Dual checkpoint blockade of CD47 and LILRB1 enhances CD20 antibody-dependent phagocytosis of lymphoma cells by macrophages

Abstract

Antibody-dependent cellular phagocytosis (ADCP) by macrophages, an important effector function of tumor targeting antibodies, is hampered by ‘Don´t Eat Me!’ signals such as CD47 expressed by cancer cells. Yet, human leukocyte antigen (HLA) class I expression may also impair ADCP by engaging leukocyte immunoglobulin-like receptor subfamily B (LILRB) member 1 (LILRB1) or LILRB2. Analysis of different lymphoma cell lines revealed that the ratio of CD20 to HLA class I cell surface molecules determined the sensitivity to ADCP by the combination of rituximab and an Fc-silent variant of the CD47 antibody magrolimab (CD47-IgGσ). To boost ADCP, Fc-silent antibodies against LILRB1 and LILRB2 were generated (LILRB1-IgGσ and LILRB2-IgGσ, respectively). While LILRB2-IgGσ was not effective, LILRB1-IgGσ significantly enhanced ADCP of lymphoma cell lines when combined with both rituximab and CD47-IgGσ. LILRB1-IgGσ promoted serial engulfment of lymphoma cells and potentiated ADCP by non-polarized M0 as well as polarized M1 and M2 macrophages, but required CD47 co-blockade and the presence of the CD20 antibody. Importantly, complementing rituximab and CD47-IgGσ, LILRB1-IgGσ increased ADCP of chronic lymphocytic leukemia (CLL) or lymphoma cells isolated from patients. Thus, dual checkpoint blockade of CD47 and LILRB1 may be promising to improve antibody therapy of CLL and lymphomas through enhancing ADCP by macrophages.

Enhanced potency of immunotherapy against B‐cell precursor acute lymphoblastic leukemia by combination of an Fc‐engineered CD19 antibody and CD47 blockade

“…15,16 In vitro phagocytosis was measured by fluorescence microscopy or live cell imaging using the IncuCyte System (Sartorius). 15,16 Target cells were coincubated with macrophages at a 2:1 target cell to macrophage ratio. Target cells were labeled with CFSE (Biolegend, microscopy) or with pHrodo (Sartorius, IncuCyte).…”

“…Cell surface expression of CD47 and CD19 was analyzed by flow cytometry using the Qifikit to determine specific antibody binding capacities (SABC) per cell. 16 Xenografts NSG mice (Charles River Laboratories) were maintained in IVCs. BCP-ALL PDX cells of seven patients were injected into the tail vein (four mice/PDX) of 6-8 weeks-old female mice.…”

Immune cells in the B-cell lymphoma microenvironment: From basic research to clinical applications

“…[64] Therefore, blocking the MHC-I/LILRB1 axis may be an effective innate immune drug target. Zeller et al [65] reported that the dual checkpoint blockade of CD47 and LILRB1 has the potential to improve antibody therapy for lymphomas by enhancing antibody-dependent cellular phagocytosis by macrophages in vitro .…”

Inhibitory receptors of plasmacytoid dendritic cells as possible targets for checkpoint blockade in cancer

“…In addition, co-treatment with temozolomide strengthened the antitumor capacity of immune cells treated with anti-ILT2 ( 171 ). Also, Fc-silent antibodies against ILT2 significantly enhanced antibody-dependent phagocytosis of lymphoma cell lines when combined with both rituximab and blockade of CD47 ( 172 ). These findings suggest that the blocking of ILT2 may be an interesting strategy to improve tumor immunotherapy.…”