both Ba/F3 EGFR 19Del/C797S and Ba/F3 EGFR 19Del/T790M/C797S xenografts grew continuously, and osimertinib did not block the tumor growth even at 25 mg/kg, which indicates osimertinib was not active for EGFR 19Del/C797S and EGFR 19Del/T790M/C797S.

The antitumor efficacy of BBT-176 against EGFR 19Del/T790M/C797S was also confirmed in an LD1–0025–200717 EGFR 19Del/T790M/C797S PDX model. The LD1–0025–200717 PDX model was derived from a patient who went through seven lines of therapy, including chemotherapy, erlotinib, and osimertinib. As shown in Fig. 6B (left), once-daily dosing of BBT-176 induced significant tumor growth inhibition, with complete tumor growth inhibition observed at dose of 90 mg/kg/day and only a small decline in body weight (Supplementary Fig. S6A). The tumor regression with only a small decline in body weight was also observed after the administration of 60 mg/kg of BBT-176 in an LU1235 EGFR 19Del PDX model (derived from a patient with poorly differentiated adenocarcinoma) for 21 days (Supplementary Fig. S6B). As shown in Fig. 6B (right), 66.5% to 77.5% of inhibition of p-EGFR was observed in tumor samples from 90 mg/kg of BBT-176 or 90 mg/kg of BBT-176 and 5 mg/kg of osimertinib combination groups. These results clearly indicate BBT-176 has potent therapeutic efficacy against EGFR 19Del/C797S, EGFR 19Del/T790M/C797S, and EGFR 19Del disease models.

An earlier summary of phase I data of BBT-176 in patients has been reported previously (27). At the time of the present publication, 25 patients were treated with BBT-176 in dose-escalation cohorts of a once-daily schedule, with good overall tolerance and a safety profile comparable to other members of the EGFR inhibitor class (Supplementary Table S2). Treatment-related adverse events of grade ≥3 occurred in patients treated with BBT-176 320 mg once daily. Gastrointestinal toxicities (diarrhea, nausea, vomiting) were the most common adverse events. Skin toxicities were mild, with no grade ≥3 events reported. Asymptomatic hematologic toxicities of grade ≥3 were observed in high doses (≥480 mg once daily). At the time of publication, dose escalation is still in progress starting from a dose of 160 mg taken twice daily. Baseline characteristics and treatment response data are provided in Supplementary Table S3 and Supplementary Fig. S7, respectively. Supplementary Table S4 shows the study representativeness of the target population.

A 52-year-old female patient from Korea had been treated with gefitinib, erlotinib, and osimertinib since April 2019 for stage IV lung adenocarcinoma. At diagnosis, EGFR 19Del was found, and subsequently T790M was also detected. She was enrolled in the phase I trial (dose-escalation phase) and was allocated to the BBT-176 320 mg once-daily cohort. The dose was reduced to 160 mg once daily because of an erythematous skin rash on her forearms. Analysis of ctDNA showed a triple-mutant clone of EGFR E746_A750Del/T790M/C797S (Supplementary Fig. S8A and S8B). Unexpectedly, JAK2 V617F was also found in blood, which was attributable to the coincidental hematologic disease of myeloproliferative neoplasm. Platelet count was more than 500,000/mm³ in this patient prior to the administration of BBT-176; therefore, this condition was judged to be concomitant unrelated disease, rather than a BBT-176–related adverse event. Considering the potential gains and risks of treatment, the investigator opted to continue, and after 6 weeks of treatment, a CT scan confirmed considerable improvement in nontarget lesions (left pleural effusion and consolidation in the left lung upper lobe; Fig. 7A), while ctDNA analysis found allelic frequency values were stable. A CT scan at week 12 showed further improvements, the investigator's response evaluation indicated a status of stable disease, and the independent central radiology reviewers confirmed partial response since the first follow-up (target lesion shrinkage in the left lung mediastinal lymph node) by Response Evaluation Criteria in Solid Tumors (RECIST) v.1.1 standards.

