Selective Bruton's Tyrosine Kinase Inhibitor Research Articles

Loxo-305, a Highly Selective and Non-Covalent Next Generation BTK Inhibitor, Inhibits Diverse BTK C481 Substitution Mutations

Introduction: Bruton's Tyrosine Kinase (BTK) is an essential component of normal and malignant B-cell receptor signaling. Covalent BTK inhibitors have transformed the treatment of B-cell malignancies but are limited by off-target toxicity and acquired resistance, leading to eventual treatment discontinuation and disease progression. Emerging evidence suggests that acquired resistance is mediated predominantly by BTK C481 substitution mutations at the covalent BTK inhibitors' binding site. There is significant unmet clinical need for new treatment approaches that overcome acquired resistance and minimize toxicity. LOXO-305 is a highly selective, non-covalent, next generation BTK inhibitor. We previously showed that LOXO-305 potently inhibited both wild-type (WT) BTK and BTK C481S -mediated kinase activity in enzyme and cell-based assays with nanomolar potency, caused regression of BTK-dependent lymphoma mouse xenograft models, and was more than 300-fold selective for BTK over 98% of 370 other kinases tested and showed no significant inhibition of non-kinase off targets at 1 mM (Brandhuber et al. SOHO 2018). In addition, ADME and pharmacokinetic experiments in two preclinical species predicted that LOXO-305 will have high human exposure and sustained BTK C481S target coverage in patients at clinically achievable doses. Here we describe the activity of LOXO-305 against additional BTK C481 substitution mutations, including mutations identified in patients with acquired resistance to covalent BTK inhibitors. We further determine equilibrium-binding affinities for LOXO-305 for diverse mutant BTK enzymes in comparison to other clinically available BTK inhibitors. Methods: To assess cellular BTK inhibitor potency, HEK293T cell lines transiently expressing wild-type BTK and BTK C481 substitution mutations were serum starved and incubated with LOXO-305 overnight. Cells were next incubated with serum and orthovanadate for 5 min and the phosphorylated Y223 BTK was analyzed by immunoblot. Bands were quantified and the IC50 values calculated with GraphPad Prism. The equilibrium-binding affinities for targeted BTK inhibitors to BTK enzyme variants were determined by surface plasmon resonance (SPR) using the Biacore T200. Biotinylated BTK variants were immobilized on a docked streptavidin coated sensor chip. Five concentrations of each inhibitor plus blank controls were analyzed. Association/dissociation rate constants were calculated by global fitting of the data to a 1:1 binding interaction model. Results: While BTK C481S possessed similar levels of basal Y223 autophosphorylation as wild-type BTK in cells, BTK C481T autophosphorylation was reduced by ~50%, C481R by ~90%, and mutants C481F, and C481Y were inactive in HEK293T cells. LOXO-305 inhibited Y223 phosphorylation of all active mutants with similar nanomolar potency. In contrast, autophosphorylation of all BTK C481 mutants were resistant to both Ibrutinib and acalabrutinib. Equilibrium-binding affinities of LOXO-305 for select BTK C481 substitution mutations confirmed LOXO-305's superior potency versus commercially available BTK inhibitors (ibrutinib and acalabrutinib). Conclusions: The next generation, non-covalent, highly selective BTK inhibitor LOXO-305 potently inhibited the cellular activity of BTK C481S, T and R mutations and displayed strong equilibrium binding to WT BTK and several BTK C481 substitution mutations. Together with high selectivity and significant BTK target coverage in vivo, these results indicate that LOXO-305 may overcome acquired resistance to covalent BTK inhibitors in patients without significant off-target toxicity. A phase 1 clinical trial of LOXO-305 is currently underway. Disclosures Gomez: LOXO Oncology Inc.: Employment, Equity Ownership. Isabel:Loxo Oncology: Employment. Rosendahal:Loxo Oncology: Employment. Rothenberg:LOXO Oncology Inc.: Employment. Andrews:Loxo Oncology: Employment. Brandhuber:LOXO Oncology Inc.: Employment, Equity Ownership.

The impacts of zanubrutinib on immune cells in patients with chronic lymphocytic leukemia/small lymphocytic lymphoma.

