Blocking the Don't Eat Me Signal (CD47-SIRPα Axis) to Improve Antibody-Based Immunotherapy of Multiple Myeloma
Authors: Klausz Katja , Gehlert Carina Lynn , Boje Ammelie Svea , Lustig Marta , Krohn Steffen , Ahmed Syeda Maliha , Kellner Christian , Valerius Thomas , Besse Lenka , Besse Andrej , Driessen Christoph , Gramatzki Martin , Peipp Matthias 
Published: November 2021

Abstract
The addition of monoclonal antibodies daratumumab, elotuzumab and isatuximab to the treatment of patients with multiple myeloma significantly improved the outcome and prolonged survival. Unfortunately, although many patients benefit, depth and duration of response are a problem. In order to improve efficacy of antibody-based immunotherapy, we aimed to combine CD38-directed antibodies daratumumab and isatuximab as well as SLAMF7-targeting elotuzumab with a CD47 blocking antibody to enhance phagocytosis of myeloma cells. Antibody-dependent cellular phagocytosis (ADCP) of malignant plasma cells is described to be one important mode of action of daratumumab, isatuximab and elotuzumab, respectively. Of note, CD47 is highly expressed on myeloma cells and allows evading immune recognition by myeloid cells, i.e. monocytes, macrophages and neutrophils. Binding of CD47 to SIRPα expressed on myeloid cells provides a strong 'don't eat me' signal and diminishes phagocytosis of tumor cells. Blocking the CD47-SIRPα axis, by a monoclonal antibody against CD47 or a SIRPα-Fc fusion protein can restore recognition of tumor cells by macrophages and enhance phagocytosis. In patients with Non-Hodgkin's lymphoma the combination of CD20 antibody rituximab with CD47 antibody magrolimab was clinically successful (Advani et al., NEJM 379:1711, 2018).
To test the applicability of blocking the CD47-SIRPα axis and improve ADCP of myeloma cells by CD38-targeting or SLAMF7-directed myeloma antibodies, we generated a CD47 IgG2σ antibody carrying an engineered Fc domain not binding to Fcγ receptors (FcγR). This CD47 antibody was subsequently used in phagocytosis experiments in combination with antibodies daratumumab, isatuximab as well as elotuzumab and various myeloma cell lines. The cell lines AMO-1, JK-6L, L363, RPMI-8226, and U266 express different levels of CD47, CD38 and SLAMF7 as determined by quantitative flow cytometry. M0 macrophages expressing FcγRs were generated from healthy donor PBMC monocytes by cultivation with M-CSF for 10-14 days prior use in 6 hour real-time live cell imaging phagocytosis experiments with pHrodo-labeled myeloma cells - turning red only when engulfed by macrophages. Macrophages and myeloma cells were used at an effector-to-target cell ratio of 1:1.
Importantly, ADCP of myeloma cells induced by all three monoclonal antibodies, daratumumab, isatuximab or elotuzumab, can be enhanced by the addition of the CD47 blocking antibody. However, improvement in phagocytosis strongly differs between myeloma cell lines although all have high CD47 level on their cell surface. In responsive myeloma cell lines, ADCP mediated by CD38 antibodies daratumumab or isatuximab was found more efficient than that by SLAMF7 antibody elotuzumab. This may be related to the significantly higher CD38 than SLAMF7 expression at the myeloma cell surface. Our findings demonstrate that ADCP of approved IgG antibodies targeting CD38 or SLAMF7 can be enhanced by blocking the CD47-SIRPα axis and this may depend on the particular malignant plasma cell phenotype. The inhibition of this myeloid 'don't eat me' signal with a CD47 blocking antibody may open a new avenue for powerful myeloma immunotherapy. Since combination treatments with proteasome inhibitors and IMiDs are commonly used, these interactions also require attention. Initial data indicate that pre-treatment of myeloma cells with proteasome inhibitor carfilzomib did not negatively impact improvement of ADCP by blocking the CD47-SIRPα axis in responsive cell lines. Taken together, particularly CD38-targeting antibodies may have a significant potential to further improve immunotherapy in multiple myeloma patients.

Disclosures
No relevant conflicts of interest to declare.