Purpose: Approved therapies for EGFR exon 20, ERBB2 mutations, and NRG1 fusions are currently lacking for non–small cell lung cancer and other cancers. Tarloxotinib is a prodrug that harnesses tumor hypoxia to generate high levels of a potent, covalent pan-HER tyrosine kinase inhibitor, tarloxotinib-effector (tarloxotinib-E), within the tumor microenvironment. This tumor-selective delivery mechanism was designed to minimize the dose-limiting toxicities that are characteristic of systemic inhibition of wild-type EGFR. Experimental Design: Novel and existing patient-derived cell lines and xenografts harboring EGFR exon 20 insertion mutations, ERBB2 mutations and amplification, and NRG1 fusions were tested in vitro and in vivo with tarloxotinib to determine its impact on cancer cell proliferation, apoptosis, and cell signaling. Results: Tarloxotinib-E inhibited cell signaling and proliferation in patient-derived cancer models in vitro by directly inhibiting phosphorylation and activation of EGFR, HER2, and HER2/HER3 heterodimers. In vivo, tarloxotinib induced tumor regression or growth inhibition in multiple murine xenograft models. Pharmacokinetic analysis confirmed markedly higher levels of tarloxotinib-E in tumor tissue than plasma or skin. Finally, a patient with lung adenocarcinoma harboring an ERBB2 exon 20 p.A775_G776insYVMA mutation demonstrated a dramatic clinical response to tarloxotinib. Conclusions: Experimental data with tarloxotinib validate the novel mechanism of action of a hypoxia-activated prodrug in cancer models by concentrating active drug in the tumor versus normal tissue, and this activity can translate into clinical activity in patients.

AA and CPG are co-inventors on patents assigned to Health Innovation Ventures (PCT/WO 2014031012 A1). AH, JT, and LD have shares in the company Convert Pharmaceuticals, and LD has a non-issue, non-licensed patent on LSRT (N2024889). JBS and AVP have served as scientific consultants to Convert Pharmaceuticals. AH, ST, and SD have been prior employees of Convert Pharmaceuticals. PL reports -within and outside the submitted work -grants or sponsored research agreements from Varian Medical, Oncoradiomics, ptTheragnostic/DNAmito, Convert Pharmaceuticals, and Health Innovation Ventures. He received an advisor/presenter fee and/or reimbursements of travel costs/external grant writing fee and/or kind manpower contribution from Oncoradiomics, BHV, Merck, Varian, Elekta, ptTheragnostic, and Convert Pharmaceuticals. PL has shares in the company Oncoradiomics SA, Convert Pharmamaceuticals, LivingMed Biotech, and Comunicare Solutions, and is co-inventor of two issues patents with royalties on radiomics (PCT/NL2014/050248, PCT/NL2014/050728) licensed to Oncoradiomics, one issue patent on mtDNA (PCT/EP2014/059089) licensed to ptTheragnostic/DNAmito, three non-patented inventions (software) licensed to ptTheragnostic/DNAmito and Oncoradiomics and Health Innovation Ventures, and three non-issues, non-licensed patents on Deep Learning-Radiomics and LSRT (N2024482, N2024889, N2024889).