Ibrutinib, a first-generation Bruton's tyrosine kinase (BTK) inhibitor, could improve immunity of relapsed or refractory (R/R) chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) patients. Whether zanubrutinib, a second-generation selective BTK inhibitor, has similar effects as ibrutinib remains to be determined. Dynamics of number and immunophenotype of immune cells during zanubrutinib treatment in 25 R/R CLL/SLL patients were examined by flow cytometry and blood routine tests. The expression intensity of programmed death-1 (PD-1) on total CD4+ (P < .01), total CD8+ (P < .01), and T helper cells (P < .05) and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) on total CD4+ (P = .010) and regulatory T cells (P < .05) reduced after treatment. There were significant differences in expression intensity of CD19 (P < .01), C-X-C chemokine receptor type 5 (CXCR5) (P < .01), and CD49d (P < .05) on B cells before and after treatment. Downregulation of PD-1 on T cells and CXCR5 and CD19 on B cells were observed in nearly all patients after zanubrutinib treatment. Programmed death-ligand 1 expression downregulated, especially in the female, CLL, normal spleen, normal β2-macroglobulin (β2-MG) and abnormal lactate dehydrogenase (LDH) subgroups, and CTLA-4 expression on CD4+ T cells tended to decrease in the male, old, CLL, splenomegaly, abnormal β2-MG, normal LDH, IGHV-mutated and wild-type tumor protein 53 subgroups after zanubrutinib treatment. These findings suggest that zanubrutinib can regulate immunity primarily by improving T cell exhaustion, inhibiting suppressor cells and disrupting CLL cells migration through downregulation of adhesion/homing receptors. Furthermore, favorable changes in cell number and immunophenotype were preferably observed in patients without adverse prognostic factors.

Phase 1 study of the selective BTK inhibitor zanubrutinib in B-cell malignancies and safety and efficacy evaluation in CLL

Zanubrutinib is a potent and highly selective inhibitor of Bruton tyrosine kinase (BTK). In this first-in-human, open-label, multicenter, phase 1 study, patients in part 1 (3 + 3 dose escalation) had relapsed/refractory B-cell malignancies and received zanubrutinib 40, 80, 160, or 320 mg once daily or 160 mg twice daily. Part 2 (expansion) consisted of disease-specific cohorts, including treatment-naive or relapsed/refractory chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). The primary end points were safety and tolerability, and definition of the maximum tolerated dose (part 1). Additional end points included pharmacokinetics/pharmacodynamics and preliminary efficacy. Reported herein are results from 144 patients enrolled in the dose-finding and CLL/SLL cohorts. No dose-limiting toxicities occurred in dose escalation. Median BTK occupancy in peripheral blood mononuclear cells was >95% at all doses. Sustained complete (>95%) BTK occupancy in lymph node biopsy specimens was more frequent with 160 mg twice daily than 320 mg once daily (89% vs 50%; P = .0342). Consequently, 160 mg twice daily was selected for further investigation. With median follow-up of 13.7 months (range, 0.4-30.5 months), 89 CLL/SLL patients (94.7%) remain on study. Most toxicities were grade 1/2; neutropenia was the only grade 3/4 toxicity observed in >2 patients. One patient experienced a grade 3 subcutaneous hemorrhage. Among 78 efficacy-evaluable CLL/SLL patients, the overall response rate was 96.2% (95% confidence interval, 89.2-99.2). Estimated progression-free survival at 12 months was 100%. Zanubrutinib demonstrated encouraging activity in CLL/SLL patients, with a low incidence of major toxicities. This trial was registered at www.clinicaltrials.gov as #NCT02343120.

Design of Potent and Selective Covalent Inhibitors of Bruton's Tyrosine Kinase Targeting an Inactive Conformation.

Bruton's tyrosine kinase (BTK) is a member of the TEC kinase family and is selectively expressed in a subset of immune cells. It is a key regulator of antigen receptor signaling in B cells and of Fc receptor signaling in mast cells and macrophages. A BTK inhibitor will likely have a positive impact on autoimmune diseases which are caused by autoreactive B cells and immune-complex driven inflammation. We report the design, optimization, and characterization of potent and selective covalent BTK inhibitors. Starting from the selective reversible inhibitor 3 binding to an inactive conformation of BTK, we designed covalent irreversible compounds by attaching an electrophilic warhead to reach Cys481. The first prototype 4 covalently modified BTK and showed an excellent kinase selectivity including several Cys-containing kinases, validating the design concept. In addition, this compound blocked FcγR-mediated hypersensitivity in vivo. Optimization of whole blood potency and metabolic stability resulted in compounds such as 8, which maintained the excellent kinase selectivity and showed improved BTK occupancy in vivo.

