Ibrutinib Off-Target Inhibition Inhibits Antibody-Dependent Cellular Phagocytosis but Not Efferocytosis of CLL Cells
Authors: Chu Charles C. , Pinney Jonathan J. , Blick-Nitko Sara K. , Baran Andrea M. , Peterson Derick R. , Whitehead Hannah E. , Izumi Raquel , Munugalavadla Veerendra , Barr Paul M. , VanDerMeid Karl R. , Elliott Michael Rusty , Zent Clive S. 
Published: November 2020

Background
Combinations of different targeted therapies, including Bruton tyrosine kinase (BTK) inhibitors and anti-CD20 monoclonal antibodies (mAbs) could improve treatment for CLL. Unexpectedly, the combination of ibrutinib (IBR) with rituximab did not show additional clinical benefit. However, IBR inhibits many off-target molecules that may limit therapeutic mAb clinical effectiveness and a more selective BTK inhibitor, such as acalabrutinib (ACALA), could be more effective in combination with mAb therapy. Initial data from the ELEVATE TN trial support this possibility. IBR off-target effects on antibody-dependent cellular phagocytosis (ADCP), the major mechanism of therapeutic mAb activity could explain this difference. Additionally, IBR induces a higher and longer duration increase in circulating lymphocytes than ACALA. IBR off-target effects on efferocytosis, another phagocytic process involved in apoptotic cell removal, might explain this difference.
Methods
Using state-of-the-art direct kinetic measurements of phagocytosis by time-lapse video, (Chu et al. J Cell Sci 2020;133:jcs237883) we investigated the effects of IBR and ACALA on phagocytosis (ADCP or efferocytosis) by human monocyte-derived macrophages (hMDM) in vitro. Live cell time-lapse video of 10 μg/ml rituximab (Genentech) mediated ADCP of CLL cells by CellTracker Deep Red (CTDR, Thermofisher) labeled hMDM (20:1 CLL:hMDM cell ratio) either untreated or treated with IBR or ACALA (3-fold serial dilutions from 100 to 0.41 μM) was imaged in a stage-top environmental chamber (37°C and 5% CO2) mounted onto a Nikon Ti-Eclipse inverted microscope with an ELWD 20x/0.45NA S Plan Fluor Ph1 objective and an Andor Zyla 5.5 sCMOS camera. Images were captured sequentially every 4 min over 2.8 h. For each experiment (n = 18), duplicate or triplicate wells for each drug concentration were imaged. For efferocytosis, live cell time-lapse video imaging of phagocytosis of pHrodo iFL Red STP ester (pHrodo Red, Thermo Fisher Scientific) labeled apoptotic CLL cells by CTDR-labeled hMDM (20:1 CLL:hMDM cell ratio) either untreated or treated with IBR or ACALA (2-fold serial dilutions from 10 to 1.25 μM) was collected every 4 min over 2.8 h. For each experiment (n = 7), duplicate or triplicate wells for each drug concentration was imaged and analyzed. Finally, for efferocytosis, the intensity of pHrodo Red dye, a pH-sensitive dye that increases in intensity with acidic pH, as found in the endolysosomes, was measured in the pHrodo Red color channel and analyzed.
Results
IBR significantly inhibited ADCP at all measured drug concentrations (0.41 μM, p &lt; 0.05; 1.2 μM, p &lt; 0.01; 3.7 - 100 μM, p &lt; 0.001). The mean peak free drug concentration (Cmax) achieved clinically by standard doses for IBR is ~0.5 μM. ACALA only significantly inhibited ADCP at the highest concentration (100 μM, p &lt; 0.001). The Cmax achieved clinically by standard doses for ACALA is ~1.2 μM. ACALA did not inhibit efferocytosis or subsequent transition to endolysosomal compartment at all tested concentrations (p &gt; 0.05). IBR did not inhibit efferocytosis (p &gt; 0.05) and only inhibited transition to endolysosomal compartment at highest concentration tested (10 μM, p &lt; 0.01)
Conclusion
Our study shows that BTK inhibition does not block ADCP and a more selective BTK inhibitor may prove effective in combination with therapeutic anti-CD20 mAbs. IBR off-target inhibition specifically blocks ADCP and not efferocytosis. Thus, IBR off-target inhibition of ADCP should be via proximal signaling by antibody Fc receptors and not subsequent downstream phagocytic mechanisms in common with efferocytosis. These results also imply the lack of BTK and IBR off-target molecules involvement in efferocytosis. Finally, the increased lymphocytosis seen with IBR compared to ACALA treatment in CLL cannot be explained by IBR off-target effects on efferocytosis. These findings provide a critical understanding of macrophage phagocytosis reduction by BTK inhibitor selectivity that will have important consequences for the development of combination targeted therapies with mAbs.

Disclosures
Chu: Acerta Pharma/AstraZeneca: Research Funding; Pfizer: Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months; TG Therapeutics: Research Funding. Izumi:AstraZeneca: Current equity holder in publicly-traded company; Acerta Pharma: Current equity holder in private company, Ended employment in the past 24 months, Patents & Royalties: Acalabrutinib patents (no royalties). Munugalavadla:Gilead Sciences: Current equity holder in publicly-traded company; AstraZeneca: Current equity holder in publicly-traded company; Acerta Pharma: Current Employment. Barr:Gilead: Consultancy; Morphosys: Consultancy; TG therapeutics: Consultancy, Research Funding; Seattle Genetics: Consultancy; Celgene: Consultancy; AstraZeneca: Consultancy, Research Funding; Janssen: Consultancy; Merck: Consultancy; Genentech: Consultancy; Abbvie/Pharmacyclics: Consultancy, Research Funding; Verastem: Consultancy. VanDerMeid:Acerta Pharma / AstraZeneca: Research Funding. Elliott:Acerta Pharma / AstraZeneca: Research Funding. Zent:Mentrik Biotech: Research Funding; TG Therapeutics, Inc: Research Funding; Acerta / Astra Zeneca: Research Funding.