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Background Neuroblastoma is the most common pediatric solid cancer, outside of brain cancers, with ~800 new cases annually in the US. The current standard of care for relapsed high-risk neuroblastoma includes chemo-immunotherapy: Temozolomide, Irinotecan, and anti-GD2 antibody dinutuximab (TIG). This regimen results in an overall response for ~40% of patients, but most responders still relapse. Combination therapies that engage the innate and adaptive immune system are needed to improve patient survival as neuroblastoma tumors are immunologically cold. Agonist anti-CD40 (aCD40) monoclonal antibody has been shown to promote the maturation of dendritic cells and activate macrophages. Here, we combine aCD40 with standard of care chemo-immunotherapy to ask if we can improve the anti-tumor efficacy and enhance immune cell activity. We also utilize a macrophage diphtheria toxin receptor mouse model to investigate the role of macrophages in this regimen.Methods We used a clinically relevant neuroblastoma mouse cell line that mimics the patient neuroblastoma tumor microenvironment including GD2 positivity, high MYCN expression, low MHC expression, and low tumor mutational burden. We implanted syngeneic 9464D-GD2 cells into the flanks of C57BL/6 mice or mCherry+ diphtheria toxin receptor+ macrophage mice to induce tumors. When tumors reached ~20 mm3 mice were randomized and treated with TIG plus aCD40. One group was also given diphtheria toxin one day prior and throughout therapy to deplete macrophages. Tumor growth and survival were followed. Tissues were also harvested for flow cytometry.Results Mice treated with TIG alone did not respond to therapy, recapitulating what occurs in ~60% of high-risk neuroblastoma patients. In contrast, treatment with TIG+aCD40 led to complete tumor regression in most mice and significantly improved survival compared to control and TIG alone. When T cells were depleted during TIG+aCD40, the anti-tumor effect remained the same while when macrophages were depleted during TIG+aCD40, the anti-tumor effect was completely lost – suggesting a key role of macrophages in driving this response and low importance for T cells.Conclusions These results show that TIG alone is not sufficient to cure 9464D-GD2 tumors. However, the addition of aCD40 to TIG induced tumor eradication and improved survival, with macrophages playing a key role. This work suggests that aCD40 may be a good candidate to combine with standard of care for relapsed high-risk neuroblastoma in children. Additional efforts are being made to better understand macrophage metabolism and function during this 9464D-GD2 response.Ethics Approval All animal work was approved by the University of Wisconsin Institutional Animal Care and Use Committees.