Discovery of HBW-3-10: A potent, orally active, reversible Bruton’s tyrosine kinase (BTK) inhibitor with antitumor activity in mice.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. e15062-e15062
Author(s):  
Ning Lee ◽  
Yingfu Li ◽  
Chester Yuan ◽  
Guanfeng Liu ◽  
Chunchao Yue
Keyword(s):  
B Cell ◽  
Second Generation ◽  
Tumor Model ◽  
B Cell Lymphoma ◽  
Lymphocytic Leukemia ◽  
Tumor Growth Inhibition ◽  
Wild Type ◽  
Btk Inhibitor ◽  
Btk Inhibitors

e15062 Background: The first generation irreversible BTK inhibitor ibrutinib has been approved for the treatment of B cell-related diseases, including chronic lymphocytic leukemia (CLL), for several years. However, CLL patients who used ibrutinib may develop drug resistance due to acquired mutations, in particular BTK C481S that directly impacts the binding of ibrutinib. In recent years, efforts have been made to develop the second generation reversible BTK inhibitors that are effective against both wild-type and C481S mutated B-cell malignancies. LOXO-305 and ARQ-531 are two examples of the second generation reversible BTK inhibitors in advanced clinical trials for ibrutinib-resistant CLL. Methods: New reversible BTK inhibitors were designed, synthesized and tested for enzymatic activities against wild-type and C481S-mutated BTK. Highly active compounds were confirmed for growth effects in diffuse large B-cell lymphoma derived TMD8 cells. Their microsomal stability and ADME properties were also assessed. Potent and bioavailable compounds were further evaluated in vivo efficacy using a TMD8 xenograft tumor model in mice. A 14-day toxicity study in rats was performed correspondingly. Results: As shown in the table, HBW-3-10 has greater potency and pharmacokinetic profile (rats, 10mg/kg PO) than ARQ-531. In a TMD8 mouse xenograft study, HBW-3-10, ARQ-531 and ibrutinib were compared directly, all dosed at 10mg/kg QD, and the resulting tumor growth inhibition rates (TGI) are 38.3%, 9.3% and 22.5%, respectively. Based on our data, HBW-3-10 is more efficacious than ARQ-531 and ibrutinib. In a 14-day preliminary toxicity experiment in rats, both HBW-3-10 and ARQ-531 were dosed 100mg/kg QD. Animals in ARQ531 group started showing signs of sickness on day 3, and all died on day 6 due toxicity; in contrast, no animal in HBW-3-10 group showed any sign of sickness or died during the entire course of study. Conclusions: We have discovered a novel second generation reversible BTK inhibitors HBW-3-10, that has a superior preclinical profile, efficacy and safety than other known ones. HBW-3-10 provides a valuable clinical candidate for treating Ibrutinib resistant CLL and beyond![Table: see text]

scholarly journals Assessing Hematologist's Knowledge of the Use of Ibrutinib and Acalabrutinib in B-Cell Malignancies

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5861-5861
Author(s):  
Lauren Willis ◽  
Melinda Tanzola ◽  
Richard R. Furman
Keyword(s):  
B Cell ◽  
Second Generation ◽  
Trial Data ◽  
Lymphocytic Leukemia ◽  
Clinical Practices ◽  
B Cell Malignancies ◽  
Previously Treated ◽  
Btk Inhibitor ◽  
First And Second Generation ◽  
Btk Inhibitors

