Preclinical Evaluation of the Novel BTK Inhibitor Acalabrutinib in Canine Models of B-Cell Non-Hodgkin Lymphoma

Abstract

Acalabrutinib (ACP-196) is a second-generation inhibitor of Bruton agammaglobulinemia tyrosine kinase (BTK) with increased target selectivity and potency compared to ibrutinib. In this study, we evaluated acalabrutinib in spontaneously occurring canine lymphoma, a model of B-cell malignancy similar to human diffuse large B-cell lymphoma (DLBCL). First, we demonstrated that acalabrutinib potently inhibited BTK activity and downstream effectors in CLBL1, a canine B-cell lymphoma cell line, and primary canine lymphoma cells. Acalabrutinib also inhibited proliferation in CLBL1 cells. Twenty dogs were enrolled in the clinical trial and treated with acalabrutinib at dosages of 2.5 to 20mg/kg every 12 or 24 hours. Acalabrutinib was generally well tolerated, with adverse events consisting primarily of grade 1 or 2 anorexia, weight loss, vomiting, diarrhea and lethargy. Overall response rate (ORR) was 25% (5/20) with a median progression free survival (PFS) of 22.5 days. Clinical benefit was observed in 30% (6/20) of dogs. These findings suggest that acalabrutinib is safe and exhibits activity in canine B-cell lymphoma patients and support the use of canine lymphoma as a relevant model for human non-Hodgkin lymphoma (NHL).

Fig 1. Effects of acalabrutinib inhibitors on canine lymphoma cells.

Dose-dependent inhibition of BTK autophosphorylation, in addition to downstream targets, was observed via immunoblot at drug concentrations as low as 0.01μM following 1 hour of treatment with acalabrutinib in the canine B-cell lymphoma CLBL1 cell line (representative of 3 repetitions) (A) and primary canine lymphoma cells treated ex vivo (representative of 4 patients, separate from the clinical study population) (B). C. Densitometry quantification of the western blots from B. Bands of phospho-proteins are normalized to respective total proteins and loading control. There was a significant dose-dependent decrease in phosphorylation for p-ERK (P = 0.0028) and p-AKT (p = 0.0019). D. Dose-dependent reductions in cell proliferation following daily treatment with acalabrutinib in the canine CLBL1 B-cell lymphoma cell line. Results are the mean of 5 independent experiments. Raw data were log transformed to reduce variance and skewness. Linear mixed effects models were applied to apoptosis data and the log-transformed proliferation data to account for the correlation of the observations from the same batch. p = 0.012 E. Representative histograms showing a dose-dependent reduction in Edu incorporation from a single day at the 72 hour timepoint. F. Dose-dependent trend toward reductions in cell viability in CLBL1. Results are the mean of 3 independent experiments. Effects not statistically significant in a linear mixed effects model.

Fig 2. Pharmacokinetic data.

Plasma levels of acalabrutinib were measured at 0.5, 1, 2, 4, 6, 8, 12 and 24 hours after oral dose administration from 7 patients on day 14. 1000 ng/mL = 2.1 μM.

Fig 3. Reduced target lesion size in acalabrutinib treated dogs.

Waterfall plot showing percentage decrease in mean sum of longest diameter of index lymph nodes as compared to baseline measurements.

Fig 4

PFS. Kaplan-Meier curves showing overall PFS (A) and PFS of dogs achieving SD compared with PR (B). Dogs achieving PR survived significantly longer than those achieving SD (p = 0.010).