Discovery of Zanubrutinib (BGB-3111), a Novel, Potent, and Selective Covalent Inhibitor of Bruton's Tyrosine Kinase.

Aberrant activation of Bruton's tyrosine kinase (BTK) plays an important role in pathogenesis of B-cell lymphomas, suggesting that inhibition of BTK is useful in the treatment of hematological malignancies. The discovery of a more selective on-target covalent BTK inhibitor is of high value. Herein, we disclose the discovery and preclinical characterization of a potent, selective, and irreversible BTK inhibitor as our clinical candidate by using in vitro potency, selectivity, pharmacokinetics (PK), and in vivo pharmacodynamic for prioritizing compounds. Compound BGB-3111 (31a, Zanubrutinib) demonstrates (i) potent activity against BTK and excellent selectivity over other TEC, EGFR and Src family kinases, (ii) desirable ADME, excellent in vivo pharmacodynamic in mice and efficacy in OCI-LY10 xenograft models.

Discovery and biological evaluation of N-(3-(7-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-4-methyl-2-oxo-2H-pyrimido[4,5-d][1,3]oxazin-1(4H)-yl)phenyl)acrylamide as potent Bruton's tyrosine kinase inhibitors.

Bruton’s tyrosine kinase (BTK) is a key component of the B cell receptor (BCR) signaling pathway and plays a crucial role in B cell malignancies and autoimmune disorders; thus, it is an attractive target for the treatment of B cell related diseases. Here, we evaluated the BTK inhibitory activity of a series of pyrimido[4,5-d][1,3]oxazin-2-one derivatives. Combining this evaluation with structure-activity relationship (SAR) analysis, we found that compound 2 exhibited potent BTK kinase inhibitory activity, with an IC50 of 7 nM. This derivative markedly inhibited BTK activation in TMD8 B cell lymphoma cells and thus inhibited the in vitro growth of the cells. Further studies revealed that compound 2 dose dependently arrested TMD8 cells at G1 phase, accompanied by decreased levels of Rb, phosphorylated Rb, and cyclin D1. Moreover, following treatment with compound 2, TMD8 cells underwent apoptosis associated with PARP and caspase 3 cleavage. Interestingly, the results of the kinase activity assay on a small panel of 35 kinases showed that the kinase selectivity of compound 2 was superior to that of the first-generation inhibitor ibrutinib, suggesting that compound 2 could be a second-generation inhibitor of BTK. In conclusion, we identified a potent and highly selective BTK inhibitor worthy of further development.

Inhibiting Bruton's Tyrosine Kinase in CLL and Other B-Cell Malignancies.

Inhibitors of Bruton's tyrosine kinase (BTK), a major kinase in the B-cell receptor (BCR) signaling pathway, mediating B-cell proliferation and apoptosis, have substantially altered the management, clinical course, and outcome of patients with B-cell malignancies. This is especially true for patients with previously limited treatment options due to disease characteristics or coexisting diseases. Ibrutinib was the first orally available, nonselective and irreversible inhibitor of BTK approved for the treatment of patients with various B-cell malignancies. Newer and more selective BTK inhibitors are currently in clinical development, including acalabrutinib, which is currently US FDA approved for previously treated mantle cell lymphoma. Significant efforts are underway to investigate the optimal combinations, timing, and sequencing of BTK inhibitors with other regimens and targeted agents, and to capitalize on the immunomodulatory modes of action of BTK inhibitors to correct tumor-induced immune defects and to achieve long-lasting tumor control. This review describes the major milestones in the clinical development of BTK inhibitors in chronic lymphocytic leukemia and other B-cell malignancies, highlights the most recent long-term follow-up results, and evaluates the role of BTK inhibitors and their combination with other agents in B-cell malignancies and other indications.