Abstract Background: The objective of this study was to assess current clinical practices of hematology/oncology (hem/onc) specialists related to BTK inhibitor use in the treatment of patients with mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL), in order to identify knowledge, competency, and practice gaps and barriers to optimal care. Methods: A continuing medical education (CME)-certified clinical practice assessment consisting of 25 multiple choice questions that were designed to measure knowledge, skills, attitudes, and competence of hem/onc specialists regarding BTK inhibitor use. The survey instrument was made available online to physicians without monetary compensation or charge. Respondent confidentiality was maintained and responses were de-identified and aggregated prior to analyses. The activity launched on May 31, 2018 (after the approval of acalabrutinib for MCL) and participant responses are still being collected at the time of abstract submission. Results: At the time of this report there are 124 hem/onc activity participants, collection is on-going. Demographics are listed in Table 1 and levels of confidence and barriers to incorporating BTK inhibitors are listed in Table 2.Differentiating Between Ibrutinib and AcalabrutinibVery low recognition of the differences between first and second generation BTK inhibitors.Only 16% identified that both ibrutinib and acalabrutinib target BTK at the same amino acid residue.Barely over a quarter (27%) understand that acalabrutinib irreversibly inhibits BTK, similar to ibrutinib.Higher level of knowledge related to the specificity of second generation BTK inhibitors and the differences in dosing frequency compared to first generation BTK inhibitors, but still room for improvement.Half correctly identified acalabrutinib is potentially better tolerated than ibrutinib due to greater relative selectivity for BTK over EGFR, ITK, and TEC kinases.Over half (56%) demonstrated knowledge of the dosing schedule for ibrutinib and acalabrutinib, while 33% incorrectly think both BTK inhibitors are dosed once a day.Strong recognition of the FDA-approved indication for ibrutinib in MCL (74%) with much less recognition of the FDA-approved indication for acalabrutinib in MCL (35%).BTK MutationsLack of knowledge about how the C481S mutation causes resistance to ibrutinib and acalabrutinib.A little over half (54%) understand that a BTK mutation at position 481 from cysteine to serine results in resistance to both ibrutinib and acalabrutinib.Only 33% demonstrated knowledge of the C481S resistance mechanism.Clinical Trial DataMore familiarity with pivotal phase 3 clinical trial data for ibrutinib, indicating a need for further education about phase 2 acalabrutinib data.69% correctly identified the OS outcome with ibrutinib in previously treated CLL43% correctly identified the ORR with acalabrutinib in previously treated CLL26% correctly identified the ORR with acalabrutinib in previously treated MCL Conclusions: This activity indicates there are significant gaps in knowledge and confidence as well as barriers impeding hem/onc ability to incorporate BTK inhibitors into the treatment plans for their patients with B-cell malignancies. There is sub-optimal awareness of differences between first and second generation BTK inhibitors as well as trial data for second generation BTK inhibitors. Additional education is needed to improve the knowledge and confidence of academic and community hem/onc specialists who care for patients with CLL and MCL so they are better equipped to utilize BTK inhibitors. Disclosures Furman: Acerta: Consultancy, Research Funding; Sunesis: Consultancy; Verastem: Consultancy; TG Therapeutics: Consultancy; Genentech: Consultancy; Incyte: Consultancy, Other: DSMB; Gilead: Consultancy; Loxo Oncology: Consultancy; Pharmacyclics LLC, an AbbVie Company: Consultancy; Janssen: Consultancy; AbbVie: Consultancy.

Abstract 4182: The two novel BTK-inhibitors M2951 and M7583 show in vivo anti-tumor activity in pre-clinical models of B cell lymphoma

2017 ◽  
Author(s):  
Eugenio Gaudio ◽  
Chiara Tarantelli ◽  
Emanuele Zucca ◽  
Davide Rossi ◽  
Anastasios Stathis ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Clinical Models ◽  
Anti Tumor Activity ◽  
Btk Inhibitors

Anti-Tumor Activity of the Aurora a Inhibitor MLN8237 in Diffuse Large B-Cell Lymphoma Preclinical Models.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1592-1592 ◽  
Author(s):  
Jessica J Huck ◽  
Mengkun Zhang ◽  
Marc L Hyer ◽  
Mark G Manfredi
Keyword(s):  
Tumor Growth ◽  
Growth Inhibition ◽  
Tumor Model ◽  
B Cell Lymphoma ◽  
Tumor Growth Inhibition ◽  
Aurora A ◽  
Hct 116 ◽  
Xenograft Models

Abstract Aurora A kinase is a serine/threonine protein kinase that is essential for normal transit of cells through mitosis. In many tumor types the Aurora A gene is amplified and/or the protein is over-expressed. The Aurora A small-molecule inhibitor MLN8237 demonstrated robust tumor growth inhibition in xenograft models of solid tumors grown subcutaneously (S.C.) in immunocompromised mice. Here we explored the antitumor activity of MLN8237 in models of diffuse large B-cell lymphoma (DLBCL) both in vitro and in vivo. In vivo three established DLBCL xenograft models (OCI-Ly7, OCI-Ly19, and WSU-DLCL2; all cells expressing luciferase) and a primary DLBCL tumor model PHTX-22-06 were tested using MLN8237 at different doses. Rituximab, an anti-CD20 monoclonal antibody that is active against CD20+ malignant B cells and is a standard of care agent was used for comparison. Using these model systems, tumor cells were injected either I.V. (to evaluate disseminated disease), or S.C. in severe combined immunodeficient mice (SCID). Animals were dosed orally for 21 days with MLN8237 (QD or BID) at various doses, or Rituximab dosed at 10mg/kg IV (once/week) and tumor growth inhibition was monitored using either bioluminescent imaging for the disseminated models or vernier calipers for the S.C. models. Tumor growth inhibition by MLN8237 was dose dependent with 20 mg/kg bid being the most efficacious dose (TGI>100% in both disseminated OCI-Ly19 and WSU models). All animals in the OCI-Ly19 disseminated model 20 mg/kg BID treatment group demonstrated regressions and remained disease free until the end of the study, day 65. In this study the Rituximab treated animals were euthanized on day 31 due to a high level of tumor burden. In the primary tumor model, PHTX-22-06, MLN8237 dosed at 20 mg/kg BID was also the most efficacious with a TGI of 95%. Moreover, tumor growth inhibition was durable as determined by prolonged tumor growth delay (>50 days). Significant efficacy was achieved in all models tested, whether grown as disseminated or subcutaneous models. A noted increase in durability of response was observed with MLN8237 treatment when compared with previous data from solid tumor models. In vitro, MLN8237 treatment increased levels of apoptosis in the OCI-Ly19 cells in comparison to the solid tumor cell line HCT-116 (colon). Greater Annexin V positive cells and greater cleaved PARP and Caspase-3 signals were detected in the MLN8237 treated OCI-Ly19 cells when compared to HCT-116 cells. The demonstration of robust and durable anti-tumor activity in preclinical models treated with MLN8237 provides the basis for its clinical evaluation as a treatment option for DLBCL. MLN8237 is currently in multiple Phase I clinical trials.