A 53-year-old male patient from Korea had been treated with afatinib, osimertinib, and chemotherapy for stage IV lung adenocarcinoma since August 2018. At diagnosis, both EGFR 19Del and T790M were detected. He was enrolled in the phase I trial and was allocated to BBT-176 480 mg QD. Analysis of ctDNA reported the existence of EGFR variant alleles of E746_A750del, T790M, L792H, and C797S, and allelic context analysis revealed that both L792H and C797S were linked mutually exclusively to T790M (Supplementary Fig. S9A and S9B). These data show that two clones of triple mutants—EGFR E746_A750del/T790M/L792H and EGFR E746_A750del/T790M/C797S—coexisted in this patient's blood. After 6 weeks of treatment, CT scan showed shrinkage of the target lesion (subcarinal lymph node conglomerates, 26.3% reduction by the short axis diameter, as assessed by the independent central radiology reviewers) with central density reduction; another upper mediastinal lymph node lesion showed similar changes (Fig. 7B). In this case, molecular changes were more prominent than radiologic tumor shrinkage. To investigate the sensitivity of this mutant to BBT-176 in vitro, we also conducted an enzyme-based assay for L792H-containing triple mutant EGFR (19Del/T790M/L792H), which showed an IC₅₀ value of 13.1 nmol/L (Supplementary Fig. S9C).

Plasma concentration profiles of BBT-176 in cases 1 and 2 are shown in Fig. 7C. The plasma concentrations of BBT-176 at steady state exceeded IC₉₀ (in vitro Ba/F3 cell IC₉₀) for 24 hours, with the AUC_last of 17,000–58,000 ng.hr/mL.

Osimertinib, a third-generation EGFR inhibitor, is indicated as a first- or second-line therapy for untreated NSCLC patients or those who have experienced progression on EGFR inhibitors due to EGFR T790M-mediated resistance, respectively. However, its clinical efficacy is limited by the development of acquired drug resistance, of which EGFR C797S is one of the common mechanisms identified to date (13). Even though the sequence of mutations may differ between patients receiving gefitinib/erlotinib or osimertinib as first-line treatment, both patient populations are expected to eventually develop the C797S-containing triple mutants (EGFR 19Del/T790M/C797S or EGFR L858R/T790M/C797S). For such patients, there are no effective treatment options; in addition to BBT-176, other EGFR C797S inhibitors in development include TQB3804 (35), CH7233163 (36), JBJ-04–125–02 (37), BLU-945 (38) and BLU-701 (39).

Here we describe the characterization and early clinical development of BBT-176, a novel oral, reversible, fourth-generation EGFR TKI. Both preclinical and preliminary clinical data suggest a BBT-176 may have favorable efficacy in patients with EGFR C797S–mediated resistance. We demonstrated that BBT-176 is a highly potent inhibitor of EGFR C797S mutants in biochemical and cellular assays and has broad selectivity over wild-type EGFR. BBT-176 showed significant antitumor activities in various in vivo cell-derived xenografts and PDX models harboring the EGFR 19Del/T790M/C797S mutation. Furthermore, we have also observed tumor shrinkages in two patients from the 320-mg once-daily and 480-mg once-daily cohorts after 6- and 12-week treatments of BBT-176, respectively. In ctDNA analyses (liquid biopsy) of the two cases presented, we could not find non-EGFR mechanisms of acquired resistance. Therefore, it is unknown whether BBT-176 may work against tumors with non-EGFR mechanisms of acquired resistance. In the ongoing phase I trial, we are assessing subjects to detect other druggable mutations. Further research is needed to understand the potential ability of BBT-176 to overcome clonal heterogeneity in acquired resistance to osimertinib; however, our current research is focused on EGFR mutations.

