ALBERT

Description: Introduction The B-cell receptor (BCR) and its downstream effectors have emerged as important therapeutic targets in B-cell malignancies. CC-292 is a novel, potent, covalent, and highly selective inhibitor of Btk (IC50apparent of 0.5 nM, kinact/KI ratio of 7.69 × 104 M-1s-1), that does not appreciably inhibit other kinases involved in BCR signaling (eg, IC50 Lyn kinase, 4401 nM) (Evans et al., J Pharmacol Exp Ther. 2013). Here, we report preclinical characterization and clinical data in CLL from a single-agent phase 1 dose-escalation trial of CC-292 in B-cell malignancies, with a focus on how target engagement and downstream events correlate with clinical activity. Results Pharmacodynamic effects of Btk inhibition by CC-292 can be monitored by occupancy of the Btk catalytic site, Btk autophosphorylation on Y223, and downstream phosphorylation of Plc-γ2 and Erk. We developed a sensitive (10 pg/mL lower limit of quantification) and quantitative assay to measure covalent binding of CC-292 to Btk (Evans et al., J Pharmacol Exp Ther. 2013), as well as Western and novel phos-flow assays to probe downstream signal transduction. These methods showed that CC-292 treatment blocks Btk autophosphorylation and downstream pathway activation in both tumor cells and human peripheral blood mononuclear cells (PBMCs). The extent of CC-292 binding to Btk correlated with its in vitro and in vivo effects. The occupancy assay demonstrated that CC-292 effectively targets Btk in tumor cell lines, PBMCs, spleen, and lymph nodes (LNs) in animal models, and in PBMC and lymph node samples from clinical trial subjects. In rats and non-human primates treated with CC-292, Btk occupancy in spleen and LNs was dose-dependent. Measured occupancy in rat spleen and axillary, mesenteric, and superficial cervical LNs was 94%, 92%, 90%, and 76% respectively, 4 hours (hrs) after a single 30-mg/kg dose. Interim data from the phase 1 CLL trial showed that PBMC Btk was completely occupied in the majority of subjects 4 hrs post-dose with both QD and BID dosing. Twenty-four hrs post-dose at 750 and 1000 mg QD, CC-292 exhibited 83% ± 17% Btk occupancy, whereas with BID dosing at 375 and 500 mg, occupancy was 94% ± 16% at the corresponding time point (12 hrs after the second dose). Thus, while both schedules achieved extensive and sustained Btk occupancy, residual free Btk levels were lower with the BID schedule, offering a rationale for an early trend towards more rapid nodal responses, lymphocytosis, and partial responses on the BID schedule observed to date in the phase 1 study. In the 10 clinical LN biopsies tested to date, no measurable levels of unoccupied Btk have been detected, although Btk protein was present as determined by Western blotting, showing that CC-292 was able to penetrate LNs and inhibit Btk in human subjects as it did in preclinical models. For monitoring downstream signal transduction, we developed reagents and assays including a phos-flow assay based on a novel rabbit monoclonal antibody to detect Btk pY223 levels in PBMC subsets. CC-292 effectively inhibited constitutive and induced phosphorylation of Btk and Plc-γ2 at low nanomolar concentrations. CC-292 also inhibited BCR activation and nurse-like cell–supported survival of CLL cells. Furthermore, CC-292 reduced CLL cell migration and actin polymerization in response to chemokines (CXCL12, CXCL13) and inhibited secretion of the chemokines CCL3 and CCL4 by CLL cells. These chemokines are essential for migration and retention of normal and neoplastic B cells in the marrow and secondary lymphatic tissues. Consistent with this preclinical data, CC-292 treatment resulted in rapid reductions in circulating CCL3 and CCL4 levels. In subjects treated at the 750 mg QD, 1000 mg QD, 375 mg BID, and 500 mg BID dose levels, plasma CCL3 was reduced from 99 ± 16 pg/ml before treatment to 28 ± 5 pg/ml (N = 48, mean ± SEM) at 24 hrs after the first dose, while