Self-Enforcing Feedback Activation Between BCL6 and Tonic Pre-B Cell Receptor Signaling in Acute Lymphoblastic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 284-284
Author(s):  
Huimin Geng ◽  
Christian Hurtz ◽  
Dirk Baumjohann ◽  
Zhengshan Chen ◽  
Wei-Yi Chen ◽  
...  
Keyword(s):  
B Cell ◽  
Tyrosine Kinases ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
Bcr Signaling ◽  
Btk Inhibitor ◽  
Bcl6 Expression ◽  
Targeting Strategy

Abstract Background and hypothesis: Like mature B cell lymphoma, pre-B ALL originates from B cell precursors that critically depend on survival signals emanating from a functional (pre-) B cell receptor (BCR). While recent work successfully introduced BCR signaling inhibitors into patient care for various subtypes of mature B cell lymphoma, it is not known whether pre-BCR signaling represents a therapeutic target in pre-B ALL and in which cytogenetic subsets targeting of pre-BCR signaling will be effective. In this study we demonstrated that ALL can be subdivided into two groups that fundamentally differ with respect to pre-BCR signalling. We identified a novel mechanism of self-enforcing feedback activation between the transcription factor BCL6 and tonic pre-BCR signaling in pre-BCR+ ALL and proposed a dual targeting strategy of both BCL6 and pre-BCR related tyrosine kinases for the treatment of patients with pre-BCR+ ALL. Results: Flow cytometry analysis of surface pre-BCR expression (λ5, VpreB), cytoplasmic μ heavy chain (μHC) expression and intracellular Ca2+ signal in 29 patient-derived pre-B ALL xenograft samples and cell lines showed pre-BCR expression and activity in a subset of pre-B ALL, including all TCF3-PBX1 cases studied (n=4) and two cases with deletions at 6q21. Studying 830 pre-B ALL cases from four clinical trials (MDACC, St. Jude, COG P9906 and ECOG E2993), tonic pre-BCR signaling and constitutive PI3K-AKT activation was found in 112 cases (13.5%), including 93% TCF3-PBX1 (53 of 57), del (6)(q21) (7 of 7), PBX1 (1q23) duplication (4 of 4), MLL-rearrangement (3 of 86), hyperdiploid (2 of 43) and other (43 of 406) pre-B ALL cases. In other major ALL subtypes, we found no evidence for pre-BCR expression and activity, including BCR-ABL1 (0 of 196) and ETV6-RUNX1 (0 of 31). We found frequent 1q23 (PBX1) duplication, TCF3-PBX1 or other PBX1-rearrangement, 6q21 (PRDM1) deletion in ALL cells with tonic pre-BCR signaling. Development of a genetic mouse model for inducible ablation of Bcl6. Pre-BCR-induced activation of BCL6 relieves PRDM1-mediated repression of pre-BCR signaling components and positively regulates pre-BCR signaling output at the transcriptional level. The clinical data (COG P9906, ECOG E2993) revealed that high mRNA levels of BCL6 at the time of diagnosis is predictive of poor clinical outcome specifically in patients with pre-BCR+ ALL but not ALL cells lacking pre-BCR expression. These findings suggest an important role of BCL6 as a cofactor of pre-BCR signaling in a large subset of ALL. To directly test the role of Bcl6- and pre-BCR interactions, we generated a novel mouse model for inducible Cre-mediated deletion of Bcl6 exons 5-10, flanked by loxP sites. For lineage-specific deletion in vivo, we crossed these mice with an Mb1-Cre deleter strain, in which Bcl6 was deleted in pro-B cells, resulting in a differentiation block at the pre-B cell stage. Deletion of Bcl6 in mouse pre-BCR+ ALL and expression of a dominate-negative form of BCL6 in human primary pre-BCR+ALL cells, both rapidly induced cell death, indicating BCL6 cooperates with the pre-BCR in leukemic transformation. Cooperation between pre-BCR and BCL6 signaling. Inhibition of BCL6 via the specific BCL6 inhibitor RI-BPI showed compromised colony formation and induced cell cycle arrest. Interestingly, constitutive BCL6 expression was sensitive to inhibition of SYK and SRC tyrosine kinases downstream of the pre-BCR. Treating 6 pre-BCR+ and 8 pre-BCR- patient-derived ALL samples with the SYK inhibitor (PRT06207), BTK inhibitor (Ibrutinib) or a broader SRC and BTK inhibitor Dasatinib, we observed remarkably decreased BCL6 expression and increased apoptosis in pre-BCR+ but not pre-BCR- ALL cells. In vivo treatments with Dasatinib prevented leukemia initiation and significantly prolonged survival of the recipient mice that were injected with primary pre-BCR+ ALL cells, compared to non-treatment or Nilotinib-treatment. These data demonstrate that both inhibition of BCL6 and pre-BCR signaling selectively killed patient-derived pre-BCR+ ALL cells. Conclusions: Our study identified two distinct subtypes of pre-B ALL that fundamentally differ with respect to pre-BCR signaling. Tonic pre-BCR signaling engages a BCL6-dependent, self-enforcing amplification loop. Based on these findings, we propose a dual targeting strategy of BCL6 and pre-BCR tyrosine kinases for the treatment of patients with pre-BCR+ALL. Disclosures No relevant conflicts of interest to declare.

