Osimertinib is a next-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) with activity against both the activating and the ‘gatekeeper’ T790M EGFR mutations. An acquired EGFR C797S mutation has been reported to mediate osimertinib resistance in approximately 15% and 7% of patients in second-line and first-line treatment respectively. This percentage in the first-line setting will likely evolve as the first line data mature. The C797S mutation leads to the loss of covalent binding of osimertinib to mutant EGFR. The high affinity of the EGFR triple mutant for ATP presents a challenge for reversible inhibitor design, particularly as the loss of the cysteine at position 797 precludes the previously exploited covalent approaches. We have explored various approaches to address this challenge, including an effort to maximise reversible affinity to target the C797S mutation without requiring a covalent bond. We describe herein the therapeutic potential of reversible phosphine oxide pyrazole inhibitors in tumors harboring C797S.

Using structure-based design, we were able to design a series of phosphine oxide pyrazole inhibitors that displayed exceptionally high biochemical potency against EGFR C797S mutation, which translated into good activity in cell-based assays. Using CRISPR-Cas 9 genome editing technology, we engineered cellular disease-relevant models to express the C797S mutation to evaluate potency in vitro and in vivo.

By modulating the physicochemical properties of our in vitro leads, we were able to achieve good oral exposure of cellularly active EGFR C797S inhibitors such as AZ’7608. We showed that AZ’7608 inhibits signalling pathways and cellular growth of C797S EGFR cell lines in vitro and demonstrated an improved WT EGFR margin. This translated into 52% (p<0.05, at day 14) tumor growth inhibition in vivo when compared to the control group. The efficacy of AZ’7608 is enhanced by its combination with anti-EGFR antibody, showing tumor regression (82%, p<0.001). In addition, we performed pharmacodynamic studies to explore the relationship between efficacy and target/pathway modulation. These studies establish a clear relationship between depth and duration of inhibition of the phosphorylation of EGFR and anti-tumor efficacy.

The work presented herein shows a proof of concept for reversible phosphine oxide pyrazole inhibitors to target tumors harboring C797S. The emergence of the C797S EGFR mutation remains a key area of unmet need and warrants further efforts in drug discovery.

Citation Format: Nicolas Floch, M. Raymond V. Finlay, Ambra Bianco, Sue Bickerton, Nicola Colclough, Darren A. Cross, Emanuela M. Cuomo, Carine M. Guerot, David Hargreaves, Matthew J. Martin, Darren McKerrecher, Daniel J. O’Neill, Jonathan P. Orme, Amar Rahi, Paul D. Smith, Richard A. Ward. Evaluation of the therapeutic potential of phosphine oxide pyrazole inhibitors in tumors harboring EGFR C797S mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4451.