Optimizing combinatorial macrophage induced innate immunotherapy against Ewing sarcoma

Abstract Background We previously reported that Ewing sarcoma (ES), a pediatric bone and soft tissue tumor, escapes macrophage phagocytosis by upregulating the CD47 “don’t eat me” signal and downregulating the cell-surface calreticulin (csCRT) “eat me” signal. Targeting this dual mechanism by combining the CD47 blockade magrolimab (Mag) with doxorubicin (Dox), a chemotherapy that enhances csCRT expression, significantly enhanced macrophage phagocytosis of ES cells and inhibited xenograft tumor growth and metastasis in an orthotopic mouse model. However, Dox is known to cause cardiotoxicity, and Mag induces anemia due to targeting of CD47-expressing erythrocytes. Alternative, less toxic regimens are needed to optimize this combinatorial therapy. Methods Single-cell RNA sequencing (scRNA-seq) on cells isolated from ES xenograft tumors treated with PBS, Dox, Mag or Dox + Mag was performed to better understand the effects of Dox and Mag on ES tumor and its immune microenvironment. Cyclophosphamide (Cyclo), another standard chemotherapy regimen for ES, was evaluated as an alternative to Dox for inducing csCRT expression on ES cells. Using in vitro phagocytosis assays, lemzoparlimab (Lem), a next-generation anti-CD47 antibody designed to spare erythrocytes, was compared to Mag, alone and in combination with Cyclo. Orthotopic ES xenograft mouse models in both NSG and humanized NSG hosts were utilized to evaluate the safety and efficacy of Cyclo combined with Lem or Mag. Results scRNA-seq results revealed that Dox or Dox + Mag treatment upregulated the cellular pathways and functions associated with dilated cardiomyopathy in macrophages. Cyclo significantly enhanced csCRT expression on ES cells in a dose dependent manner. In vitro phagocytosis assays demonstrated that combining Cyclo with either Lem or Mag markedly increased macrophage phagocytosis of ES cells. Furthermore, in the NSG xenograft mouse model, both Cyclo + Mag and Cyclo + Lem significantly reduced tumor growth and lung metastasis while prolonging animal survival. Importantly, in humanized NSG mouse models, Cyclo + Mag induced severe anemia and animal death, whereas Cyclo + Lem combination was well-tolerated, significantly reduced tumor burden, and extended animal survival. Conclusions Our preclinical data demonstrate that the combination of Cyclo and Lem is a safe and effective alternative therapeutic strategy with high potential for clinical translation in patients with ES.