scholarly journals Dual Conditional Loss of BLIMP-1 and p53 in B-Cells Drives B-Cell Lymphomagenesis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4169-4169
Author(s):  
Antonio Sacco ◽  
Yawara Kawano ◽  
Michele Moschetta ◽  
Jihye Park ◽  
Oksana Zavidij ◽  
...  
Keyword(s):  
B Cells ◽  
Lymph Nodes ◽  
B Cell ◽  
Southern Blotting ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Advisory Board ◽  
Whole Exome ◽  
Btk Inhibitor

Abstract Background. p53 is a well defined tumor suppressor involved in the modulation of cell proliferation, cell cycle progression and programmed cell death. BLIMP-1 plays a crucial role in modulating B-cell differentiation towards Ig-secreting plasma cells, and it acts as a tumor suppressor, as documented in both diffuse large B-cell lymphoma and Burkitt lymphoma. Whether B-cell specific loss of both p53 and BLIMP-1 may favor a B-cell lymphoma phenotype remains unanswered. We therefore aimed to generate in vivo dual p53/BLIMP-1-floxed conditional inactivation in B-cells, and to define the functional relevance of both p53 and BLIMP-1 n B-cell lymphomagenesis in vivo Methods.Cre recombinase under the control of CD19 promoter (C57BL/6 CD19Cre/Cre) mice were crossed with either C57BL/6 BLIMPflox/flox or C57BL/6 p53flox/flox mice to achieve deletion of BLIMP or p53, respectively, in B cells. Secondly, CD19Cre/Cre BLIMPflox/flox mice were crossed with CD19Cre/Cre p53flox/flox to achieve concomitant deletion of both BLIMP and p53 in B cells (CD19Cre/Cre BLIMPflox/flox p53flox/flox), referred as CD19/Bl-/p53- mice. Transgenic experimental mice (CD19/Bl-/p53-) where characterized for B cell infiltration using immunohistochemistry, flow cytometry; clonotypic immunoglobulin heavy-chain rearrangement was assessed by Southern Blotting. Whole exome sequencing was performed using DNA isolated from B220+ selected cells obtained from pathological lymph nodes of CD19/Bl-/p53- mice and from matched tail-derived tissues, used as germline (Illumina HiSeq 2500 platform; Agilent SureSelectXT). MTT assay was used to BTK-inhibitor-dependent cytotoxicity using CD19/Bl-/p53-derived B220 cells. Results.We generated dual p53/BLIMP-1-floxed conditional inactivation in B-cells, using mice expressing Cre recombinase under the control of CD19 promoter. 100% of the CD19/Bl-/p53- mice presented with diffuse lymphadenomegalies, and splenomegaly, hepatomegaly (90.3% and 77.4%, respectively). Other clinical manifestations included presence of ascites and hind lymb paralysis (12.9% and 19.3%, respectively). The CD19/Bl-/p53- showed worse survival compared to Bl-/p53- mice non-expressing the CD19/Cre recombinase, CD19/p53-, or CD19/Bl- (363, 469.5, 460.5, and 770 days, respectively). H.E. staining of CD19/Bl-/p53--derived lymph nodes, defined a nodal architecture with a monomorphic population of large sized atypical lymphoid cells with finely clumped and dispersed chromatin, and multiple basophilic medium sized, paracentrally situated nucleoli. A "starry sky" pattern was also observed. Overall, these features are compatible with a high-grade lymphomas. IHC analysis confirmed a marked positivity for B220 staining (TdT, Bcl6, CD138 and CD4, CD8 negative). Tumors were confirmed to be B220+/IgM+, with either Igk- or Ig-lambda-restriction as demonstrated by flow cytometry; and either mono- or bi-clonal, as demonstrated by Southern blotting, thus further confirming the clonal transformation induced by dual BLIMP/p53 deletion in B cells. Whole exome sequencing was performed from B220+ selected cells obtained from pathological lymph nodes of CD19/Bl-/p53- mice and identified 143 SNVs. Among them, non-synonymous somatic mutations were mapped on genes involved in the regulation of focal adhesion, PDGF signaling, p53-downstream pathway, and lipoprotein metabolism. B220+ cells selected from CD19/Bl-/p53--derived lymph nodes were implanted subcutaneously into recipient SCID/Bg mice (n: 10), and presented with 100% engraftment, with a monomorphic lymphoid infiltration of B220+ and IgM+ cells. B220 positive cells were selected from the s.q. tumor and intravenous injected into recipient SCID/Bg (n: 10) and BL/6 mice (n: 10). Engraftment was demonstrated in all the mice, where hepatomegaly, splenomegaly and hind lymb paralysis were observed. Infiltration of B220+ cells was documented within bone marrow, liver and spleen. We next investigated the anti-tumor activity of BTK-inhibitor, and found that B220+ cells selected from lymph nodes harvested from CD19/Bl-/p53-mice were sensitive to ibrutinib treatment. Conclusion. These studies demonstrate that the specific dual inactivation of p53 and BLIMP in B-cells promotes oncogenic transformation, resulting in aggressive B-cell lymphoma development. Disclosures Ghobrial: Celgene: Other: Advisory Board; BMS: Other: Advisory Board; Amgen: Other: Advisory Board; Takeda: Other: Advisory Board; Janssen: Other: Advisory Board. Roccaro:Takeda Pharmaceutical Company Limited: Honoraria.

