Injectable chitosan thermogels adapted for cytotoxic T lymphocytes immunotherapy

摘要

Introduction: Adoptive T cell therapy (ACT) is an emerging form of cancer immunotherapy, showing tremendous potential in clinical studies aimed at treating advanced or inoperable cancers. Unfortunately, it is limited by the large numbers of anti-tumor cytotoxic T lymphocytes (cTL) that must be expanded before intravenous systemic delivery (20-150 billion) due to their loss to non-cancerous sites of inflammation. We here propose a new approach consisting of encapsulating cTL in an injectable scaffold to increase their concentration and retention close to the tumor, and improve the outcomes of this therapy. To that purpose, new chitosan-based thermosensitive hydrogels (CTGels) were developed. These present high mechanical strength and excellent cytocompatibility due to low salt concentration and the absence of chemical crosslinker. The goal of the present work was to fine-tune these hydrogels and demonstrate that they can provide a microenvironment allowing the three-dimensional (3D) proliferation, release and bioactivity of cTL in immunotherapy context. Method: Chitosan (CH; Mw 250kDa, DDA 94%) thermogels were prepared by mixing CH acidic solution with a combination of phosphate buffer (PB) and sodium hydrogen carbonate (SHC) as gelling agents. They were optimized in regards to their rheological, mechanical properties and morphology and ability to sustain human cTL (8M/mL) growth. One formulation was then selected to characterize and quantify cell release, phenotype and bioactivity by flow cytometry. Finally, transwell-mediated recognition/killing assay using CTGel-α-gp100 cTL clone and specific or non-specific melanoma cancer cell lines were performed. Their phenotype was determined by flow cytometry and induction of annexin V expression in cancer cell lines was quantified by ELISA. Results and Discussion: Rheological tests confirmed the thermoresponsive behavior of the new hydrogels. Compressive tests showed drastic enhancement of mechanical properties (Figure 1), with about 14-fold increase of Young modulus compared with conventional CH-giycerophosphate thermogels. One formulation outperforms the others by providing an environment suitable for the encapsulation of viable CD8+ T lymphocytes, supporting their proliferation in 3D colonies and their gradual release. Encapsulated a-gp100 cTL expressed cytotoxic markers, released IFN-γ and induced annexin V expression when co-cultured with specific melanoma cells (Figure 2). Conclusion: This new chitosan hydrogel is thermosensitive and allows easy injection and cell encapsulation. It allows local growth in 3D colonies and delivery of T cells and presents sufficient mechanical properties to reach high retention and withstand in vivo stresses. It is expected to increase the number of patients who can benefit from ACT whilst minimizing side effects. It could also reduce treatment costs by replacing repeated injections with a single one.