Breaking immune silence: Tumor-targeted molecular therapy reprograms the tumor-immune interface in progressive neuroblastoma

Progressive neuroblastoma (pNB) that defies intensive multimodal clinical therapy remains a challenging pediatric cancer, characterized by swift clinical deterioration, recalcitrance, and a high degree of immune evasion. Our recent investigations recognized retinal degeneration 3 (RD3) deficiency as a central molecular driver of malignant characteristics, correlating with unfavorable outcomes and dictating immune evasion. Herein, we developed precision-engineered nano-immunotherapeutics targeting RD3-deficient (RD3 -/- ) NB. Five different RD3 peptides were strategically designed and enhanced via chemical stabilization through capping modifications and were labeled with a fluorescent marker to assess biodistribution and stability in vivo . These peptides were encapsulated into GD2-conjugated immunoliposomes (IL), archiving five RD3[GD2]IL formulations tailored for selective delivery to NB. ILs were assessed for uniform nanoscale distribution (NT Analyzer NS300), encapsulation efficiency, and structural integrity via TEM. Therapeutic efficacy was evaluated using an in vivo mouse model established using patient-derived stage 4 progressive pNB cells from a retroperitoneal tumor mass. Systemic administration (IV) of RD3[GD2] ILs (5 μM, thrice weekly × 4 weeks) inflicted immune signatures that were compared to the vehicle control (plain liposomes). High-resolution multiplex immune profiling was performed to characterize the tumor immune landscape, assessing the distribution (CD4⁺ and CD8⁺) and activation status (GITR⁺) of immune subsets. Among the five different formulations, RD3[GD2]IL-1 and RD3[GD2]IL-2 exhibited greater immunogenicity, restoring immune surveillance in RD3 -/- tumors. RD3[GD2]IL-2 enhanced the infiltration of CD4⁺ helper and CD8⁺ cytotoxic T cells, which was associated with a significant increase in the co-stimulatory receptor GITR, an important factor for T-cell activation and survival. Remarkably, RD3[GD2]IL-2 countered T-cell activation and boosted multifunctional T-cell populations, including CD4⁺GITR⁺, CD8⁺GITR⁺, and a rare, highly functional subset of the CD4⁺CD8⁺GITR⁺ triple-positive population. Particularly, RD3[GD2]IL-2 demonstrated impressive homing and tolerability, with no observable systemic toxicity or off-target effects. Collectively, our findings highlight RD3[GD2]IL-2 as a promising therapeutic candidate that synergizes precision tumor targeting with immune reinstatement. This next-generation immunoliposomal delivery reinstates immune responsiveness and treatment sensitivity in refractory pNB, offering a scheme for RD3-based therapies in immunosuppressive “cold” tumors, beyond NB. Funding: This work was funded by DoD-CA-210339, OCAST-HR19-045, and NIH P20GM103639 to Dr. Aravindan and NCI-P30 CA225520, and NIGMS P30GM154635 awarded to the OU Health SCC.