Strategies for improving T-cell manufacturing for adoptive immunotherapies

Abstract

Immunotherapy is a novel approach to disease treatment and has shown numerous clinical successes. In brief, immunotherapy exploits the principles of immunology to generate an anti-viral or anti-cancer response. One of the myriad methods that exist to stimulate an immunotherapeutic response is adoptive T-cell transfer (ACT). ACT is an immunotherapy approach whereby patient-derived antigen-specific T-cells are expanded in vitro and infused into the patient. Thus, a central factor of ACT immunotherapy is the in vitro T-cell stimulation and expansion step. In recent years, T-cell stimulation and expansion methods have focused on maximising cell yield. These expansion methods rely on the use of high-dose and often repeated cycles of stimulation. While effective in deriving large cell yields, this Tcell expansion strategy appears to compromise T-cell quality. Furthermore, various antigen sources are used to stimulate and expand antigen-specific T-cells. The impact these differing antigen sources have on T-cell quality is poorly understood. An aspect of this project aims to describe the consequences of antigen stimulation dose on T-cell quality. This project also evaluated common antigen stimulation sources, including Epstein-Barr Virus (EBV) transformed lymphoblastoid cell lines (EBV-LCLs), adenoviral vector-infected antigen presenting cells (APCs), and peptide-pulsed APCs, to determine their impact on T-cell quality. Of particular interest is the impact of EBV-LCLbased stimulation as this method is widely used for the manufacture of ACT drug products. To this end, we hypothesised that the therapeutic potential of an ACT drug product is heavily influenced by the stimulation and expansion method used. Furthermore, this project aimed to demonstrate the utility of comprehensive ACT drug product assessment. Indeed, our findings revealed that T-cell cultures stimulated with varying stimulation doses exhibit different phenotypic, transcriptional, and T cell receptor (TCR) repertoire profiles that reflect functional differences. This analysis can potentially be used to distinguish between ACT drug products of superior quality. The comprehensive analysis approach used also revealed that the dose of EBV-LCL-based stimulation had a profound impact on the overall ACT drug product composition. Specifically, it was observed that a high-dose EBV-LCL stimulation promoted a significant increase in natural killer cells but limited expansion of CD4 + T-cells. In contrast, varying stimulation dose of other antigen stimulation sourcesadenoviral vector-infected APCs and peptide-pulsed APCs-had less influence on culture composition. Overall, these two stimulation approaches resulted in ACT drug products that predominately contained T-cells. Additionally, T-cells derived from a high-dose EBV-LCL stimulation showed hallmarks of T-cell exhaustion, via the co-expression of multiple I would like to thank everyone who has helped me directly or indirectly throughout my Ph.D degree. First and foremost, I must express my sincere gratitude to Prof. R...