Secondary mutations on the target protein are frequently reported to limit the clinical benefit of TKI (40, 41). To explore mechanisms of resistance that may reduce BBT-176 activity, we performed an ENU mutagenesis screen, a technique widely used to identify potential secondary resistance mutants (25, 31). Using this method, we previously identified clinically relevant mutations driving resistance to EGFR, HER2, KRAS, and MET inhibitors and proposed treatment strategies to overcome these (26, 31, 42–45). For example, we reported that EGFR L792H and EGFR C797S mutations could occur as resistance mechanisms to afatinib and dacomitinib respectively before they were reported clinically (26, 42). In this experiment, we were unable to detect EGFR resistance mutations to BBT-176, suggesting that genomic point mutations in EGFR are not likely to induce resistance to BBT-176. The enhanced EGFR signaling is possibly due to the increased EGFR homo- or hetero dimerization. Recently, it was reported that the decrease of EGFR expression or remodeling of EGFR protein conformation by cetuximab remarkably enhanced EGFR TKI activity against C797S mutants (20, 32, 37). We therefore evaluated the combination of cetuximab and BBT-176 in BBT-176–resistant EGFR 19Del/T790M/C797S clones, where cetuximab significantly enhanced BBT-176 activity. Despite promising preclinical findings with the combination of BBT-176 with cetuximab, this was not explored in clinical studies because of the concern of overlapping toxicities of BBT-176 and cetuximab that may make the precise evaluation of safety and dose-finding difficult. Because we did not sequence the whole genome of the resistant clones, we cannot exclude other potential resistance mutations or gene alterations affecting EGFR family members, such as HER2. We are aware that HER2-targeting approaches are emerging in clinical practice in addition to EGFR-targeting therapies. Recently, trastuzumab deruxtecan was approved in the United States for HER2-mutant NSCLC (46). However, the FDA denied the request for accelerated approval of poziotinib for HER2-mutant NSCLC (47). Currently, the potential role of such HER2-targeting therapies in acquired resistance to EGFR-mutant NSCLC remains an open question. The combination of BBT-176 and osimertinib did not show synergistic antiproliferation activity against BBT-176-resistant clones of EGFR 19Del/C797S and 19Del/T790M/C797S mutants (Supplementary Fig. S10A–S10C).

Various new options to address the challenge of acquired resistance to osimertinib are being evaluated. Considering only novel drugs targeting EGFR and excluding the combination of approved drugs, there are several approaches: (i) allosteric kinase inhibitors, for example, JBJ-09–063 (48), (ii) catalytic kinase inhibitor with enhanced coverage, for example, BBT-176, (iii) antibody–drug conjugates targeting the extracellular domain of oncogenic receptor tyrosine kinase, for example, patritumab deruxtecan (49), and (iv) combinations of antibody and small-molecule kinase inhibitors, for example, amivantamab with lazertinib (50). At the time of this writing, there are no drugs approved by the FDA specifically for the treatment of EGFR mutation–positive NSCLC previously treated with a third-generation EGFR TKI.

In our study, we have observed that drug response correlated with ctDNA response. The interpretation of findings from ctDNA and their application to clinical decision-making is an area of emerging interest. In addition to detection of EGFR T790M and C797S, which are the targets, dynamic monitoring of allele frequency may also assist clinicians. Currently, there is no consensus about how to interpret changes in allelic frequency during treatment, although a number of clinical trials have reported such data (51, 52).

Despite the promising early clinical data of BBT-176, unanswered questions remain. The dose-finding study of BBT-176 is still ongoing, and although early data show encouraging signals of efficacy in a small population, the dosing regimen that produces the optimal balance of deep and durable responses with an acceptable tolerability profile is yet to be determined. Currently, dose-escalation with a twice-daily dosing sequence is ongoing, and we expect further clinical insights to come from that study. Following determination of the optimal dosing schedule, an expansion phase of the clinical study is planned.

The ongoing clinical trial with BBT-176 will provide insights into its potential role, and the preliminary clinical data included in this article justify further exploration in a larger population of molecularly defined disease entities.

