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Summary of EGFR inhibitor resistant patient-derived xenografts.

Antibodies used in the study

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) are the standard-of-care treatment for EGFR-mutant non–small cell lung cancers (NSCLC). However, most patients develop acquired drug resistance to EGFR TKIs. HER3 is a unique pseudokinase member of the ERBB family that functions by dimerizing with other ERBB family members (EGFR and HER2) and is frequently overexpressed in EGFR-mutant NSCLC. Although EGFR TKI resistance mechanisms do not lead to alterations in HER3, we hypothesized that targeting HER3 might improve efficacy of EGFR TKI. HER3–DXd is an antibody–drug conjugate (ADC) comprised of HER3-targeting antibody linked to a topoisomerase I inhibitor currently in clinical development. In this study, we evaluated the efficacy of HER3–DXd across a series of EGFR inhibitor–resistant, patient-derived xenografts and observed it to be broadly effective in HER3-expressing cancers. We further developed a preclinical strategy to enhance the efficacy of HER3–DXd through osimertinib pretreatment, which increased membrane expression of HER3 and led to enhanced internalization and efficacy of HER3–DXd. The combination of osimertinib and HER3–DXd may be an effective treatment approach and should be evaluated in future clinical trials in EGFR-mutant NSCLC patients.Significance:EGFR inhibition leads to increased HER3 membrane expression and promotes HER3–DXd ADC internalization and efficacy, supporting the clinical development of the EGFR inhibitor/HER3–DXd combination in EGFR-mutant lung cancer.See related commentary by Lim et al., p. 18

Allelic Frequencies in PC-9 vs PC-9 T790M/C797S cells.

Supplementary Figures and legends

Introduction: EphA2 regulates cell migration, adhesion, proliferation and differentiation, and is overexpressed in human cancers which have been shown to correlate with tumor progression. BCY6033 contains a bicyclic peptide targeting EphA2, linked to the cytotoxin Monomethyl auristatin E via a molecular spacer and cleavable linker designed to target EphA2 expressing tumors. BCY6033 is analogous to BT5528, a BTC™ currently in phase I/II clinical trial in patients with advanced malignancies associated with EphA2 expression. Recent literature indicate that tumor EphA2 expression may be up regulated in EGFR mutant lung models following 1st, 2nd and possibly 3rd generation EGFR inhibitors. Here we report on EphA2 expression in well characterized and clinically annotated patient derived xenografts (PDX) of NSCLC including EGFR mutant NSCLC derived either from erlotinib or osimertinib resistant patients. In vivo activity of BCY6033 is presented in two EphA2 expressing PDX models. Experimental procedures: In vivo, an initial tolerability study was conducted with once weekly administration of BCY6033 by intravenous injection in NSG mice. The activity of BCY6033 was evaluated in EphA2 expressing EGFR mutant PDX models, DFCI-161 and DFCI-220 in NSG mice. A TMA was constructed containing FFPE derived from 69 PDX models (NSCLC n=61, SCLC-transformed n=5, de novo SCLC n=3). Of the 69 models, 35 were EGFR mutant. The TMA was subsequently stained via immunohistochemistry (IHC) using an α-EphA2 (R&D Systems) primary antibody. Tumor membranous H-score was assigned by a pathologist and a score of ≥50 was considered positive. Results: Of the 69 PDXs, 16 were determined to be EphA2 positive. No correlation with genotype or clinical history was observed with EphA2 expression. EphA2 IHC was repeated on at least 5 different tumors for 5 PDX models with fast and robust in vivo growth latency. Two models, DFCI-161 and DFCI-220 with the most reproducible EphA2 staining were selected. Treatment with BCY6033 showed that a dose of 3 mg/kg once weekly administered intravenously was well tolerated in female NSG mice. BCY6033 treatment led to significant tumor growth inhibition with >80% tumor regressions in both DFCI-161 and DFCI-220 PDX models. Re-challenge of tumor out-growth showed that they retain sensitivity to BCY6033 treatment. Conclusion: BCY6033 is a potentially promising drug effective against EphA2 expressing PDX models. Future studies and clinical trials will seek to determine the efficacy of BT5528 in EphA2 expressing tumors. Citation Format: Kenneth Ngo, Elena V. Ivanova, Tyler J. Teceno, Carly Campbell, Johanna Lahdenranta, Stephen J. Blakemore, Gavin Bennett, Pasi A. Jänne, Cloud P. Paweletz, Prafulla C. Gokhale. Activity of the erythropoietin-producing hepatocellular A2 receptor (EphA2) targeting Bicycle® Toxin Conjugate (BTC™) BCY6033 in EGFR inhibitor resistant non-small lung cancer (NSCLC) patient derived xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 333.