Abstract B099: BTK-EndoMut Platform: Engineered Resistance Models Accelerating BTK-Targeted Drug Discovery

Abstract Background: Kyinno provides TMD8 cell lines with endogenous BTK resistance mutations (e.g., C481S, L528W, V416L, T474I, A428D) under native regulation, enabling detailed studies of covalent and non-covalent inhibitor responses. These models faithfully recapitulate clinical escape alleles, supporting both mechanistic investigations and drug-resistance profiling. Methods: High-fidelity CRISPR/Cas9 RNP electroporation with 200-bp ssODN donors and proprietary HDR enhancers routinely yields >80 % BTK knock-in efficiency. Following optimized limiting-dilution cloning, each clone undergoes genomic PCR, Sanger sequencing, and RT-PCR to confirm on-target integration, allelic zygosity. Results: Mutant TMD8 lines display broad IC50 shifts: A428D and L528W confer >100-fold resistance to both ibrutinib and pirtobrutinib, C481S remains nanomolar to pirtobrutinib, whereas T474I/V416L stay responsive to zanubrutinib; all variants expect A428D retain low-nanomolar BGB-16673 and NX-5948 susceptibility. In vivo, C481F tumors show modest ibrutinib delay, whereas L528W xenografts resist ibrutinib yet regress under daily NX-5948 without overt weight loss. Proliferation assays mirror these drug-response patterns across the panel. Impact: Kyinno’s BTK-mutant lines accelerate high-throughput screening of small-molecule inhibitors and targeted degraders, delineate resistance circuitry via isogenic comparisons, and improve translational relevance by faithfully modeling patient-derived escape variants. This platform supports rapid go/no-go decision-making in early drug discovery and biomarker identification for next-generation BTK therapies. Citation Format: Jinying B. Ning, feng B. hao, yue B. huang, Thomas A. Strong. BTK-EndoMut Platform: Engineered Resistance Models Accelerating BTK-Targeted Drug Discovery [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr B099.