Chitosan Thermogels for Local T Lymphocyte Delivery in Cancer Immunotherapy

Transcription

1 Chitosan Thermogels for Local T Lymphocyte Delivery in Cancer Immunotherapy Ceccaldi Caroline (Laboratory of Endovascular Biomaterials (LBeV), Montréal, QC, Canada.) Monette Anne (Laboratoire d'immuno-oncologie, ICM, U de Montréal/CRCHUM, Montréal, QC, Canada.) Assaad Elias (Laboratory of Endovascular Biomaterials (LBeV), Montréal, QC, Canada.) Lapointe Rejean (Laboratoire d'immuno-oncologie, ICM, U de Montréal/CRCHUM, Montréal, QC, Canada.) Lerouge Sophie (Laboratory of Endovascular Biomaterials (LBeV), Montréal, QC, Canada.) Introduction Systemic adoptive T lymphocyte transfer is an emerging cancer immunotherapy showing tremendous promise in clinical studies, and its success lies in the ability of the antigenexperienced cytotoxic T lymphocytes (ctl) to access and to persist in the tumour microenvironment [1]. The mimicking of tertiary lymphoid structures (TLS) that persist and promote a protective immune response against cancer can be achieved using an injectable biocompatible matrix releasing antitumour proliferating ctl. A potential candidate for this application is chitosan thermogel. Chitosan is a natural polymer that can be combined with a weak base such a betaglycerophosphate (BGP) to form a

2 thermosensitive hydrogel at physiological ph which is liquid at room temperature and is able to gelify at 37 C [2]. However, its potential is limited by a slow gelation kinetic, low mechanical properties and hyperosmolality. Our laboratory is working on the development of novel chitosan hydrogel formulations having high potential in terms of cytocompatibility, mechanical properties and porosity for cell encapsulation and delivery. Recently, new combinations of gelling agents including phosphate buffer (PB) and sodium hydrogen carbonate (SHC) were found to generate injectable scaffolds with high mechanical properties after gelation. Moreover, we have shown that this combination permits to decrease the concentration of salts implied in the gelation process. Supported by these promising characteristics we hypothesize that the implantation TLS-like structures composed of these new hydrogels and ctl into the tumour microenvironment will provide a means for the delivery of a continuous feed of these cells against the tumour for the reprogramming of inflammation mechanisms and the reduction of tumour burden. Therefore, our aim was to fine-tune a chitosanbased hydrogel formulation that would provide an

3 environment allowing the three-dimensional (3D) proliferation and release of ctl whose activation state can be influenced by the surrounding conditions. For such applications, the hydrogel should present good compatibility with cells, as well as adequate rheological properties allowing it to be injectable and to rapid solidify into a 3D scaffold of adequate porosity. Materials and Methods Chitosan (Mw 250kDa, DDA 94%) thermogels were prepared with novel combinations of gelling agents (composed with PB and SHC) with the goal to reduce total salts concentration and thus improve the cytocompatibility of hydrogels. Their rheological properties and mechanical strengths were evaluated by rheometry and unconfined compression tests (up to 50% deformation) respectively. The ph and osmolilaty of the hydrogels were measured, and their morphology was observed by scanning electron microscopy. Two formulations, differing in PB and SHC concentrations presenting appropriated gelation kinetic, mechanical properties and low ionic strength were selected for further

4 experiments. Human T-cells were encapsulated in different hydrogel formulations. The biocompatibility was observed after live/dead staining by fluorescent microscopy. Gel- and supernatant-derived cells were marked and quantified by flow cytometry to evaluate their phenotype and activation status over time. Results Rheological tests permitted to highlight the thermoresponsiveness behavior of both formulations. The storage modulus remained low at 22 C and rapidly increased at 37 C. Compressive tests showed that they present excellent mechanical properties after gelation (Figure 1), with 10 to 20 fold increase of Young modulus compared with conventional CH-BGP hydrogels. However, minute differences in hydrogel formulation have dramatic effects on survival and proliferation potential of encapsulated primary T cells in vitro. Hydrogels morphology revealed differences that could impact the affinity of cell for the structure, affect their ability to proliferate and to migrate through the gel. Flow cytometry and microscopy

5 demonstrated that T cells encapsulated in Gel 1 presented a lower viability than those encapsulated in Gel 2. In addition, only Gel 2 permitted the growth of cells, where flow cytometry provided evidence of increased cellular proliferation and escape from the gel over time, along with the maintenance of cellular phenotype and activation status. Finally, microscopy proved the continuous development and expansion of T cells colonies over time (Figure 2). Gel2 outperforms the others by providing an environment favoring the growth of CD8+ T lymphocytes and allowing the encapsulated cellular phenotype to be influenced by surrounding conditions. Discussion and Conclusion The synergic effect of gelling agents permitted to obtain injectable chitosan hydrogels with strong and cohesive structures after gelation. We have developed a hydrogel formulation that acts as a scaffold for the growth of a 3D Tcell culture. Encapsulated cells maintain their phenotype and is influenced by surrounding conditions, suggesting that their will retain their therapeutic functions.

6 This locally injectable hydrogel can serve as a TLSlike T cell delivery vehicle to complement adoptive cell transfer therapies.

7 Acknowledgements Funding: CRC in biomaterials and endovascular implants to S.L. and the CCSRI to R.L. E.A also

8 acknowledges FRQ-NT scholarship. References 1. Restifo et al., Nat Rev Immunol, (4): p Chenite, et al., Biomaterials, (21): p