In Vivo Targeting of Intratumor Regulatory T Cells Using PEG-Modified Single-Walled Carbon Nanotubes

Recent evidence regarding the role of regulatory T cells (T-reg) in tumor development has suggested that the manipulation of T-reg function selectively in the tumor microenvironment would be a desirable immunotherapy approach. Targeting intratumor immune populations would reduce side effects on peripheral healthy cells and increase antitumor efficacy of immunotherapies. However, no current approaches are available which enable selective in vivo targeting of intratumor T-reg or other immune cell subpopulations. Herein, we investigated the ability of ligands against T-reg-specific receptors to drive selective internalization of PEG-modified single-walled carbon nanotubes (PEG-SWCNTs) into T-reg residing in the tumor microenvironment. We focused our attention on the glucocorticoid-induced TNFR-related receptor (GITR), as it showed higher overexpression on intratumor vs peripheral (i.e., splenic) T-reg compared to other reported T-reg-specific markers (folate receptor 4, CD103, and CD39). Ex vivo investigations showed that the T-reg efficiency and selectivity of PEG-SWCNTs depended on incubation time, dose, number of ligands per nanotube, and targeted surface marker. In vivo investigations showed that PEG-SWCNTs armed with GITR ligands targeted T-reg residing in a B16 melanoma more efficiently then intratumor non-T-reg or splenic T-reg. The latter result was achieved by exploiting a combination of passive tumor targeting due to enhanced tumor vascular permeability, naturally increased intratumor T-reg vs effector T cell (T-eff) ratio, and active targeting of markers that are enriched in intratumor vs splenic T-reg, We also found that PEG-SWCNTs loaded with GITR ligands were internalized by T-reg through receptor-mediated endocytosis and transported into the cytoplasm and nucleus ex vivo and in vivo. This is the first example of intratumor immune cell targeting and we hope it will pave the way to innovative immunotherapies against cancer.