The Bruton tyrosine kinase inhibitor PCI-32765 blocks B-cell activation and is efficacious in models of autoimmune disease and B-cell malignancy

Abstract

Activation of the B-cell antigen receptor (BCR) signaling pathway contributes to the initiation and maintenance of B-cell malignancies and autoimmune diseases. The Bruton tyrosine kinase (Btk) is specifically required for BCR signaling as demonstrated by human and mouse mutations that disrupt Btk function and prevent B-cell maturation at steps that require a functional BCR pathway. Herein we describe a selective and irreversible Btk inhibitor, PCI-32765, that is currently under clinical development in patients with B-cell non-Hodgkin lymphoma. We have used this inhibitor to investigate the biologic effects of Btk inhibition on mature B-cell function and the progression of B cell-associated diseases in vivo. PCI-32765 blocked BCR signaling in human peripheral B cells at concentrations that did not affect T cell receptor signaling. In mice with collagen-induced arthritis, orally administered PCI-32765 reduced the level of circulating autoantibodies and completely suppressed disease. PCI-32765 also inhibited autoantibody production and the development of kidney disease in the MRL-Fas(lpr) lupus model. Occupancy of the Btk active site by PCI-32765 was monitored in vitro and in vivo using a fluorescent affinity probe for Btk. Active site occupancy of Btk was tightly correlated with the blockade of BCR signaling and in vivo efficacy. Finally, PCI-32765 induced objective clinical responses in dogs with spontaneous B-cell non-Hodgkin lymphoma. These findings support Btk inhibition as a therapeutic approach for the treatment of human diseases associated with activation of the BCR pathway.

Fig. 1.

Chemical structure of Btk inhibitors. Chemical structures of the irreversible Btk inhibitor PCI-32765, the irreversible Btk inhibitor PCI-33380 (probe), and the reversible Btk inhibitor PCI-29732.

Fig. 2.

Selective irreversible targeting of Btk. (A–E) (Upper) Fluorescent gel scans of lysates from cells that were incubated with the affinity probe PCI-33380. Arrows indicate the predominant band labeled by the probe (approximately 76 kDa, the expected MW of Btk) and this band was confirmed to align with Btk as detected by Western blot of the same gel (Lower). (A) 293H cells transfected with WT Btk, K430A, C481A, or Y551A Btk mutants. Nontransfected (NT) 293H cells were included as a negative control. (B) DOHH2 cells incubated with increasing concentrations of PCI-33380 for 1 h. Densitometry values for the band corresponding to Btk are shown below the gel. (C) Btk immunoprecipitated (IP) from affinity probe labeled DOHH2 lysates. Total lysate (10 μg), and mock IP or Btk IP (from 50 μg of total lysate) were analyzed. A fluorescent gel scan of the immunodepleted supernatants (Sup) is also shown. (D) Affinity probe labeling of DOHH2 or Jurkat cells with and without 1 μM PCI-32765 pretreatment. (E) DOHH2 cells incubated with increasing concentrations of PCI-32765 for 1 h before labeling with affinity probe. By densitometry, the IC₅₀ for active site occupancy by PCI-32765 is 4 nM.

Fig. 3.

Inhibition of B-cell receptor signaling. (A) Concentration-dependent inhibition of BCR stimulation-induced phosphorylation events in DOHH2 cells. Cells were exposed to PCI-32765 and then drug was washed out before stimulation with anti-IgG. Blots were probed with the indicated antibodies. (B) Comparison of the reversible Btk inhibitor PCI-29732 to the irreversible Btk inhibitor PCI-32765 on the transcriptional response of six genes induced by BCR stimulation. Purified human peripheral B cells were treated with vehicle or 1 μM inhibitor for 1 h and then stimulated for 6 h with anti-IgM. In the washout condition, inhibitor-containing cell media was replaced with fresh media before addition of anti-IgM. Gene expression levels (mean ± SD) were measured by TaqMan RT-PCR and normalized to unstimulated cells. Btk expression is not affected by BCR stimulation or drug treatment. (C) Concentration-dependent inhibition of antigen receptor stimulation induced cell surface expression of the lymphocyte activation marker CD69 (mean ± SD). Purified human B or T cells were treated with PCI-32765 for 1 h and then stimulated for 18 h. In the washout condition, inhibitor-containing cell media was replaced with fresh media before stimulation. (D) Concentration-dependent covalent binding of PCI-32765 to Btk in purified human B cells as measured by the ability of Btk to bind to the fluorescent affinity probe PCI-33380. Total Btk levels are measured by Western blot (Bottom).

Fig. 4.

Btk inhibition by PCI-32765 inhibits collagen-induced arthritis in mice. (A) Mean clinical arthritis scores ± SEM (n = 5) from daily oral treatment for 11 d with different doses of PCI-32765 or dexamethasone as indicated. Disease control mice received vehicle only. Mean clinical scores from dexamethasone or PCI-32765 treatments at 3.125, 12.5, and 50 mg/kg were significantly different when compared with vehicle treatment (***P < 0.001, repeated-measures ANOVA). (B and C) Antibody levels from experiment shown in A. Data are mean ± SEM (**P < 0.01 and ***P < 0.001, ANOVA). (D) PCI-32765 occupancy of splenocyte Btk following a single oral dose in DBA/1 mice. Binding of PCI-32765 to Btk prevents binding of PCI-33380, detected by fluorescent gel scanning of PCI-33380-labeled lysates (Upper). Subsequent Western blotting confirms the presence of Btk (Lower).

Fig. 5.

Inhibition of Btk reduces renal disease and autoantibody production in MRL-Fas(lpr) mice. (A) Eight-week-old MRL-Fas(lpr) mice (n = 12) were randomized and treated orally with PCI-32765 or vehicle once daily for 12 wk at different concentrations as indicated. Range of urine protein concentration is calculated as a proteinurea score. Proteinurea scores from PCI-32765 treatments were significantly lower than vehicle group (**P < 0.01 for 3.125 mg/kg and ***P < 0.001 for 12.5 and 50 mg/kg treatments; repeated-measure ANOVA). (B) Serum BUN in PCI-32765–treated and vehicle-treated mice (***P < 0.001). (C) dsDNA-specific IgG in PCI-32765 treated and vehicle treated mice. (D) Histopathology scores at week 20 from the animals shown in A. Scores are mean ± SEM (n = 12; *P < 0.05).

Fig. 6.

Orally-dosed PCI-32765 leads to sustained occupancy of Btk in dogs with lymphoma. PBMCs and biopsy specimens from affected lymph nodes (LN) were collected from dogs (Table 1) treated with PCI-32765 (oral capsule formulation). Tissue samples were then treated with PCI-33380 to determine Btk occupancy by PCI-32765. Shown are predose (P), 4 h, 24 h, and predose d 7 occupancy data for all available week 1 samples in the study. Arrow marked “P” indicates fluorescent probe (PCI-33380) signal, arrow marked “Btk” indicates Btk protein level by Western blot. Full occupancy was achieved in all dogs. Western blots indicate that some LN biopsies did not contain Btk, presumably as a result of sample collection heterogeneity.