Priming and Cloning of Antigen-Specific T Cells for Adoptive Therapy

Abstract

Immunotherapy has emerged as a promising approach to the treatment of cancer, utilising and enhancing the natural potential of the adaptive immune system to identify transformed tumour cells, and specifically target them for cytotoxic lysis. Research has resulted in multiple immunotherapies including anti-cancer vaccines, adoptive T cell therapies (ACT), and anti-PD-1 and anti-CTLA-4 checkpoint blockade which has become the most successful therapy for late-stage melanoma. Resistance to checkpoint blockade has stimulated interest in ACT due to a dearth of tumour-specific T cells being an important reason for the failure of this therapy, raising the possibility of a powerful synergistic approach for melanoma and many other cancers. In this thesis we examine various aspects of a new approach to adoptive cell immune therapy: priming naïve T cells to cancer specific antigens, aiming to optimise and validate new approaches to improve performance and translatability from research lab to clinic. We first explored new methods to expand in vitro rare naïve antigen-specific T cells to the model antigen Melan-A, showing that monocytes could be successfully used instead of monocyte-derived dendritic cells (moDCs). We detected melan-A specific T cell expansion within 12 days, potentially representing a reduction in both time and complexity of culture methods to prime T cells. We showed that the synthetic TLR ligand Pam2Cys could serve as a clinically acceptable maturation signal for APCs, but surprisingly found it unnecessary for successful priming. We used similar protocols to successfully prime melan-A-specific T cells from whole PBMC without purifying monocytes, further accelerating and simplifying the protocol compared with current moDC-based priming methods. However, this could not be extended to naïve T cells specific for NY-ESO-1, suggesting further optimisation is needed to prime rarer naïve T cell than those specific for melan-A. We then FACS sorted primed melan-A-specific T cells and grew a range of clonal cell lines for characterisation, confirming that they were at an early stage of differentiation and hence suitable for use in ACT. Finally, we used similar cell sorting and cloning techniques to generate T cell clones specific for the cancer-testis antigen PRAME from a healthy donor, including demonstrating the ability to kill PRAME-positive melanoma cell lines.