CCL4 was reduced from 235 ± 59 pg/ml to 74 ± 16 pg/ml (N = 51). Conclusions These data demonstrate that CC-292 achieves significant and durable occupancy of Btk in vitro and in vivo, inhibits Btk-mediated downstream signaling events and chemokine production, and that these preclinical activities have translated into the clinic. Taken together, these results argue that Btk inhibition is necessary and sufficient for clinical activity in CLL. These emerging data support continued development of CC-292 for the treatment of B-cell malignancies. Disclosures: Pierce: Celgene: Employment, Equity Ownership. O'Brien:Genentech: Consultancy, Research Funding; Emergent: Consultancy, Research Funding; CLL Global Research Foundation: Membership on an entity’s Board of Directors or advisory committees; Celgene: Consultancy; Gilead Sciences: Consultancy, Research Funding; Infinity: Consultancy, Research Funding; MorphoSys: Research Funding; Pharmacyclics: Consultancy, Research Funding; Talon: Consultancy, Research Funding; Teva/Cephalon: Consultancy. Heise:Celgene: Employment, Equity Ownership. Nacht:Celgene: Employment, Equity Ownership. Aslanian:Celgene: Employment, Equity Ownership. Liu:Celgene: Employment, Equity Ownership. Hong:Celgene: Employment, Equity Ownership. Wu:Celgene: Employment, Equity Ownership. Zavodovskaya:Celgene: Employment, Equity Ownership. Marine:Celgene: Employment, Equity Ownership. Barnett:Celgene: Employment, Equity Ownership. Nava-Parada:Celgene: Employment, Equity Ownership. Mei:Celgene: Employment, Equity Ownership. Chopra:Celgene: Employment, Equity Ownership. Burger:Pharmacyclics: Research Funding; Gilead: Research Funding. Singh:Celgene: Employment, Equity Ownership.

Description: Abstract 3485 Targeted therapies that suppress B cell receptor (BCR) signaling have emerged as promising agents in the treatment of several B cell malignancies. Bruton's tyrosine kinase (Btk) plays a key role in promoting B cell proliferation and survival through participation in the BCR signaling pathway and represents a promising new drug target. AVL-292 is a covalent, highly selective, orally active small molecule inhibitor of Btk currently being evaluated in a Phase 1b clinical trial in relapsed, refractory B cell malignancies including Chronic Lymphocytic Leukemia (CLL) and non-Hodgkin lymphomas. AVL-292 forms a covalent bond with Cys481 in Btk and potently inhibits Btk in biochemical (IC50 〈 0.5nM) and cellular assays (EC50 1–10 nM) including anti-IgM stimulation of BCR signaling, B cell proliferation and activation. A quantitative pharmacodynamic assay to determine the level of AVL-292 bonded to Btk in vitro or in vivo was developed and this drug-target engagement by AVL-292 was shown to correlate directly with inhibition of Btk enzyme activity and substrate phosphorylation. To rationally determine the dose and dose frequency of AVL-292 most likely to benefit patients and to reduce the potential for sub-therapeutic dosing in initial oncology patient cohorts, AVL-292 was administered to healthy adult subjects in a double-blind, placebo controlled, single ascending dose study. This study assessed safety, pharmacokinetics, and quantitatively measured Btk protein levels and AVL-292-Btk engagement in freshly isolated peripheral B lymphocytes. In healthy human subjects, AVL-292 was found to be safe and well tolerated following oral administration at dose levels ranging from 0.5–7.0 mg/kg. AVL-292 plasma levels and pharmacodynamic measurement of Btk engagement was dose-proportional across cohorts. All subjects that received 1.0 mg/kg AVL-292 achieved 〉80% Btk engagement and mean peak plasma levels (Cmax 365 ng/mL) of AVL-292 were rapidly achieved (Tmax median 40 min). Subjects receiving 2.0 mg/kg AVL-292 had a mean peak plasma concentration of 542 ng/mL. All subjects demonstrated 〉84% Btk engagement at this dose, with 5 of 6 subjects achieving 〉98% drug-target engagement. Although AVL-292 