Separation of Bruton’s tyrosine kinase inhibitor atropisomers by supercritical fluid chromatography

Bruton's tyrosine kinase (BTK) plays an essential role in multiple cell types responsible for numerous autoimmune diseases, thus inhibition of BTK is anticipated to provide an effective strategy for the clinical treatment of autoimmune diseases. Preparative-scale super/subcritical fluid chromatography (SFC) separation methods for four groups of highly potent and selective BTK inhibitor atropisomers were successfully developed. Depending on the rotation barrier around the chiral axis, the compounds were prepared as a single stereochemically stable atropisomer or as an atropisomeric mixture. Among the four, compound 2 with one rotationally stable atropisomeric center (carbazole/quinazolinedione based) was resolved as a mixture of two atropisomers, while compound 3 (carbazole-chlorine/quinazolinedione based) and 4 (tetrahydrocarbazole-fluorine/quinazolinedione based) with two rotationally stable atropisomeric centers were resolved into a single stable atropisomer. This article discusses the challenges and strategies in preparing large quantities of these atropisomeric active pharmaceutical ingredients (APIs) in support of the BTK program discovery efforts.

Ibrutinib significantly inhibited Bruton’s tyrosine kinase (BTK) phosphorylation,in-vitro proliferation and enhanced overall survival in a preclinical Burkitt lymphoma (BL) model

ABSTRACT Pediatric and adult patients with recurrent/refractory Burkitt lymphoma (BL) continue to have poor outcomes, emphasizing the need for newer therapeutic agents. Bruton’s tyrosine kinase (BTK) is activated following B-cell receptor stimulation and in part regulates normal B-cell development. Ibrutinib, a selective and irreversible BTK inhibitor, has been efficacious in chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), Waldenström’s macroglobulinemia, and marginal zone lymphoma. In this study, we investigated the efficacy of ibrutinib alone and in selective adjuvant combinations against BL in-vitro and in a human BL xenografted immune-deficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mouse model. Our data demonstrated that phospho-BTK level was significantly reduced in BL cells treated with ibrutinib (p < 0.001). Moreover, we observed a significant decrease in cell proliferation as well as significant decrease in IC50 of ibrutinib in combination with dexamethasone, rituximab, obinutuzumab, carfilzomib, and doxorubicin (p < 0.001). In-vivo studies demonstrated ibrutinib treated mice had a significantly prolonged survival with median survival of mice following ibrutinib treatment (32 days) (24 days) (p < 0.02). In conclusion, our findings demonstrate the significant in-vitro and preclinical in-vivo effects of ibrutinib in BL. Based on our preclinical results in this investigation, there is an on-going clinical trial comparing overall survival in children and adolescents with relapsed/refractory BL treated with chemoimmunotherapy with or without ibrutinib (NCT02703272).

Design and Synthesis of Novel Amino-triazine Analogues as Selective Bruton's Tyrosine Kinase Inhibitors for Treatment of Rheumatoid Arthritis.

Bruton's tyrosine kinase (BTK) is a promising drug target for the treatment of multiple diseases, such as B-cell malignances, asthma, and rheumatoid arthritis. A series of novel aminotriazines were identified as highly selective inhibitors of BTK by a scaffold-hopping approach. Subsequent SAR studies of this series using two conformationally different BTK proteins, an activated form of BTK and an unactivated form of BTK, led to the discovery of a highly selective BTK inhibitor, 4b. With significant efficacy in models in vivo and good ADME and safety profiles, 4b was advanced into preclinical studies.

ABBV-105, a selective and irreversible inhibitor of Bruton’s tyrosine kinase, is efficacious in multiple preclinical models of inflammation

Objectives: Bruton’s tyrosine kinase (BTK) is a non-receptor tyrosine kinase required for intracellular signaling downstream of multiple immunoreceptors. We evaluated ABBV-105, a covalent BTK inhibitor, using in vitro and in vivo assays to determine potency, selectivity, and efficacy to validate the therapeutic potential of ABBV-105 in inflammatory disease.Methods: ABBV-105 potency and selectivity were evaluated in enzymatic and cellular assays. The impact of ABBV-105 on B cell function in vivo was assessed using mechanistic models of antibody production. Efficacy of ABBV-105 in chronic inflammatory disease was evaluated in animal models of arthritis and lupus. Measurement of BTK occupancy was employed as a target engagement biomarker.Results: ABBV-105 irreversibly inhibits BTK, demonstrating superior kinome selectivity and is potent in B cell receptor, Fc receptor, and TLR-9-dependent cellular assays. Oral administration resulted in rapid clearance in plasma, but maintenance of BTK splenic occupancy. ABBV-105 inhibited antibody responses to thymus-independent and thymus-dependent antigens, paw swelling and bone destruction in rat collagen induced arthritis, and reduced disease in an IFNα-accelerated lupus nephritis model. BTK occupancy in disease models correlated with in vivo efficacy.Conclusion: ABBV-105, a selective BTK inhibitor, demonstrates compelling efficacy in pre-clinical mechanistic models of antibody production and in models of rheumatoid arthritis and lupus.

