Pharmacodynamic Analysis of BTK Inhibition in Patients with Chronic Lymphocytic Leukemia Treated with Acalabrutinib

Abstract

Purpose: To determine the pharmacodynamic relationship between target occupancy of Bruton tyrosine kinase (BTK) and inhibition of downstream signaling.

Patients and methods: Patients with chronic lymphocytic leukemia (CLL) enrolled in a phase II clinical trial (NCT02337829) with the covalent, selective BTK inhibitor acalabrutinib donated blood samples for pharmacodynamic analyses. Study design included randomization to acalabrutinib 100 mg twice daily or 200 mg once daily and dose interruptions on day 4 and 5 of the first week. BTK occupancy and readouts of intracellular signaling were assessed sequentially between 4 and 48 hours from last dose.

Results: Four hours from last dose, BTK occupancy exceeded 96% and at trough, was higher with twice daily, median 95.3%, than with once daily dosing, median 87.6% (P < 0.0001). By 48 hours from last dose, median free BTK increased to 25.6%. Due to covalent binding of acalabrutinib, free BTK is generated by de novo synthesis. The estimated rate of BTK synthesis varied widely between patients ranging from 3.6% to 31.4% per day. Acalabrutinib reduced phosphorylation of BTK and inhibited downstream B-cell receptor (BCR) and NFκB signaling. During dosing interruptions up to 48 hours, expression of BCR target genes rebounded, while phosphorylation of signaling molecules remained repressed. In vitro cross-linking of IgM on CLL cells obtained 36 to 48 hours from last dose upregulated CD69, with high correlation between cellular free BTK and response (R = 0.7, P ≤ 0.0001).

Conclusions: Higher BTK occupancy was achieved with twice daily over once daily dosing, resulting in deeper and more sustained inhibition of BCR signaling.

Conflict of interest statement

Conflicts of Interest

J.C., M.G., A.H., R.I., E.B., P.P., and T.C. were or currently are full-time employees and shareholders of Acerta Pharma, BV. AW received research funding from Acerta Pharma, a member of the Astra-Zeneca group; Pharmacyclics, an Abbvie company; Merck; and Nurix. All other authors declare no competing interests.

©2020 American Association for Cancer Research.

Figures

Figure 1:. Immediate inhibition of BCR signaling with deepening response over time.

A-F, Levels of phosphorylated signaling molecules measured by flow cytometry on day3 (D3) and day28 (D28) of acalabrutinib therapy are displayed as % change from baseline. Red circles represent 200mg qd dosing; blue circles represent 100mg bid dosing. Box and Whisker plots display median (±IQR) for all patients analyzed (n=21). A, pBTKY551; B, pBTKY223; C, pPLCG2Y759; D, pP38Y182; E, pERKT202/Y204; F, pNF-κB p65S529. Statistical significance by Wilcoxon matched-pairs signed-rank test between baseline and treatment timepoints is indicated: **P<0.01, ***P<0.001, and ****P<0.0001.

Figure 2:. Inhibition of downstream effects of BCR activation by acalabrutinib.

A-B, Change in gene signature score in CD19+ selected CLL cells collected on day 3 (D3) and day 28 (D28) normalized to pre-treatment (n=11). Red circles represent 200mg qd dosing (n=6); blue circles represent 100mg bid dosing (n=5). Bars represent median (±IQR). (A) BCR signature score. (B) NF-κB signature score. C, Pearson correlation between change in BCR and NF-κB signature scores. Each dot represents one patient, circles represents day 3, squares represent day28. R and P values of Pearson correlation are displayed. D, Nuclear expression of NF-κB p50 in CD19+ selected CLL cells by ELISA (n=11). E, CCL4 serum levels measured by Luminex Assays at baseline (Pre), day 3, and day 28 (n=22). F-G Median (±IQR) percent change in MFI measured by flow cytometry (n=21) at day 3 and day 28 compared to baseline for (F) CD69; (G) CD86. Statistical significance by Wilcoxon matched-pairs signed-rank test between baseline and treatment timepoints is indicated: **P<0.01, ***P<0.001 and ****P<0.0001.

Figure 3:. Interpatient variability in BTK synthesis rates.

A, percent free BTK on day 3 (Peak), day 4 (Trough) and day 5 (Trough + 24 hours). Black line represents the median. Each symbol represents one patient. Red symbols represent qd dosing; blue symbols represent bid dosing. B, Rate of change in percent free BTK per day for all evaluated patients aligned with different baseline factors; red bars represent 200mg qd patients; blue bars represent 100mg bid patients. Range 3.5 – 31.4%. Linear regression was used to calculate slopes for the return of free BTK and to determine the BTK synthesis rate per day. Statistical significance by Wilcoxon matched-pairs signed-rank test between baseline and treatment timepoints, and Wilcoxon rank-sum test between dose groups is indicated: ****P<0.0001.

Figure 4:. On-target effects up to 48 hours from last dose.

A-D, Levels of phosphorylated signaling molecules measured by flow cytometry at 36 and 48 hours from the last dose of acalabrutinib therapy are displayed as % point change from peak. (A) pBTKY551, (B) pBTKY223(C) pERKT202/Y204 and (D) pNF-κB p65S529. Only subjects with a significant reduction at peak were included here; Box and Whisker plots display median (±IQR) for all patients analyzed. E, Change in gene signature average in CD19+ selected CLL cells collected 12, 24, 36, and 48 hours from the last dose of acalabrutinib therapy normalized to peak (n=11). Red bars represent 200mg qd dosing (n=6); blue bars represent 100mg bid dosing (n=5). Bars represent median (±IQR). F, Median (±IQR) percent point change in CD69 MFI measured by flow cytometry at 36 and 48 hours from the last dose of acalabrutinib therapy compared to peak (n=21). G, CCL4 serum levels measured by Luminex Assays at 36 and 48 hours from the last dose of acalabrutinib therapy are displayed as % point change from peak (n=22). Green circles represent patients with measured free BTK ≥30%; orange circles represent patients with measured free BTK ≥20% but <30 %; black circles represent patients with measured free BTK <20. Statistical significance by Wilcoxon matched-pairs signed-rank test is indicated: *P<0.05.

Figure 5:. Rebound of BCR signaling occurs in the presence of activation signals and correlates with level of free BTK.

A-B, levels of (A) CD69 expression and (B) pBTKY223 at 24 and 48 hours (qd) from the last dose of acalabrutinib are displayed as % point change from peak. C-D, Levels of (C) CD69 expression and (D) pBTKY223 measured by flow cytometry at 12 and 36 hours (bid) from the last dose of acalabrutinib are displayed as % point change from peak. Statistical significance by Wilcoxon matched-pairs signed-rank test between treatment timepoints is indicated: **P<0.01, ***P<0.001. PBMCs collected at the indicated time points were stimulated with anti-IgM in vitro. Only subjects with day 1 predose ≥ 1.5 fold change over exogenous treatment control (+ 1μM acalabrutinib) were included in the CD69 analysis. In all panels, green circles represent patients with free BTK ≥30%; orange circles represent patients with measured free BTK ≥20% but <30%; black circles represent patients with measured free BTK <20%. E, Pearson correlation of free BTK and % point change in CD69 expression in matched patient samples. Each dot represents one patient, squares represent trough (12h-24h), circles represents trough + 24h (36h-48h).