Abstract P141: Preclinical characterization of LOX-24350, a highly potent and isoform-selective FGFR3 inhibitor
Authors: Ballard Joshua A. , Kercher Timothy , Abraham David , Brecht Ryan , Brooks Nathan A. , Buckles Thomas , Bume Desta , Busha David , Cedervall Ernst Peder , Condroski Kevin , Ebata Kevin , Gharbi Severine Isabelle , Hazlitt Robert , Morales Tony , Patel Nisha , Podoll Jessica , Urkalan Kaveri , Villalain Sandra Gomez , Walls Shane , Watson Faith , Yang Peiyi , Brandhuber Barbara J. , Andrews Steven W. 
Published: December 2021

Abstract
Alterations in the fibroblast growth factor receptors (FGFRs) have been identified as oncogenic drivers in many human cancers. Specifically, activating FGFR3 gene alterations are found in ~15% of metastatic bladder cancers. One pan-FGFR inhibitor has been approved for patients with FGFR3-altered bladder cancer and others are in clinical development. Importantly, all of these agents inhibit FGFR1-3 with approximate equal potency. Consequently, these agents are associated with toxicities driven by off-target inhibition of FGFR1 and FGFR2, potentially limiting efficacy. Additionally, existing drugs lose potency in the setting of FGFR3 gatekeeper mutations and acquired resistance due to gatekeeper mutations has been described. LOX-24350 is a highly potent and isoform-selective FGFR3 inhibitor with activity against wild-type FGFR3, FGFR3 activating mutations such as S249C, and FGFR3 gatekeeper (V555M) mutations. Here, we describe the preclinical profile of LOX-24350.
Compound potency and selectivity were measured using enzyme fluorescent activity assays, and cell-based assays using in-cell western and cell-titer Glo methods. Tumor growth inhibition and PK/PD studies were performed in mice.
LOX-24350 showed greater than 56-fold selectivity for FGFR3 S249C over wild-type FGFR1 in mechanistic cellular inhibition assays, while maintaining potency for the V555M gatekeeper mutation. In HEK293 cells stably expressing FGFR3 S249C and FGFR3 S249C/V555M, LOX-24350 inhibited FGFR3 phosphorylation with IC50 values of 3.1 and 5.0 nM, respectively, as compared to FGFR1 and FGFR2 IC50 values of 174.5 and 90.7 nM, respectively. Similarly, in NIH3T3 cells engineered to express FGFR3 S249C or FGFR3 S249C/V555M, LOX-24350 inhibited cell growth with IC50 values of 12.2 and 22.9 nM, respectively. LOX-24350’s isoform-selectivity was best exemplified in cancer cell line models, with IC50 values of 15.1 and 12.6 nM in RT112 (FGFR3-TACC3) and UMUC14 (FGFR3 S249C) cell lines, respectively, as compared to 4712.6 nM in DMS114 (FGFR1 amp). LOX-24350 demonstrated high oral bioavailability in preclinical species as well as favorable in vitro ADME properties. In vivo, LOX-24350 demonstrated tumor regressions in FGFR3-driven tumor models on par with pan-FGFR inhibitors, without body weight loss or hyperphosphatemia seen with pan-FGFR inhibitors. This wider therapeutic index is predicted to allow for greater efficacy in patients.
These data demonstrate that LOX-24350 potently and selectively inhibits FGFR3, the S249C activating mutation, and its gatekeeper mutation, V555M, while sparing FGFR1, FGFR2, and other problematic off-targets. We hypothesize that this profile will lead to differentiated efficacy and tolerability for patients with FGFR3-driven cancers. An IND submission is planned for 2022.
Citation Format: Joshua A. Ballard, Timothy Kercher, David Abraham, Ryan Brecht, Nathan A. Brooks, Thomas Buckles, Desta Bume, David Busha, Ernst Peder Cedervall, Kevin Condroski, Kevin Ebata, Severine Isabelle Gharbi, Robert Hazlitt, Tony Morales, Nisha Patel, Jessica Podoll, Kaveri Urkalan, Sandra Gomez Villalain, Shane Walls, Faith Watson, Peiyi Yang, Barbara J. Brandhuber, Steven W. Andrews. Preclinical characterization of LOX-24350, a highly potent and isoform-selective FGFR3 inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P141.