Discovery of potent, highly selective covalent irreversible BTK inhibitors from a fragment hit

Bruton's Tyrosine Kinase (BTK) is a member of the TEC kinase family that is expressed in cells of hematopoietic lineage (e.g., in B cells, macrophages, monocytes, and mast cells). Small molecule covalent irreversible BTK inhibitor targeting Cys481 within the ATP-binding pocket, for example ibrutinib, has been applied in the treatment of B-cell malignancies. Starting from a fragment hit, we discovered a novel series of potent covalent irreversible BTK inhibitors that occupy selectivity pocket of the active site of the BTK kinase domain. Guided by X-ray structures and a fragment-based drug design (FBDD) approach, we generated molecules showing comparable cellular potency to ibrutinib and higher kinome selectivity against undesirable off-targets like EGFR.

Targeting Bruton's tyrosine kinase for the treatment of B cell associated malignancies and autoimmune diseases: Preclinical and clinical developments of small molecule inhibitors.

B cell receptor (BCR) signaling plays a key role in B cell development and function. Aberrant BCR signaling has been confirmed as a central driver for the pathogenesis of various B cell malignancies. Bruton's tyrosine kinase (BTK) is a vital component of BCR signaling and exhibits overexpression in various B cell leukemias and lymphomas. Inhibiting BTK has been proved as an efficient way for B cell malignancy intervention. Remarkable achievements have been made in the pursuit of selective BTK inhibitors, represented by the success of the irreversible BTK inhibitors, ibrutinib and acalabrutinib. Constantly emerging agents exhibiting superior efficacy and safety in preclinical and clinical studies provide promising therapeutics for the treatment of B cell malignancies.

Updates from the 2017 American Society of Hematology Annual Meeting: Practice-Changing Studies in Relapsed and Refractory Mantle Cell Lymphoma

The 2017 annual meeting of the American Society of Hematology took place 9–12 December in Atlanta, Georgia. At the meeting, results from key studies in the treatment of relapsed and refractory mantle cell lymphoma were presented. Of those studies, oral presentations focused on the efficacy and safety of therapy with Bruton tyrosine kinase (BTK) inhibitors. One study presented pooled data from three trials using ibrutinib, with a median follow-up of 3.5 years. A second phase ii study presented data on the efficacy and safety of acalabrutinib, a highly selective BTK inhibitor with minimal off-target activity. The final study presented early phase ib data on the efficacy and safety of zanubrutinib, a novel, highly selective BTK inhibitor, in patients with non-Hodgkin lymphoma. Our meeting report describes the foregoing studies and presents interviews with investigators and commentaries by Canadian hematologists about potential effects on Canadian practice.

Acalabrutinib, A Second-Generation Bruton's Tyrosine Kinase Inhibitor.

The Bruton's tyrosine kinase (BTK) is an essential in the B-cell receptor (BCR) signaling pathway which was identified as crucial in the pathogenesis of B-cell malignancies. Ibrutinib, a first-in-class BTK inhibitor, has been approved for the treatment of distinct B-cell malignancies. To overcome off-target side effects of and emerging resistances to ibrutinib, more selective second-generation BTK inhibitors were developed. Acalabrutinib is a novel second-generation BTK inhibitor and has shown promising safety and efficacy profiles in phase 1/2 clinical trials in patients with relapsed CLL and pretreated MCL. Recently, acalabrutinib was approved by the FDA for treatment of adult patients with MCL who received at least one prior therapy. However, clinical trials on a direct comparison between ibrutinib and acalabrutinib and on combination treatment options with other agents as CD20 antibodies are warranted.