scholarly journals Tisagenlecleucel cellular kinetics, dose, and immunogenicity in relation to clinical factors in relapsed/refractory DLBCL

2020 ◽  
Vol 4 (3) ◽  
pp. 560-572 ◽  
Author(s):  
Rakesh Awasthi ◽  
Lida Pacaud ◽  
Edward Waldron ◽  
Constantine S. Tam ◽  
Ulrich Jäger ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
Lymphoblastic Leukemia ◽  
Quantitative Polymerase Chain Reaction ◽  
Statistical Significance ◽  
B Cell Lymphoma ◽  
Lymphocytic Leukemia ◽  
Extrinsic Factors ◽  
Cellular Kinetics

Abstract The anti-CD19 chimeric antigen receptor (CAR)–T cell therapy tisagenlecleucel was evaluated in the global, phase 2 JULIET study in adult patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL). We correlated tisagenlecleucel cellular kinetics with clinical/product parameters in 111 patients treated in JULIET. Tisagenlecleucel persistence in responders and nonresponders, respectively, was demonstrated for 554 and 400 days maximum by flow cytometry and for 693 and 374 days maximum by quantitative polymerase chain reaction (qPCR). No relationships were identified between cellular kinetics (qPCR) and product characteristics, intrinsic/extrinsic factors, dose, or immunogenicity. Most patients with 3-month response had detectable transgene at time of response and continued persistence for ≥6 months. Expansion (maximal expansion of transgene/CAR-positive T-cell levels in vivo postinfusion [Cmax]) was potentially associated with response duration but this did not reach statistical significance (hazard ratio for a twofold increase in Cmax, 0.79; 95% confidence interval, 0.61-1.01). Tisagenlecleucel expansion was associated with cytokine-release syndrome (CRS) severity and tocilizumab use; no relationships were observed with neurologic events. Transgene levels were associated with B-cell levels. Dose was associated with CRS severity, but this was not statistically significant after adjusting for baseline tumor burden. In contrast to the results from B-cell precursor acute lymphoblastic leukemia (B-ALL) and chronic lymphocytic leukemia, similar exposure was observed in DLBCL in this study regardless of response and expansion was lower in DLBCL than B-ALL, likely from differences in cancer location and/or T-cell intrinsic factors. Relationships between expansion and CRS severity, and lack of relationships between dose and exposure, were similar between DLBCL and B-ALL. Tisagenlecleucel cellular kinetics in adult relapsed/refractory DLBCL improve current understanding of in vivo expansion and its relationships with safety/efficacy endpoints. This trial was registered at www.clinicaltrials.gov as #NCT02445248.

scholarly journals Macrophage-Mediated Antibody Dependent Effector Function in Aggressive B-Cell Lymphoma Treatment is Enhanced by Ibrutinib via Inhibition of JAK2

Cancers ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 2303
Author(s):  
Verena Barbarino ◽  
Sinika Henschke ◽  
Stuart Blakemore ◽  
Elena Izquierdo ◽  
Michael Michalik ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
B Cell ◽  
Cell Lymphoma ◽  
Antibody Therapy ◽  
B Cell Lymphoma ◽  
Lymphocytic Leukemia ◽  
Janus Kinase ◽  
Monoclonal Antibody Therapy ◽  
Antibody Treatment ◽  
Btk Inhibitors

Targeted inhibition of Bruton’s Tyrosine Kinase (BTK) with ibrutinib and other agents has become important treatment options in chronic lymphocytic leukemia, Waldenström’s Macroglobulinemia, Mantle cell lymphoma, and non-GCB DLBCL. Clinical trials combining small molecule inhibitors with monoclonal antibodies have been initiated at rapid pace, with the biological understanding between their synergistic interactions lagging behind. Here, we have evaluated the synergy between BTK inhibitors and monoclonal antibody therapy via macrophage mediated antibody dependent cellular phagocytosis (ADCP). Initially, we observed increased ADCP with ibrutinib, whilst second generation BTK inhibitors failed to synergistically interact with monoclonal antibody treatment. Kinase activity profiling under BTK inhibition identified significant loss of Janus Kinase 2 (JAK2) only under ibrutinib treatment. We validated this potential off-target effect via JAK inhibition in vitro as well as with CRISPR/Cas9 JAK2−/− experiments in vivo, showing increased ADCP and prolonged survival, respectively. This data supports inhibition of the JAK-STAT (Signal Transducers and Activators of Transcription) signaling pathway in B-cell malignancies in combination with monoclonal antibody therapy to increase macrophage-mediated immune responses.

