Abstract 2587: A novel covalent inhibitor of mutant but not wild-type (WT) epidermal growth factor receptor (EGFR) has activity in vitro and in vivo in non-small cell lung cancer (NSCLC) models
Authors: Beltran Pedro J. , Zhan Jinghui , Mitchell Petia , Wurz Ryan P. , Pettus Liping , Wu Tian , Chaves Mary , Reid Darren L. , Radinsky Robert , Cooke Keegan , Tasker Andrew 
Published: August 2015

Abstract
Background: Mutations in the EGFR kinase are common in NSCLC. Patients with first-line EGFR mutations (exon 19 deletions, L858R mutations, exon 20 variants, codon 719 variants) initially respond to the reversible EGFR inhibitors erlotinib and gefitinib, but then relapse as additional mutations emerge. EGFR T790M is the most common of these mutations and is found in &gt; 50% of refractory tumors. We assessed the in vitro and in vivo activity of CPD 24, a newly developed, covalent, small-molecule inhibitor of both first-line and T790M mutant EGFR, but not WT EGFR. Inhibition of WT EGFR in normal tissues can be a source of dose-limiting toxicities.
Methods: The effects of CPD 24 were evaluated on H1975 (EGFR T790M/L858R), HCC827 (EGFR exon 19 deletion), and A431 (WT) cell lines. Inhibition of EGFR phosphorylation was measured in serum-starved cells using an MSD assay. Viability was assessed by measuring ATP in cells using a CellTiter-Glo® assay. Dose- and time-dependent pharmacodynamic effects were evaluated by treating mice bearing matrigel plugs containing H1975 cells. Drug levels were measured in plasma samples by LC-MS. CPD 24 was administered to athymic nude mice bearing H1975, HCC827, or A431 xenografts (3, 10, and 30 mg/kg, QD, PO) and tumor growth was measured twice per week with digital calipers. CPD 24 was also formulated in PLGA microspheres and administered subcutaneously (SC) as a sustained release formulation to mice bearing H1975 or A431 xenografts (30 and 100 mg/kg, Q3D, SC). Proliferation of H1975 and HCC827 cells in the presence of CPD 24, growth factors, and a MET inhibitor was measured with an IncuCyte live-cell imaging system.
Results: The IC50 of CPD 24 for p-EGFR inhibition was 4 nM on L858R/T790M mutant EGFR (H1975 cells), 17 nM on the exon 19 deletion EGFR (HCC827 cells), and 510 nM on WT EGFR (A431 cells). CPD 24 inhibited the growth of tumor cells in vitro and led to both dose- and time-dependent p-EGFR inhibition in vivo. Phosphorylation of EGFR in H1975 cells growing in matrigel plugs was inhibited by &gt; 75% for 12 hours by a single 30 mg/kg dose. Tumor growth inhibition (TGI) was observed in two EGFR mutant NSCLC xenograft models (H1975, 89% TGI; HCC827, 139% TGI) but not in a WT EGFR model (A431). CPD 24 formulated in PLGA microspheres achieved drug levels above the IC50 for 72 hours after a single SC dose. Tumor regression was observed with this formulation in H1975 xenografts but not in WT tumors. HGF was able to confer resistance to CPD 24 inhibition in both H1975 and HCC827 cells in vitro. This resistance was prevented by the addition of a selective MET inhibitor.
Conclusion: CPD 24 is a covalent inhibitor of both first-line and T790M mutant EGFR while maintaining &gt; 100-fold selectivity over WT EGFR. The antiproliferative effects of CPD 24 can be reversed by HGF, and this resistance can be prevented by treatment with a MET inhibitor.
Citation Format: Pedro J. Beltran, Jinghui Zhan, Petia Mitchell, Ryan P. Wurz, Liping Pettus, Tian Wu, Mary Chaves, Darren L. Reid, Robert Radinsky, Keegan Cooke, Andrew Tasker. A novel covalent inhibitor of mutant but not wild-type (WT) epidermal growth factor receptor (EGFR) has activity in vitro and in vivo in non-small cell lung cancer (NSCLC) models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2587. doi:10.1158/1538-7445.AM2015-2587