Abstract B152: CB1763, a highly selective, novel non-covalent BTK inhibitor, targeting ibrutinib-resistant BTK C481S mutant

Abstract Introduction: Bruton's tyrosine kinase (BTK) is a member of the Tec family of cytoplasmic tyrosine kinase. BTK plays a crucial role in the BCR signaling, essential for B-cell development, and BTK has been recognized as a validated therapeutic target for B-cell malignancies. Ibrutinib, the first FDA-approved BTK inhibitor, has been appreciated as a promising targeted therapy for patients with B-cell malignancies. Ibrutinib covalently binds to the Cys481 residue of BTK to inhibit BTK enzymatic activity, but recent studies suggested that the C481S mutation in BTK disrupts the irreversible binding of ibrutinib, and resulted in ibrutinib-resistance in patients. Therefore a non-covalent BTK inhibitor is highly demanded to overcome ibrutinib resistance. We identified a highly selective, orally available non-covalent inhibitor of BTK, CB1763. Here, we report the characterization of CB1763, capable of potently inhibiting BTK [C481S] mutant, both in vitro and in vivo. Material and Methods: We produced ibrutinib-resistant BTK [C481S] mutant recombinant enzyme to assess efficacy of compounds against the drug-resistant enzyme. To evaluate cellular potency, phosphorylations of BTK and PLC- γ were analyzed by Western blotting in C481S BTK mutant transfected HEK293 cells. ABC-type DLBCL cell line, OCI-Ly10 was used to evaluate antitumor efficacy of BTK inhibitors. Kinase selectivity profiling was performed to confirm inhibitor selectivity. Results: In addition to optimization of CB988, previously reported by our group, we identified CB1763 as a highly selective non-covalent BTK inhibitor. CB1763 exhibited strong inhibitory activity for both wild type and C481S mutant BTKs with sub-nanomolar enzyme inhibitory potency in a reversible manner (IC50 = 0.85 and 0.99 nM for wild type and C481S, respectively). In cellular assays, CB1763 significantly reduced Tyr223 autophosphorylation of BTK protein at nano-molar concentration in C481S BTK mutant transfected HEK293 cells. Interestingly, persistent inhibition of autophosphorylation was observed after washout of CB1763, while no prolonged inhibition was observed by ibrutinib treatment. CB1763 demonstrated excellent antitumor activity in the BTK driven OCI-Ly10 xenograft mouse model. Conclusions: BTK [C481S] mutation has been reported in ibrutinib-relapsed CLL and MCL patients. Orally available novel non-covalent BTK inhibitor, CB1763, strongly inhibited C481S mutant BTK in vitro and in vivo, suggesting that CB1763 has the potential to treat patients who are relapsed/refractory to ibrutinib. Citation Format: Tokiko Asami, Wataru Kawahata, Shigeki Kashimoto, Masaaki Sawa. CB1763, a highly selective, novel non-covalent BTK inhibitor, targeting ibrutinib-resistant BTK C481S mutant [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B152.

Comparison of Acalabrutinib, A Selective Bruton Tyrosine Kinase Inhibitor, with Ibrutinib in Chronic Lymphocytic Leukemia Cells.

Purpose: Ibrutinib inhibits Bruton tyrosine kinase (BTK) by irreversibly binding to the Cys-481 residue in the enzyme. However, ibrutinib also inhibits several other enzymes that contain cysteine residues homologous to Cys-481 in BTK. Patients with relapsed/refractory or previously untreated chronic lymphocytic leukemia (CLL) demonstrate a high overall response rate to ibrutinib with prolonged survival. Acalabrutinib, a selective BTK inhibitor developed to minimize off-target activity, has shown promising overall response rates in patients with relapsed/refractory CLL. A head-to-head comparison of ibrutinib and acalabrutinib in CLL cell cultures and healthy T cells is needed to understand preclinical biologic and molecular effects.Experimental Design: Using samples from patients with CLL, we compared the effects of both BTK inhibitors on biologic activity, chemokine production, cell migration, BTK phosphorylation, and downstream signaling in primary CLL lymphocytes and on normal T-cell signaling to determine the effects on other kinases.Results: Both BTK inhibitors induced modest cell death accompanied by cleavage of PARP and caspase-3. Production of CCL3 and CCL4 chemokines and pseudoemperipolesis were inhibited by both drugs to a similar degree. These drugs also showed similar inhibitory effects on the phosphorylation of BTK and downstream S6 and ERK kinases. In contrast, off-target effects on SRC-family kinases were more pronounced with ibrutinib than acalabrutinib in healthy T lymphocytes.Conclusions: Both BTK inhibitors show similar biological and molecular profile in primary CLL cells but appear different on their effect on normal T cells. Clin Cancer Res; 23(14); 3734-43. ©2016 AACR.

