Objective: Osimertinib is the 3rd generation EGFR inhibitor, which has been approved for the treatment of NSCLC patients with EGFRT790M. More recently, a tertiary EGFRC797S mutation was reported as the dominant resistance (40~20%) mechanism to Osimertinib. The emergence of C797S mutation prevent covalent bond formation with Osimertinib, and caused the drug resistance. So, it’s an urgent demand for new EGFR inhibitors that can effectively inhibit EGFR triple mutant, d746-750/T790M/C797S & L858R/T790M/C797S. Here, we disclose our clinical candidate, TQB3804, as a novel 4th generation inhibitor that potently inhibits triple mutants.

Methods: The enzyme activities of TQB3804 for EGFRd746-750/T790M/C797S, EGFRL858R/T790M/C797S, EGFRd746-750/T790M, EGFRL858R/T790M, and EGFRWT were measured with corresponding kinase assays. The anti-proliferative activity was evaluated in Ba/F3 (EGFRd746-750/T790M/C797S), NCI-H1975 (EGFRd746-750/T790M/C797S), PC9 (EGFRd746-750), and A431 (EGFRWT) cell lines, and the phosphorylation of EGFR was also evaluated in Ba/F3 (EGFRd746-750/T790M/C797S) cell line. Antitumor activity of TQB3804 was evaluated in three triple mutant cell-derived tumor xenograft (CDX) models Ba/F3 (EGFRd746-750/T790M/C797S), NCI-H1975 (EGFRd746-750/T790M/C797S), and PC9 (EGFRd746-750/T790M/C797S) and one Osimertinib resistant patient-derived xenograft (PDX) model of NSCLC (LUPF104, EGFRd746-750/T790M/C797S).

Results: TQB3804 displayed potent enzymatic activities for EGFRd746-750/T790M/C797S, EGFRL858R/T790M/C797S, EGFRd746-750/T790M, and EGFRL858R/T790M with IC50 of 0.46, 0.13, 0.26, and 0.19 nM respectively, and has similar enzymatic activity for EGFRWT (IC50 = 1.07) to Osimertinib. It also showed expected anti-proliferative activity in 4 cell lines, Ba/F3 (EGFRd746-750/T790M/C797S), NCI-H1975 (EGFRd746-750/T790M/C797S), PC9 (EGFRd746-750), and A431 (EGFRWT), with IC50 of 26.8, 163, 45, and 147 nM, respectively. The phosphorylation for EGFR in Ba/F3 (EGFRd746-750/T790M/C797S) cell line was potently inhibited with IC50 =18.5 nM. TQB3804 significantly inhibited tumor growth in the triple mutant Ba/F3 (EGFRd746-750/T790M/C797S), NCI-H1975 (EGFRd746-750/T790M/C797S), and PC9 (EGFRd746-750/T790M/C797S) CDX models, as well as in the LUPF104 PDX model. Western blot analysis of the tumor samples in the Ba/F3 (EGFRd746-750/T790M/C797S) CDX model showed that TQB3804 inhibited p-EGFR, p-AKT and p-ERK indicating that the tumor growth inhibition was through inhibition of the resistant triple mutant EGFR.

Conclusions: We have identified a potent orally active 4th generation EGFR inhibitor, TQB3804. It can inhibit the activity of Osimertinib resistant triple mutant EGFR, and showed strong antitumor activity in corresponding in vitro and in vivo preclinical assays. These results are considered highly promising and warrant moving the compound forward to clinical investigation.

Citation Format: Xile Liu, Xiquan Zhang, Ling Yang, Xin Tian, Tiantian Dong, Charles Z Ding, Lihong Hu, Lingyu Wu, Lele Zhao, Jun Mao, Qusheng Ji, Shaoyu Yan, Zhenzhen Zhu, Yuanfeng Xia, Chichung Chan, Shuhui Chen. Preclinical evaluation of TQB3804, a potent EGFR C797S inhibitor [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 1320.