(P 76) CD47 BLOCKADE SYNERGIZES WITH CHEMOTHERAPY IN ENHANCING MACROPHAGE PHAGOCYTOSIS OF EWING SARCOMA CELLS

Objective: Macrophages are known to harbor phagocytic activity against cancer cells, yet they fail to phagocytose Ewing sarcoma (ES) cells and confer a poor prognosis for ES patients. We previously found that ES cells utilize dual mechanisms, that is, up-regulation of CD47 “don’t eat me” signal and down-regulation of cell surface calreticulin (csCRT) “eat me” signal, to resist macrophage mediated phagocytosis. We demonstrated that enhancing csCRT by chemotherapy doxorubicin (DOX) and blocking CD47 by magrolimab (MAG) significantly enhanced macrophage phagocytosis of ES cells and had synergistic anti-tumor effects against ES xenograft tumors. However, the cumulative total lifetime dose of DOX is limited, and MAG is associated with side effects especially anemia due to CD47 expression on erythrocytes. To better translate the CD47 blockade and chemotherapy combinatorial therapy to the clinic, we sought to identify and evaluate alternative regimens in enhancing macrophage phagocytosis and anti-tumor activity against ES.

Methods: Macrophages were derived from human PBMC by M-CSF (100 ng/mL). Cyclophosphamide (CP), another common chemotherapy regimen for ES and was reported to enhance csCRT level on cancer cells, was utilized to enhance csCRT on ES cells (0-20 mg/mL for 24 hrs). Lemzoparlimab (LEM) (1 μg/mL), a next generation therapeutic CD47 blockade that does not produce substantial hematologic toxicity, was used to block CD47 signal. Flow cytometry based in vitro phagocytosis assay was performed to evaluate and compare macrophage phagocytosis of ES cells induced by CP+LEM and DOX+MAG.

Results: We found that csCRT levels on ES (A673, TC32, EWS502) cells were increased by CP in a dose-dependent manner with 20 mg/mL as the most effective dose (Fig.1A). CP or LEM alone significantly enhanced (p < 0.01), and the combination of the two further significantly enhanced (p < 0.01) macrophage phagocytosis of ES cells (Fig.1B). Furthermore, we found that CP and/or LEM induced comparable levels of increase in macrophage phagocytosis of ES cells as DOX and/or MAG (Fig.1C).

Conclusion: Our data demonstrates a synergistic effect of CP and LEM in increasing macrophage phagocytosis of ES cells that is comparable to the combination of DOX and MAG. This provides a rationale to move this combinatorial therapy to preclinical investigation.