The Bruton Tyrosine Kinase (BTK) Inhibitor Acalabrutinib Demonstrates Potent On-Target Effects and Efficacy in Two Mouse Models of Chronic Lymphocytic Leukemia.

Purpose: Acalabrutinib (ACP-196) is a novel, potent, and highly selective Bruton tyrosine kinase (BTK) inhibitor, which binds covalently to Cys481 in the ATP-binding pocket of BTK. We sought to evaluate the antitumor effects of acalabrutinib treatment in two established mouse models of chronic lymphocytic leukemia (CLL).Experimental Design: Two distinct mouse models were used, the TCL1 adoptive transfer model where leukemic cells from Eμ-TCL1 transgenic mice are transplanted into C57BL/6 mice, and the human NSG primary CLL xenograft model. Mice received either vehicle or acalabrutinib formulated into the drinking water.Results: Utilizing biochemical assays, we demonstrate that acalabrutinib is a highly selective BTK inhibitor as compared with ibrutinib. In the human CLL NSG xenograft model, treatment with acalabrutinib demonstrated on-target effects, including decreased phosphorylation of PLCγ2, ERK, and significant inhibition of CLL cell proliferation. Furthermore, tumor burden in the spleen of the mice treated with acalabrutinib was significantly decreased compared with vehicle-treated mice. Similarly, in the TCL1 adoptive transfer model, decreased phosphorylation of BTK, PLCγ2, and S6 was observed. Most notably, treatment with acalabrutinib resulted in a significant increase in survival compared with mice receiving vehicle.Conclusions: Treatment with acalabrutinib potently inhibits BTK in vivo, leading to on-target decreases in the activation of key signaling molecules (including BTK, PLCγ2, S6, and ERK). In two complementary mouse models of CLL, acalabrutinib significantly reduced tumor burden and increased survival compared with vehicle treatment. Overall, acalabrutinib showed increased BTK selectivity compared with ibrutinib while demonstrating significant antitumor efficacy in vivo on par with ibrutinib. Clin Cancer Res; 23(11); 2831-41. ©2016 AACR.

Phase I/II, first in human trial of the Bruton’s tyrosine kinase inhibitor (BTKi) M7583 in patients with B cell malignancies.

e14101 Background: BTK is a cytoplasmic tyrosine kinase expressed in cells of hematopoietic origin that is involved in innate and adaptive immunity. BTK inhibition blocks B-cell functions including proliferation, antigen presentation, antibody production, and cell migration. The B-cell receptor pathway is implicated in the pathogenesis of B-cell malignancies such as diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), and B-cell chronic lymphocytic leukemia (CLL). Small molecule BTKis are a new class of agent against B-cell malignancies; ibrutinib has recently been approved for the treatment of resistant/refractory CLL, MCL, and Waldenström’s macroglobulinemia (WM). This ongoing phase I/II clinical trial (NCT02825836) is designed to determine the safety, tolerability, PK, BTK occupancy, and preliminary antitumor activity of the highly selective BTKi M7583 in patients (pts) with refractory/resistant B cell malignancies. Methods: The trial is in two parts: a dose-escalation part for which pts with refractory/resistant B cell malignancies are eligible; and a dose-expansion part for which pts with DLBCL (ABC subtype) or MCL who have failed 1-3 prior lines of therapy are eligible. Pts in the dose-escalation part will be enrolled to sequential dose escalation, with ascending once-daily M7583 doses starting at 80 mg (3 initial days)/160 mg (full 28-day cycle) and increasing according to an adaptive Bayesian design. Results: Three pts have been enrolled to treatment with M7583 80/160 mg (Table). All 3 patients have had an objective response or stable disease and relevant clinical benefit.Conclusions: M7583 appears to have a favorable benefit:risk profile, with an efficacy signal at a dose of 80 mg (3 initial days)/160 mg (full cycle) with objective response or clinical benefit. Clinical trial information: NCT02825836. [Table: see text]

Ibrutinib Represents a Novel Class of Immune Modulating Therapeutics That Enhances the Survival of Activated T Cells in Vitro and In Vivo through a Non-BTK Mediated Mechanism

