Background
Neuroblastoma is the most common pediatric extracranial solid tumor with ~50% of patients having high-risk disease (HR-NBL). HR-NBL patients undergo multimodal therapy that includes anti-GD2 immunotherapy. While some patients receiving this regimen respond, ~50% develop refractory or relapsed disease. Considered a ‘cold’ tumor, most HR-NBLs have infiltration of suppressive immune cells [including type-2 tumor associated macrophages (TAMs) and myeloid derived suppressor cells (MDSCs)], poor anti-tumor immune infiltrate (with minimal T cell infiltration), and low-to-no expression of MHCI. Both TAMs and MDSCs express CD40, allowing treatment with anti-CD40 agonist (aCD40) to reprogram them to have anti-tumor activity; DCs also express CD40 and are activated with aCD40. Since T cells are engaged via interactions with MHC, low expression of MHCI may contribute to reduced T cell involvement during immunotherapy. Diminished surface expression of MHCI on HR-NBLs can occur through multiple mechanisms including epigenetic modifications (EMs). Using a syngeneic murine model of ‘cold’ HR-NBL tumors (9464D-GD2+/MHCI-), we investigated whether inhibitors of EMs (EMis) together with aCD40 could improve the anti-tumor efficacy of anti-GD2. Increased anti-tumor immune infiltration may lead to upregulation of immune checkpoints (i.e., PD-L1/PD-1). To avert immune escape, inclusion of the immune checkpoint inhibitor (ICI), anti-PD1, may further improve response.
Methods
9464D-GD2+/MHCI- cells were treated with EMis and assessed for MHCI expression changes via qPCR and flow cytometry. Mice bearing 9464D-GD2+/MHCI- tumors were treated with combinations of aCD40+/-anti-GD2+/-EMi+/-anti-PD-1 and monitored for response. Flow cytometry was used to determine changes in immune infiltration.
Results
In vitro treatment of 9464D-GD2+/MHCI- with EMis showed that MHCI-related genes are expressed by qPCR, and MHCI expression is observed via flow cytometry. Tumor shrinkage is observed in mice bearing 9464D-GD2+/MHCI- only if treated with therapy that includes aCD40. The best response is achieved with therapies that include anti-GD2/aCD40/anti-PD1/EMis. Preliminary studies reveal increased involvement of anti-tumor immune cells if treated with anti-GD2/aCD40/anti-PD1/EMis.
Conclusions
Using a regimen that includes anti-GD2/aCD40/anti-PD1/EMis, we hypothesize that the immune cells in the tumor microenvironment may be reinvigorated, thereby enabling activity of T cells through EMis and reprograming of suppressive cells (TAMs and MDSCs) while simultaneously activating macrophages and DCs to enhance the efficacy of anti-GD2. Furthermore, by including anti-PD-1, we were able to circumvent the potential for immune cell exhaustion through ICIs. Therapies geared towards restoring MHCI expression, combined with effective immunotherapy regimens that allow for persistent immune responses, might augment immune responses and improve efficacy of combination immunotherapy for HR-NBL.
Acknowledgements
This work was supported by the Alex’s Lemonade Stand Foundation, the Midwest Athletes Against Childhood Cancer, the CRI, the University of Wisconsin Carbone Cancer Center and by public health service grants R35-CA197078, and P01 CA250972 from the National Cancer Institute.
Ethics Approval
All of the research conducted was approved under approved Biological Safety and Institutional Care and Animal Use Committee for the University of Wisconsin, Madison.