281 Fc gamma receptor crosslinking promotes antibody-induced LILRB4 internalization and immune regulation of acute monocytic leukemia

OBJECTIVES/GOALS: The aims of this study were to: 1) determine the involvement of each Fc gamma receptor isoform in antibody-mediated crosslinking and internalization of LILRB4 in acute monocytic leukemia and 2) elucidate the role of this crosslinking and internalization in antibody-mediated immune regulation of these malignant cells. METHODS/STUDY POPULATION: To determine the involvement of Fc gamma receptors in antibody-mediated complex internalization, we generated acute monocytic leukemia cell lines with CRISPR-Cas9 knockout of each Fc gamma receptor isoform. We tested the effects of each knockout on anti-LILRB4 antibody-mediated internalization by flow cytometry and confirmed our findings with confocal microscopy. To elucidate the role of this crosslinking and internalization in immune regulation of acute monocytic leukemia, we conducted preliminary ELISA-based studies of the inflammatory signaling and cytokine release profiles of wild-type and Fc gamma receptor knockout cells treated with the LILRB4-targeting antibody. RESULTS/ANTICIPATED RESULTS: We have concluded that Fc gamma receptor I (CD64) plays a role in LILRB4 crosslinking and internalization by our anti-LILRB4 antibody and there are also contributions from Fc gamma receptor IIA (CD32A) observed in the absence of CD64 on the cell surface. Preliminary signaling studies have demonstrated that Fc gamma receptor-mediated antibody crosslinking and internalization of LILRB4 decreases anti-inflammatory signaling downstream of LILRB4 as well as pro-inflammatory signaling downstream of Fc gamma receptors, particularly in the absence of CD64 on the cell surface. The immunomodulatory effect of antibody-mediated LILRB4 crosslinking and internalization is being confirmed in follow-up signaling, cytokine release and lymphocyte activation studies. DISCUSSION/SIGNIFICANCE: This study will improve the efficacy of LILRB4-targeting antibody therapeutics for patients suffering from acute monocytic leukemia and help characterize CD64 and CD32A as potential clinical biomarkers for patients undergoing LILRB4-targeting antibody immunotherapeutic treatment, currently in first-in-human clinical trials.