Disruption of BIRC3 associates with Fludarabine Chemorefractoriness in TP53 Wild Type Chronic Lymphocytic Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 466-466
Author(s):  
Marco Fangazio ◽  
Silvia Rasi ◽  
Tiziana Vaisitti ◽  
Sara Monti ◽  
Stefania Cresta ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Marginal Zone ◽  
B Cell Lymphoma ◽  
Lymphocytic Leukemia ◽  
Consecutive Series ◽  
Wild Type ◽  
Poor Outcome ◽  
Inactivating Mutations ◽  
Large B Cell

Abstract Abstract 466 The genetic lesions identified to date do not fully recapitulate the molecular pathogenesis of chronic lymphocytic leukemia (CLL) and do not entirely explain the development of severe complications, such as chemorefractoriness. BIRC3, along with TRAF2 and TRAF3, cooperate in the same protein complex that negatively regulates MAP3K14, the central activator of non-canonical NF-kB signaling. Following the initial observation of recurrent BIRC3 mutations in splenic marginal zone lymphomas, we performed targeted re-sequencing of the BIRC3 coding sequence and splicing sites across the spectrum of B-cell neoplasia. By this screening, BIRC3 mutations were identified only in CLL (2/20), while were absent in diffuse large B-cell lymphoma (0/30), Burkitt lymphoma (0/38), follicular lymphoma (0/20), extranodal marginal zone lymphoma (0/65), hairy cell leukemia (0/19), and multiple myeloma (0/22). This observation prompted the investigation of prevalence and clinical impact of BIRC3 genetic lesion in CLL. The study population included 4 clinical CLL panels representative of different disease phases: i) a cohort of fludarabine-refractory CLL (n=49); ii) a cohort of fludarabine-sensitive CLL (n=62); iii) clonally related Richter syndrome (RS) (n=33; all diffuse large B cell lymphomas); and iv) a consecutive series of newly diagnosed and previously untreated CLL (n=308). BIRC3 was analyzed for mutations by Sanger sequencing (exons 2–9) and for copy number abnormalities by FISH (probe RP11-177O8). BIRC3 was affected in 12/49 (24%) fludarabine-refractory CLL by inactivating mutations (7 frameshift and 1 non-sense) and gene deletions (n=7) that distributed in a mutually exclusive fashion with TP53 disruption (p=.004) (Fig 1A and 1B). Two fludarabine-refractory CLL carried a biallelic inactivation of the BIRC3 gene by mutation of one allele and deletion of the second allele. All inactivating mutations were somatically acquired, were predicted to generate aberrant transcripts carrying premature stop codons, and caused elimination or truncation of the C-terminal RING domain, whose E3 ubiquitin ligase activity is essential for MAP3K14 proteosomal degradation by BIRC3. Western blot analysis confirmed the expression of aberrant bands corresponding in size to the predicted truncated BIRC3, and revealed constitutive non-canonical NF-kB activation in fludarabine-refractory CLL harboring BIRC3 disruption by mutations or deletion, as documented by NFkB2 processing from p100 to p52 (Fig. 1). BIRC3 was the sole gene of the TRAF3/MAP3K14-TRAF2/BIRC3 complex targeted by molecular lesions after extensive mutation and FISH analysis of TRAF2, TRAF3 and MAP3K14 in the fludarabine-refractory CLL cohort (n=49). To investigate whether BIRC3 genetic lesions were restricted to chemorefractory cases, we analyzed BIRC3 for mutations and deletions in other CLL subsets. Fludarabine-sensitive CLL were consistently devoid of BIRC3 disruption in all cases (n=62), suggesting that BIRC3 genetic lesions specifically associate with a chemorefractory phenotype among CLL requiring treatment (Fig. 1A). BIRC3 disruption was restricted to 1/33 (3.0%) clonally-related RS, corroborating the notion that RS is molecularly distinct from chemorefractory progression without transformation (Fig. 1A). In a consecutive series evaluated at CLL diagnosis, BIRC3 disruption was rare (13/308, 4%, all TP53 wild type), associated with primary chemorefractoriness among cases requiring treatment, and predicted poor outcome (Fig. 1A). By univariate analysis, the crude impact of BIRC3 disruption on survival was a ∼5.3 fold increase in the hazard of death (HR: 5.31; 95% CI: 2.62–10.73) and a significant overall survival (OS) shortening (median OS in BIRC3 disrupted cases: 3.1 years vs not reached in BIRC3 wild type cases; p<.001) (Fig. 1D). Multivariate analysis selected BIRC3 disruption as an independent risk factor of OS (HR: 4.25; 95% CI: 1.74–10.36; p=.001) after controlling for confounding clinical (age and Rai stage) and genetic (IGHV mutation status, 11q22-q23 deletion, TP53 disruption, NOTCH1 mutations) variables at diagnosis. These data document that disruption of BIRC3, a critical regulator of non-canonical NF-kB signaling, associates with fludarabine-refractoriness among TP53 wild type CLL and identifies a subgroup of patients showing poor outcome. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Bruton's Tyrosine Kinase (BTK) Inhibitor ARQ 531 Effectively Inhibits Wild Type and C481S Mutant BTK and Is Superior to Ibrutinib in a Mouse Model of Chronic Lymphocytic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3232-3232 ◽  
Author(s):  
Sean D. Reiff ◽  
Rose Mantel ◽  
Lisa L. Smith ◽  
Samantha McWhorter ◽  
Virginia M. Goettl ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Mouse Model ◽  
Research Funding ◽  
Potent Inhibitor ◽  
Lymphocytic Leukemia ◽  
Transfer Model ◽  
Wild Type ◽  
Bcr Signaling ◽  
Btk Inhibitor

