Abstract 909: The role of checkpoints in Rituximab-mediated NK cell activation

Abstract Purpose: Despite the remarkable clinical success of anti-CD20 monoclonal antibody (mAb) therapy, the mechanisms of action by which such mAb mediate their anti-tumor effects are not completely understood. This includes a need to better understand the role of various NK cell subtypes and checkpoint molecules in anti-CD20 mAb therapy. Experimental procedures: Raji B cell lymphoma cells and peripheral blood mononuclear cells (PBMCs) from normal donors (n=21) were co-cultured with rituximab (RTX) or trastuzumab as a control mAb for up to 15 days. Analysis included assessment of proliferation, remaining viable cells and phenotype of various cell types. Results: As expected, RTX was superior to trastuzumab in eliminating Raji cells. RTX also induced proliferation of NK cells and a shift of NK cells from a CD56dim to CD56bright population. Both NK cell proliferation and the increase in the CD56bright population occurred at day 5-7 when elimination of Raji cells was largely complete. Addback of additional Raji cells at day 5 delayed proliferation of NK cells and the shift from a CD56dim to CD56bright population suggesting this shift occurs once activation of the NK cell via Fcγ;;R is complete. Evaluation of checkpoint molecule expression by NK cells demonstrated RTX, in the presence of Raji target cells, upregulates NK cell expression of TIGIT and TIM3, but not PD1, CTLA-4, or LAG-3. This upregulation was maintained for the full duration of incubation (up to 15 days). The level of expression of TIGIT and TIM3 on NK cells induced by RTX was dose dependent and peaked at 1-5 ug/ml. NK cells induced by RTX to express TIGIT or TIM3 degranulated (CD107a), produced cytokines (IFNγ, TNFα) and expressed activation markers (CD69 and CD25) to a greater degree than did TIGIT or TIM3 negative NK cells. Anti- TIGIT mAb enhanced CD107a expression and TNFα production by RTX-activated NK cells. Conclusion: NK cells proliferate and shift from CD56dim to CD56bright population after being activated and eliminating RTX-coated target cells. TIGIT and TIM3 are biomarkers for RTX-mediated NK cell activation. Blocking TIGIT can enhance RTX-mediated NK cell degranulation and cytokine production. Citation Format: Zhaoming Wang, George J. Weiner. The role of checkpoints in Rituximab-mediated NK cell activation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 909.