Abstract 1960: CFON-026 is a potent non-covalent BTK inhibitor suitable for combination therapy with covalent BTK inhibitors for early eradication of resistance mutations
Authors: de Man Jos , Muller Michelle , Uitdehaag Joost C.M. , van Cauter Freek , van Gemert Sander , Hoffmann Milan , van Mil Yvonne G.T.H. , Mulder Winfried R. , Prinsen Martine B.W. , Sterrenburg Jan Gerard , Vu Diep , de Wit Joeri , Ensing Erik , Buijsman Rogier C. 
Published: March 2024

Abstract
Bruton’s Tyrosine Kinase (BTK) inhibitor drugs are a mainstay in the treatment of CLL, WM and MCL. They are not curative because of the emergence of BTK inhibitor resistance mutations in as much as 67% of relapsed patients (in CLL) [1]. Frequently observed mutations include BTKC481S, which abrogates covalent binding of irreversible drugs such as acalabrutinib, ibrutinib and zanubrutinib, and BTKT474I and BTKV416L which both abrogate binding of the non-covalent inhibitor drug pirtobrutinib [1, 2]. To combat resistance, third generation BTK inhibitors are needed, which must 1. bind to unique regions in the tumor driving kinases that are not marred by resistance mutations, 2. have sufficient potency for sustained inhibition of BTK signaling, and 3. be sufficiently selective to afford efficacious doses and suitable for a combination regimen. We have developed a platform called Energetically Privileged Ligands (EPriLs), based on a macrocycle scaffold that binds non-covalently in the kinase ATP pocket. Their unique binding mode involves connecting the N-terminal and C-terminal lobes of the general kinase fold in a continuous β-sheet, in this way avoiding hotspots for kinase inhibitor resistance mutations. The potent binding of the macrocycle ensures a very long residence time. Using the EPriL platform, we have developed CFON-026, a next generation non-covalent BTK inhibitor with a covalent-like residence time as demonstrated by surface plasmon resonance (SPR) and cellular washout experiments. The biochemical binding KD to BTKWT, determined by SPR, is 0.14 nM. CFON-026 binds mutants with KDs of 0.4 nM (BTKC481S) and and 1.8 nM (BTKT474I). X-ray crystal structures of CFON-026 bound to BTKWT and BTKT474I confirm the unique binding mode and water network around the active site. CFON-026 inhibits the growth of the BTK dependent cell lines TMD8 and Rec-1 with IC50s of 0.45 nM and 1.7 nM, respectively. CFON-026 is very active on TMD8 variants containing endogenous BTK inhibitor resistance mutations (BTKC481S, BTKV416L, BTKT474I and BTKL528W, IC50 range 1-100 nM). CFON-026 has favorable metabolic stability, pharmacokinetic properties, and safety characteristics, and induces complete tumor regression in a TMD8 xenograft model. Since BTK resistance mutations can be detected diagnostically 12-18 months before clinical relapse, CFON-026 could be applied as combination partner to prolong progression free survival on first line BTK inhibitor therapy. Experiments with mixed populations of BTK mutant cell lines, to model a heterogenous tumor, show that CFON-026 can deepen the efficacy of ibrutinib, acalabrutinib and zanubrutinib. In conclusion, CFON-026 is a novel EPriL-based non-covalent BTK inhibitor that could enhance and prolong responses of existing covalent BTK inhibitor therapy. [1] Woyach et al., J Clin Oncol (2017). [2] Wang et al., N. Eng J Med (2022).
Citation Format: Jos de Man, Michelle Muller, Joost C.M. Uitdehaag, Freek van Cauter, Sander van Gemert, Milan Hoffmann, Yvonne G.T.H. van Mil, Winfried R. Mulder, Martine B.W. Prinsen, Jan Gerard Sterrenburg, Diep Vu, Joeri de Wit, Erik Ensing, Rogier C. Buijsman. CFON-026 is a potent non-covalent BTK inhibitor suitable for combination therapy with covalent BTK inhibitors for early eradication of resistance mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1960.