Abstract:
Immunotherapy has emerged as a promising tool in the treatment of cancer, enhancing the immune system’s ability to detect and target tumour cells for eradication. Research has led to the development of numerous immunotherapies such as immune checkpoint blockade (ICB), cancer vaccines, and adoptive cell transfer (ACT). However, despite positive clinical responses to ICB, many patients do not respond to treatment or relapse after an initial response. Resistance to treatment has sparked interest in ACT therapy, which provides the opportunity for a powerful synergistic approach in cancer treatment. ACT therapy aims to induce a durable tumour-specific response through administration of autologous, specific, and highly-tumour-reactive T cells that have been expanded in vitro.
While most published work in ACT has focused on infusing existing memory T cells derived from patient tumours or blood, new techniques for T cell priming in vitro offer the hope of generating ACT products targeting antigens to which patients have not previously responded. The host laboratory has been developing new protocols that will routinely generate T cell products from blood samples to target such previously unrecognised molecules within a patient’s tumours. These techniques could be used to enhance responses to ICB in patients who have “cold” tumours lacking T cell infiltrates, as well as patients who initially fail ICB.
In this thesis, we aimed to validate and optimise protocols previously developed within the host lab to prime melanoma specific CD8+ T cells from the naïve T cell pool within whole PBMC. These protocols differ substantially from existing T cell priming protocols, and have promise in facilitating translation of priming techniques to clinical use as they minimise manipulation of patient blood samples. We therefore examined various aspects of both T cell priming and expansion from whole PBMC, as well as the downstream expansion and characterisation of the resulting T cell cultures to determine their potential suitability for clinical use.
We show that the restimulation of a primed culture likely requires the administration of a combination of agents to balance the tension between T cell differentiation and proliferation/survival.
Significantly, we demonstrate that the restimulation of primed T cells in vitro using the host laboratory’s current protocols is likely to be suboptimal due to a lack of APCs capable of processing peptide antigens. Specifically, we show that CD14+ monocytes rapidly decrease in culture after the priming step, suggesting that additional PBMC containing monocytes need to be added to the cultures at the time of restimulation.
We also show that non-specific expansion of T cell cultures using CD3/CD28 Dynabeads® and the γ-chain cytokine IL-15 enhances cell expansion and survival, with greater retention of favourable phenotypes in comparison to the mitogenic lectin PHA.
Finally, we validate a method for single cell sorting and cloning T cells from a polyclonal primed culture as a method for downstream characterisation of a cell product, and refine the gating strategy necessary for efficient T cell cloning.