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

Abstract

Recent evidence regarding the role of regulatory T cells (Treg) in tumor development has suggested that the manipulation of Treg 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 Treg or other immune cell subpopulations. Herein, we investigated the ability of ligands against Treg-specific receptors to drive selective internalization of PEG-modified single-walled carbon nanotubes (PEG-SWCNTs) into Treg 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) Treg compared to other reported Treg-specific markers (folate receptor 4, CD103, and CD39). Ex vivo investigations showed that the Treg targeting 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 Treg residing in a B16 melanoma more efficiently then intratumor non-Treg or splenic Treg. The latter result was achieved by exploiting a combination of passive tumor targeting due to enhanced tumor vascular permeability, naturally increased intratumor Treg vs effector T cell (Teff) ratio, and active targeting of markers that are enriched in intratumor vs splenic Treg. We also found that PEG-SWCNTs loaded with GITR ligands were internalized by Treg 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.

GITR+ regulatory T cells in the treatment of autoimmune diseases

“…For example, Sacchetti et al demonstrated that GITR expression in Tregs residing in a B16 melanoma (CD4 + FoxP3 + ) is approximately 10-fold higher than that of Tregs in the spleens of the same animals [67]. Similarly, Wainwright et al demonstrated that GITR was expressed in all CD4 + FoxP3 + Tregs infiltrating glioblastomas but only 50% of CD4 + FoxP3 + Tregs in the spleen [4].…”

It takes two to tango: Understanding the interactions between engineered nanomaterials and the immune system

“…No antibody responses to SWCNTs themselves were detected. Using PEG-modified SWCNTs armed with the glucocorticoid-induced TNFR-related receptor (GITR), which has shown higher expression on intratumor versus peripheral regulatory T cells, Sacchetti et al [99] demonstrated in vivo targeting of regulatory T cells residing in a B16 melanoma (skin cancer) in mice. In this context, it is worth noting that innate immune cells can also enzymatically 'digest' PEGylated CNTs [100].…”

Nanoparticulate immunotherapy for cancer

“…To target intratumoral Tregs, Sacchetti et al, conjugated glucocorticoid-induced TNFR-related receptor (GITR) ligands to pegylated single-walled carbon nanotubes (PEG-SWCNTs). In vivo investigations of PEG-SWCNTs armed with GITR ligands in B16F10 melanoma model found higher accumulation of SWCNTs in intratumoral Tregs, as compared to accumulation within intratumoral non-Tregs or splenic Tregs [180]. This kind of innovative strategies to target cellular mediators in TME could pave the way for novel anti-tumor immunotherapy.…”