Abstract Introduction: The Bruton's tyrosine kinase inhibitor ibrutinib improves survival in chronic lymphocytic leukemia (CLL) compared to standard chemotherapy or immune therapy. In a subset of patients, somatic mutation (C481S) of its binding site results in acquired resistance to ibrutinib therapy with poor clinical outcome. ARQ 531 is an ATP competitive, orally bioavailable, potent inhibitor of BTK and other relevant kinases including the majority of Src and Tec family kinases. Herein we present preclinical data with ARQ 531 in CLL including C481S mutated BTK and its efficacy versus ibrutinib in the TCL1 mouse model of CLL. Methods: Potency of ARQ 531 and its binding kinetics were measured in enzymatic and Surface Plasmon Resonance (SPR) binding assays. Primary CLL B cells were negatively selected and treated with 1μM ARQ 531. BCR signaling was investigated by immunoblot following a 1 hour drug incubation. CLL cells migrating towards CXCL12 and CXCL13 after 4 hours across a 5.0 micron transwell insert were counted by flow cytometry. Annexin V and propidium iodide flow cytometry was used to measure CLL viability over a range of drug concentrations and time. CpG mediated CLL cell activation was measured by CD40 and CD86 expression by flow cytometry. In vivo investigation utilized B6 mice engrafted with 1E7 CD5+/CD19+ TCL1 lymphocytes via tail vein injection. Mice were randomized to treatment with vehicle, ARQ 531, or ibrutinib following the establishment of a CD5+/CD19+ population >10% in peripheral blood. Results: At 72 hours the viability of CLL cells treated with 0.1µM, 1.0µM, or 10.0µM ARQ 531 was found to be 94%, 67%, and 50% that of untreated samples, respectively (p=0.76, p<0.001, p=0.016) in comparison to the 71% relative viability seen with 1µM ibrutinib (p<0.001). ARQ 531 demonstrates dose dependent inhibition of BTK, AKT, and ERK phosphorylation. Stimulation through TLR9 by CpG ligand increases expression of the activation markers CD40 and CD86; ARQ 531 decreased CpG induced expression of CD40 by 25% and CD86 by 40% (p=0.022, p=0.041). ARQ 531 in the presence of CXCL12 or CXCL13 decreased migration by 51% and 66%, respectively (p=0.015, p=0.025), below baseline migration of untreated CLL. Unlike ibrutinib, ARQ 531 inhibits both wild type and C481S BTK with an IC50 less than 1nM in a biochemical assay, and inhibits phosphorylation of C481S mutated BTK in LV125 transfected HEK293T cells. Cytotoxicity was also observed in CLL cells isolated from patients with C481S BTK mutations, where the viability of cells treated with 1µM ARQ 531 was 72% that of untreated cells at 72 hours (p=0.038). SPR binding assay showed a residence time of 51 minutes in wild type BTK and 56 minutes in C481S mutated BTK. The TCL1 mouse model displays active BCR signaling and responds to treatment with the BTK inhibitors ibrutinib and acalabrutinib (Ponader S, 2012; Herman SEM, 2015). Following establishment of leukemia in the TCL1 transfer model, we randomized mice to treatment with vehicle (n=14), 25 mg/kg ibrutinib (n=6), 50 mg/kg ARQ 531 (n=14) or 75 mg/kg ARQ 531 (n=14) given by daily gavage. ARQ 531 significantly improved survival over both ibrutinib and vehicle (median survival: 36 days vehicle, 53 days ibrutinib, not reached by 74 days 50mg/kg and 75 mg/kg ARQ 531, p<0.0001). Mice receiving ARQ 531 have consistently lower lymphocyte counts on microscopic examination and reduced CD5+/CD19+ populations via flow cytometry compared to vehicle or ibrutinib. Additionally, we have observed increases in absolute neutrophil count over time in mice receiving ARQ 531. Pharmacokinetic studies are pending; however, in a single oral dose study of 10mg/kg in monkeys, the bioavailability was 72.4% with a CMax of 9µM and a half-life greater than 24 hours. Conclusion: The BTK inhibitor ARQ 531 is a potent inhibitor of BTK with promising activity both in vitro and in vivo. Multi-targeted inhibition of cytokine, chemokine, and BCR pathways by ARQ 531 decreases activation, migration, and viability of CLL cells. Unlike ibrutinib, ARQ 531 inhibits activation of C481S mutated BTK variants and maintains cytotoxicity in ibrutinib resistant clones. Additionally, ARQ 531 has demonstrated remarkable efficacy in an in vivo TCL1 adoptive transfer model, improving survival to a greater extent than ibrutinib and potentially restoring granulocyte production. These data justify continued preclinical work and development to facilitate transition to clinical trials. Disclosures Eathiraj: ArQule, Inc: Employment. Abbadessa:ArQule, Inc: Employment. Schwartz:ArQule, Inc: Employment. Woyach:Acerta: Research Funding; Morphosys: Research Funding; Karyopharm: Research Funding.

