Clinical success with the targeted Bruton Tyrosine Kinase (BTK) inhibitors ibrutinib and acalabrutinib has greatly improved the outcome of patients with relapsed chronic lymphocytic leukemia (CLL). However toxic side effects and acquired resistance due to C481S mutations in BTK remain an issue and the prognosis of those developing resistance is poor. Hence better therapeutic options are needed for these patients. Here we characterize a next generation highly selective reversible BTK inhibitor, LOXO-305, and describe its effectiveness in vitro in treatment naïve CLL patients and in those with C481S mutations. Unlike the irreversible BTK inhibitors ibrutinib and acalabrutinib, LOXO-305 does not require the C481 site for binding to the ATP binding domain of BTK. In addition, LOXO-305 is highly selective, with minimal activity against non-BTK kinase and non-kinase off targets, including ITK, TEC and EGFR.

We evaluated the efficacy of LOXO-305 in BTK wild-type B cell lymphoma lines most similar to CLL, namely MEC1 and OSU-CLL, and in CLL patient samples, by determining its effect on cell viability, apoptosis and BCR signaling. In both cell lines LOXO-305 reduced cell viability and BCR mediated activation of BTK, PLCg2, ERK and AKT similar to ibrutinib. We measured cellular apoptosis in these cell lines with Annexin V APC and PI staining and showed that after 48 hours LOXO-305 treated cells had a significantly higher percentage of apoptotic cells in comparison with both DMSO (mean increase with LOXO-305: MEC1 21% (p = 0.0005) & OSU-CLL 35% (p = 0.0016)) and ibrutinib (mean increase with LOXO305: MEC1 19% (p=0.0006), OSU-CLL 22% (p=0.0013)). Since LOXO-305's mechanism of BTK inhibition does not involve covalent binding to the C481 site, we tested its efficacy in in vitro ibrutinib resistant models of stably transfected HEK293 cells expressing either wild-type (WT) or C481S BTK. In WT BTK HEK cells, both LOXO-305 and ibrutinib showed comparable inhibitory activity in vitro against wild-type BTK, with IC50 values for phospho-BTK inhibition equal to 5.69 nM and 3.33 nM, respectively. LOXO-305 also inhibited phospho-BTK in BTK-C481S-expressing HEK293 cells at an IC50 equal to 21.2 nM, while ibrutinib had no inhibitory effect at concentrations as high as 300nM.

When we extended our analysis to treatment naïve CLL patient cells, we observed that both LOXO-305 and ibrutinib potently inhibited IgM-induced phospho-BTK with IC50 values equal to 1.34 ± 1.23 nM for LOXO-305 (n = 7, p < 0.0001) and 1.04 ± 1.26 nM for ibrutinib (n = 7, p < 0.0001). We also found a significant reduction in phosphorylation of PLCγ2 (Y1217), the immediate downstream effector of BTK (IC50 33 nM for each agent, n = 7, p = 0.02 for LOXO-305, p = 0.0017 for ibrutinib). When we compared the cellular cytotoxicity of LOXO-305 with ibrutinib and acalabrutinib, all three agents demonstrated significant induction of apoptosis (annexin V positive) in BTK wild-type cells compared to DMSO treatment: (LOXO-305: median 63.77% n=3, p = 0.0073, ibrutinib: median 57.76%, n=3, p < 0.0001, acalabrutinib: median 67.11%, n=3, p = 0.0397).

In CLL patient cells harboring C481S mutations, we observed a decrease in BCR signaling with 0.6 μM LOXO-305 treatment, with 95% reduction in phospho-BTK (Y223) and 50% reduction in phospho-PLCγ2 vs DMSO (n = 3, phospho-BTK p < 0.0001, phospho-PLCγ2 p = 0.01). In comparison ibrutinib at 0.6 μM demonstrated 50% reduction in phospho-BTK (n = 3, p = 0.01) and no significant change in phospho-PLCγ2. We also observed a 73% reduction in phospho-ERK vs DMSO with 10 μM LOXO-305 treatment (n=3, p = 0.004) while ibrutinib and acalabrutinib showed no significant change. In CLL patient cells with C481S variant allele frequencies (VAF) of 87% and 89%, LOXO-305 was 40-fold more potent at inhibiting phospho-BTK than ibrutinib (IC50 for LOXO305 0.02 μM, for ibrutinib 0.9 μM). Even in patient cells with lower C481S VAF of 9%, LOXO-305 still demonstrated 30-fold higher potency than ibrutinib (IC50 LOXO-305 0.2 μM, ibrutinib 0.6 μM).

Our findings show that LOXO-305 potently inhibits cell survival and BCR signaling in both treatment naïve and C481S CLL patient cells, and therefore may prove an effective treatment in both treatment naïve and ibrutinib-resistant CLL patients. A phase 1 clinical trial is ongoing.


Ebata:LOXO Oncology Inc.: Employment, Equity Ownership. Gomez:LOXO Oncology Inc.: Employment, Equity Ownership. Brandhuber:LOXO Oncology Inc.: Employment, Equity Ownership. Rothenberg:LOXO Oncology Inc.: Employment. Brown:Janssen, Teva Pharmaceuticals: Honoraria; Gilead, Loxo, Sun Pharmaceuticals, Verastem: Research Funding; Morphosys, Invectys (Data Safety Monitoring Board): Membership on an entity's Board of Directors or advisory committees; Abbvie, Acerta, Astra-Zeneca, Beigene, Catapult Therapeutics, Dynamo Therapeutics, Genentech/Roche, Gilead, Juno/Celgene, Kite, Loxo, Octapharma, Novartis, Pfizer, Pharmacyclics, Sunesis, TG Therapeutics, Verastem: Consultancy.

Author notes


Asterisk with author names denotes non-ASH members.

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