Project Summary Unconjugated monoclonal antibodies (mAbs) are highly effective but non-curative targeted therapies for B cell malignancies. The Bruton tyrosine kinase inhibitor (BTKi) ibrutinib improved outcomes but does not cure chronic lymphocytic leukemia (CLL), the most prevalent lymphoid malignancy in the USA. Ibrutinib mobilizes CLL cells into the circulation where they are highly sensitive to mAb targeting CD20 (αCD20 mAb). However, addition of standard doses of the αCD20 mAb rituximab (RTX) to ibrutinib showed no benefit in clinical trials. Studies by us and others suggest that limited innate immune cytotoxic capacity, decreased CLL cell CD20 after standard dose αCD20 mAb therapy, and inhibition of RTX induced cytotoxicity by off-target effects of ibrutinib are largely responsible for this negative outcome. We propose that these data on the mechanisms of action of αCD20 mAb and BTKi can be used to improve combination targeted therapy. We and others have shown that activating antibody dependent cellular phagocytosis (ADCP) by fixed macrophages is the primary mechanism of action of αCD20 mAb. We then showed that initially rapid engulfment of target lymphocytes during ADCP (< 1h) is followed by a prolonged pause of ~ 24h (hypophagia) which limits innate immune cytotoxic capacity. In addition, standard dose αCD20 mAb therapy induces sufficient non-lethal removal of B-cell membrane CD20-αCD20 mAb immune complexes from circulating CLL cells by immune effector cells (trogocytosis) to cause drug resistance. mAb activity against malignant B cells in lymphoid tissue is limited by slow tissue penetration by these large molecules, limited innate immune cytotoxic capacity, and microenvironment support. Ibrutinib can mobilize lymphoid tissue resident CLL cells into the circulation where they are sensitive to αCD20 mAb mediated cytotoxicity. Unfortunately, ibrutinib also inhibits mAb mediated cellular cytotoxicity including ADCP by off-target effects. In contrast the more specific BTKi acalabrutinib mobilizes CLL cells into the circulation without inhibiting macrophage mediated ADCP. Using these data, we have repurposed RTX in a novel high frequency low dose regimen with acalabrutinib for initial treatment of patients with CLL. The regimen is being tested in a fully enrolled (n=38) clinical trial funded by Acerta/AstraZeneca (ClinicalTrials.gov NCT03788291). We are requesting funding to perform the correlative studies required to determine if this design is achieving the stated goals: 1) Determine if sequential low dose RTX can achieve maximum clearance of circulating CLL cells and 2) Determine the effect of acalabrutinib on sensitivity of CLL cells to RTX induced cytotoxicity. Data generated by these studies are essential for designing future clinical trials to test strategies to optimize treatment efficacy, tolerance, convenience and accessibility at lower cost. A potential triplicate therapy of HFLD αCD20 mAb, BTK and BCL2 inhibitors could result ...