Background: Ibrutinib is an irreversible inhibitor of Bruton's tyrosine kinase (BTK) that induces durable remissions in chronic lymphocytic leukemia (CLL). In other systems, ibrutinib has also been shown to have an immune modulating effect on T-cells through inhibition of interleukin-associated tyrosine kinase (ITK). Our group and then others have reported that ibrutinib enhances antibacterial and antitumor T cell immune response in murine models. ITK has been demonstrated to be involved in TCR-induced up-regulation of FAS Ligand (FAS-L) and activation induced cell death (AICD) of T cells. In murine models, ITK deficiency or inhibition by specific kinase inhibitors lead to impaired AICD and a much greater persistence and accumulation of activated T cells. We therefore postulate that ibrutinib, by its capability to irreversibly inhibit ITK, may have similar effect on recusing activated T cells from AICD. To date, a comprehensive analysis of the effects on cellular immunity in humans treated with ibrutinib, or more selective BTK inhibitors has not been reported.Methods: PBMCs from previously treated CLL patients enrolled on clinical trials (NCT01589302 and NCT02029443) of ibrutinib (a BTK and ITK inhibitor) and acalabrutinib (ACP-196, a selective BTK inhibitor) were collected at baseline and during treatment at the completion of 2 months( cycle 3) and 5 months (cycle 6). Patients with persistent lymphocytosis post treatment were chosen to allow comparable tumor burden before and after therapy. T cell immune function and the immunosuppressive capacity of CLL cells were evaluated with detailed immunologic and functional studies. Mechanistic studies examining AICD with ibrutinib and acalabrutinib treatment were performed.Results: A total of 25 patients treated with ibrutinib (n=17) and acalabrutinib (n=8) were studied. Ibrutinib, but not acalabrtinib treatment promoted a significant increase (3-5 fold) in absolute CD4(P<0.01) and CD8(p=0.001) T cell numbers in CLL patients that was more prominent in effector/effector memory subsets and less in naïve and central memory subsets (Figure 1). While the number of regulatory T cells (Treg cells) remained unchanged, the ratio of Treg cells to CD4 T cells was reduced by approximately two fold (p<0.001) after ibrutinib treatment. Detailed ex vivo studies demonstrated significantly diminished activation induced cell death of activated T-cells when pre-treated with ibrutinib, but not acalabrutinib (Figure 2). Treatment with ibrutinib or acalabrutinib significantly reduced T cell PD-1 (p<0.01 for CD8 T cells, p<0.05 for CD4 T cells and CTLA-4 (p< 0.05 for CD8 T cells, p<0.001 for CD4 T cells) expression, most prominent in central memory T cell compartment. Ibrutinib and acalabrutinib both significantly decreased CD200 (p<0.001) and BTLA (p<0.001) surface expression as well as IL-10 production by CLL cells after prolonged "B10-Pro" conditioning (p<0.001 for ibrutinib, p<0.05 for acalabrutinib).Conclusions:These findings collectively differentiate ibrutinib as a therapeutic that can both expand activated T-cells and also ameliorate T-cell exhaustion thorough reversal of CLL tumor cell mediated immune suppression. While acalabrutinib reverses CLL cell mediated immune suppression, it lacks ability to expand activated T-cells. Collectively, these data provide support for dual BTK/ITK inhibitor therapeutics when applied to enhance the therapeutic efficacy of adoptive immunotherapy (e.g.chimeric antigen receptor T cells) or tumor vaccines for CLL and potentially other types of hematologic and solid cancers. [Display omitted] [Display omitted] DisclosuresJones:AbbVie: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Membership on an entity’s Board of Directors or advisory committees, Research Funding. Andritsos:Hairy Cell Leukemia Foundation: Research Funding. Awan:Novartis Oncology: Consultancy; Innate Pharma: Research Funding; Pharmacyclics: Consultancy. Fraietta:Novartis: Patents & Royalties: Novartis, Research Funding. June:Novartis: Honoraria, Patents & Royalties: Immunology, Research Funding; Tmunity: Equity Ownership, Other: Founder, stockholder ; University of Pennsylvania: Patents & Royalties; Immune Design: Consultancy, Equity Ownership; Pfizer: Honoraria; Johnson & Johnson: Research Funding; Celldex: Consultancy, Equity Ownership. Maus:Novartis: Patents & Royalties: inventor on patents related to ibrutinib use in T cell therapies. Woyach:Karyopharm: Research Funding; Morphosys: Research Funding; Acerta: Research Funding.