Blockade of the CD47/SIRPα Checkpoint Potentiates the Anti-Tumor Efficacy of Tafasitamab

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 11-12
Author(s):  
Doris Mangelberger ◽  
Christian Augsberger ◽  
Karin Landgraf ◽  
Christina Heitmüller ◽  
Stefan Steidl
Keyword(s):  
Monoclonal Antibody ◽  
Tumor Growth ◽  
B Cell ◽  
Tumor Model ◽  
B Cell Lymphoma ◽  
Checkpoint Blockade ◽  
Subcutaneous Tumor ◽  
Anti Tumor Activity

Introduction Tafasitamab (MOR208) is an Fc-enhanced, humanized, monoclonal antibody that targets CD19 and has shown promising clinical activity in patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL). CD19 is homogeneously expressed among different B-cell malignancies, and the binding of tafasitamab to CD19 directly mediates cell death, induces antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis. Aiming to potentiate the tafasitamab-mediated "eat me" signal, we tested a combination with a CD47-directed monoclonal antibody (mAb) to inhibit the CD47/SIRPα "don't eat me" signal and further enhance macrophage-mediated phagocytosis. Preclinical studies demonstrated that blocking the CD47/SIRPα checkpoint in combination with antibodies, such as rituximab, increased phagocytosis by macrophages, resulting in effective anti-tumor effects in non-Hodgkin lymphoma (NHL) (Chao, et al. 2010). Additionally, the combination of the anti-CD47, magrolimab, and the anti-CD20, rituximab, demonstrated beneficial outcomes for patients with refractory NHL (Advani, et al. 2019). Here, we present in vitro and in vivo data on the combinatory effect of tafasitamab and an anti-CD47 mAb in preclinical models of Burkitt's lymphoma (BL). Methods During in vitro studies, CD14+ monocytes were isolated from the whole blood of healthy volunteers and differentiated with 50 ng/mL M-CSF for 6 days. ADCP was analyzed by flow cytometry in co-culture experiments with Ramos cells (BL) after 3 hours of treatment with tafasitamab and anti-CD47 mAb (clone B6H12). In vivo, the combination of tafasitamab with an anti-CD47 mAb was tested in a Ramos disseminated survival and subcutaneous tumor model in SCID and NOD-SCID mice, respectively. In both models, tafasitamab was administered therapeutically twice a week either at 3 mg/kg (disseminated) or 10 mg/kg (subcutaneous) for max. 4 weeks. The anti-CD47 mAb was administered at 4 mg/kg three times per week. Main study readouts were to assess animal survival and any delays in tumor growth. Results The combination of tafasitamab + CD47/SIRPα checkpoint blockade enhanced ADCP activity of primary M2 macrophages on BL-derived Ramos cells, in comparison with the anti-CD47 mAb or tafasitamab monotherapies (Figure 1A). In vivo, a significant increase in anti-tumor activity was observed with the combination of tafasitamab + anti-CD47 mAb. In the Ramos disseminated survival model, the combination showed an increased life span (ILS) of &gt;182% compared with tafasitamab monotherapy control, with an overall survival of all animals treated with the combination (15/15) until the end of the study (Day 99 post-cell injection). Additionally, pronounced anti-tumor efficacies were detected in the Ramos subcutaneous tumor model. Here, the combination resulted in a significant delay in tumor growth compared with the tafasitamab or anti-CD47 mAb monotherapies (ILS &gt;175% tafasitamab and ILS &gt;72% anti-CD47 mAb vs tafasitamab + B6H12) (Figure 1B). Conclusions The ADCP activity of primary macrophages was increased by combining tafasitamab with an anti-CD47 mAb in vitro, resulting in enhanced anti-tumor activity compared with tafasitamab or anti-CD47 mAb monotherapies in vivo. Overall, results indicate the combination of tafasitamab with a CD47/SIRPα checkpoint blockade may be a promising novel combination approach for lymphoma therapy. Disclosures Mangelberger: MorphoSys AG: Current Employment. Augsberger:MorphoSys AG: Current Employment. Landgraf:MorphoSys AG: Current Employment. Heitmüller:MorphoSys AG: Current Employment. Steidl:MorphoSys AG: Current Employment.

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