plasma levels declined substantially by 8 hours, Btk engagement persisted throughout 24 hours, demonstrating that covalent inhibition of Btk with AVL-292 enables prolonged duration of activity without high levels of circulating drug. These results suggest that a once daily dosing schedule is sufficient for sustained Btk inhibition. Furthermore, the Btk protein level in circulating B lymphocytes from all study subjects was evaluated and the mean level was found to be 417.7 pg Btk/mg total protein. Interestingly, this finding in normal B cells correlates well with preclinical ex vivo analysis of Btk protein in primary CLL cells where comparable Btk protein levels were found. This suggests that the AVL-292 dose range and schedule identified for complete Btk engagement in this healthy volunteer trial is likely to inform appropriate dose selection in the subsequent phase 1b oncology study, allowing more rapid identification of a safe and clinically effective dose. Disclosures: Evans: Avila Therapeutics: Employment, Equity Ownership. Tester:Avila Therapeutics: Employment, Equity Ownership. Aslanian:Avila Therapeutics: Employment, Equity Ownership. Chaturvedi:Avila Therapeutics: Employment, Equity Ownership. Mazdiyasni:Avila Therapeutics: Employment, Equity Ownership. Sheets:Avila Therapeutics: Employment, Equity Ownership. Nacht:Avila Therapeutics: Employment, Equity Ownership. Stiede:Avila Therapeutics: Employment, Equity Ownership. Witowski:Avila Therapeutics: Employment, Equity Ownership. Lounsbury:Avila Therapeutics: Employment, Equity Ownership. Petter:Avila Therapeutics: Employment, Equity Ownership. Singh:Avila Therapeutics: Employment, Equity Ownership. Westlin:Avila Therapeutics: Employment, Equity Ownership.

Description: B-cell receptor (BCR) signaling is essential for normal B cell development and proliferation. In addition, functional BCR signaling contributes to the proliferation and survival of many B cell lymphomas through constitutive low level or tonic signaling. Disruption of this tonic signaling pathway results in cell death or decreased viability in many B cell lymphoma cells. Bruton’s tyrosine kinase (Btk) is a member of the Tec family of protein tyrosine kinases and plays a crucial role in the development and activation of B cells through association with the BCR signalosome. We have developed a novel, potent, irreversible inhibitor of Btk that selectively disrupts the BCR signaling pathway as measured by inhibition of both Btk Y223 autophosphorylation and phosphorylation of the Btk substrate PLCg2. AVL101 inhibits Btk but leaves upstream BCR kinase activity intact; both Syk phosphorylation at Y525/526 and transphosphorylation of Btk on residue Y551 are unaltered. By mass spectrometry, we have shown that AVL101 covalently modifies Btk specifically at Cys-481, and in cell-based assays, this irreversible binding leads to prolonged inhibition of Btk kinase activity. After removal of the compound, inhibition of Btk activity is maintained in cultured B cell lymphoma cells for 〉 8 hours. This prolonged inhibition correlates with the half-life of the Btk protein, which we have found to be greater than 8 hours, irrespective of BCR activation state or modification by the inhibitor. We have demonstrated that AVL101 functionally inhibits BCR signaling in vitro and in vivo. In vitro, AVL101 inhibits BCR ligand-induced calcium flux as well as proliferation of the B cell lymphoma cell lines DOHH2 and WSU-DLCL2. In vivo, we have shown AVL101 is orally bioavailable and can inhibit Btk-dependent B cell function. Our data demonstrate that Btk is a valid therapeutic target for B cell lymphomas and other disorders dependent on BCR signaling. Furthermore, irreversible inhibition of Btk enables complete and prolonged inactivation of the target kinase, and suggests that clinically, a rapid and sustained therapeutic response may be possible using an orally available, irreversible Btk inhibitor.