Fc gamma receptors promote antibody induced LILRB4 internalization and immune regulation of monocytic AML

Abstract Background The immune checkpoint leukocyte immunoglobulin-like receptor B4 (LILRB4) is found specifically on the cell surface of acute monocytic leukemia (monocytic AML), an aggressive and common subtype of AML. We have developed a humanized monoclonal IgG1 LILRB4-blocking antibody (h128–3), which improved immune regulation but reduced cell surface expression of LILRB4 in monocytic AML models by 40–60%. Interestingly, most of this effect was neutralized by mutation of the Fc region of the antibody (h128–3/N297A) which prevents interaction with Fc gamma receptors (FcγRs). This suggested that there is FcγR-dependent antigenic modulation underlying h128–3’s effects, a mechanism known to alter the function of antibodies targeting B-cell malignancies. Methods We disrupted the Fc-FcγR interaction pharmacologically and with stable CRISPR-Cas9-mediated genetic knockout of FcγRs in monocytic AML cell lines to investigate the role of FcγR-dependent antigenic modulation in the regulation of LILRB4 by h128–3. Results When FcγRI is inhibited or removed from the surface of monocytic AML cells, h128–3 cannot optimally perform its blocking function, resulting in activation of the LILRB4 inhibitory receptor and leading to a 15–25% decrease in T cell-mediated cytotoxicity in vitro. In the absence of FcγRI, scaffolding by FcγRIIa allows h128–3 to maintain LILRB4-blocking function. Conclusions Here we define a FcγR-dependent antigenic modulation mechanism underlying the function of an immunoreceptor blocking antibody for the first time in myeloid malignancy. This research will facilitate the development of safe, precision-targeted antibody therapeutics in myeloid malignancies with greater potency and efficacy.