T. Fujino reports grants from Bridge Biotherapeutics during the conduct of the study, grants from Grants-in-Aid for scientific research from the Japan Society for Promotion of Science, grants from Apollomics, and personal fees from Novartis outside the submitted work. C. Kim reports grants from Korea Drug Development Fund during the conduct of the study and grants from Korea Drug Development Fund outside the submitted work. Y. Lee reports grants from KDDF during the conduct of the study. D. Kim reports grants and personal fees from Bridge Bio Therapeutics during the conduct of the study; grants from Alpha Biopharma, Mirati Therapeutics, ONO Pharmaceutical, TP Therapeutics, and Xcovery; grants and nonfinancial support from Takeda, Amgen, AstraZeneca, Boehringer-Ingelheim, Daiichi-Sankyo, Janssen, Merus, Novartis, Pfizer, Roche/Genentech, Yuhan, Chong Keun Dang, GSK, and MSD outside the submitted work. S.J. Ahn reports personal fees from Boryung, BC World, Takeda Phar, Roche Korea, Menarini Korea, Pfizer, Lily Korea, Boehringer Ingelheim, Kyowa Kirin, Amgen Korea, Yuhan, AstraZeneca Korea, Bayer Korea, Novartis Korea, Hanmi, Therapex, Guardant, Immuneoncia, and PharmbioKorea outside the submitted work. T. Mitsudomi reports personal fees from AstraZeneca, BMS, MSD, Daiichi-Sankyo, Amgen, Eli-Lilly, and Takeda outside the submitted work; grants and personal fees from Boehriner Ingelheim, Chugai, Pfizer, and Taiho; grants from Bridge Biopharma; and personal fees from Novartis during the conduct of the study. S. Lee reports other support from Bridge Biotherapeutics, Inc. and grants from Korea Drug Development Fund during the conduct of the study, and has a patent for Clinical Utility Application of BBT-176 pending. S.M. Lim reports grants from Bridge Bio Therapeutics, Amgen, AstraZeneca, Boehringer-Ingelheim, Daiichi-Sankyo, Gilead, Janssen, J Ints Bio, Roche, Takeda, Therapex, and Yuhan. No disclosures were reported by the other authors.

S. Lim: Resources, supervision, writing–original draft, project administration, writing–review and editing. T. Fujino: Writing–review and editing. C. Kim: Formal analysis, validation, investigation, visualization, methodology, writing–original draft. G. Lee: Resources, data curation, investigation, visualization, methodology, writing–original draft, writing–review and editing. Y. Lee: Resources, data curation, formal analysis, supervision, visualization, methodology, writing–original draft, writing–review and editing. D. Kim: Resources, investigation, methodology, writing–original draft, project administration, writing–review and editing. J. Ahn: Resources, investigation, methodology, writing–original draft, project administration, writing–review and editing. T. Mitsudomi: Resources, formal analysis, methodology, writing–original draft, project administration, writing–review and editing. T. Jin: Resources, data curation, software, formal analysis, methodology, writing–original draft, project administration, writing–review and editing. S. Lee: Conceptualization, resources, data curation, software, supervision, validation, investigation, visualization, methodology, writing–original draft, project administration, writing–review and editing.

The research presented in this manuscript and the medical writing support received were made possible by funding from Bridge Biotherapeutics. The ongoing clinical study of BBT-176 is partially supported by the Korea Drug Development Fund (HN21C0859), which is funded by the Ministry of Science and ICT, Ministry of Trade, Industry, and Energy, and Ministry of Health and Welfare of the Republic of Korea. Additionally, a portion of the preclinical work was funded by the National Research Foundation of Korea (2022R1A2B5B02001403), which is funded by the Ministry of Science and ICT of the Republic of Korea. We thank all collaborators involved in material manufacturing as well as in vitro and in vivo experiments. We also acknowledge the medical writing assistance provided by MediPaper Medical Communications Ltd, Hong Kong SAR, China.

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