Fiona ThistlethwaiteAbdulrahman Salem bahashwan2022-05-262022-05-26https://drepo.sdl.edu.sa/handle/20.500.14154/33092Introduction: Chimeric antigen receptor (CAR) T-cell is a branch of cancer immunotherapy, whereby T-cells are modified to express variable fragments of a monoclonal antibody for targeted specificity and the cytoplasmic domain of CD3ζ for activation. These CARs represent the first generation, whereas later generations also include one or more co-stimulatory receptors in the cytoplasmic domain. The major achievements of CAR T-cells to date are in B-cell malignancies via targeting the CD19 antigen, which is restricted to B-cells. The development of CAR T-cells for the treatment of acute myeloid leukaemia (AML) is not as advanced as those for B-cell malignancies because of shared antigen expression on important tissues. Aims: This study aimed to assess the effects of various co-stimulation receptors within anti-CD19 CARs in order to improve the safety and activity of CAR T-cells. CAR components; hinge and transmembrane (TM) regions were also assessed with regard to the effects of CD80/CD86 binding and dimerisation of the CD28 co-stimulatory receptor, and the effects of the two regions within other co-stimulation receptors on T-cell activity. The study also sought to determine the expression of the oncofoetal 5T4 antigen as a possible target for AML therapy and investigated the potential of targeting AML using anti-5T4 CAR T-cells. Methods: Using restriction endonucleases, anti-CD19 and anti-5T4 CARs, with different co-stimulation molecules, were generated. T-cells expressing the CARs were assessed for in vitro activity using different functional assays. Using flow cytometry, the expression of 5T4 antigen in AML patient samples and cell lines was assessed. Results: Three panels of anti-CD19 and anti-5T4 CARs were generated: (1) CD28, (2) other co-stimulation molecules, and (3) CD8 TM. CD28 panel involves CARs with ability to bind CD80/CD86 ligands and/or to form dimers, and those without these abilities. Among the CD28 CARs, the level of surface CAR expression correlated to the length and dimerisation of CD28 hinge; the shorter and monomer CARs were expressed at lower levels than their parent full-length dimer CARs. The parent CD28 CARs were also involved in increased T-cell activation and cytokine production. Interestingly, the CD28 activation motif MYPPPY seemed less involved in CD28 CAR T-cell activation. The other two CAR panels included OX40, 4-1BB, and CD2, in which hinge and TM regions were derived from the co-stimulation receptor or from CD8α. The importance of hinge and TM regions of the co-stimulation receptors was demonstrated by the low CAR expression and T-cell function when these regions were replaced with CD8α. However, this observation was variable between the receptors, where CD28 and CD2 CAR expression and T-cell function were significantly affected, 4-1BB CAR T-cells demonstrated similar activity regardless of the origin of the two regions. Furthermore, CD2 CAR T-cells showed higher levels of activity than OX40, 4-1BB, and CD8 TM CAR T-cells but with higher rates of T-cell death. Expression of 5T4 was dim to negative in the majority of clinical samples, except for those with mixed-lineage rearrangement, which demonstrated > 60% expression. Variable expression, from 0% to almost 100%, was seen in the cell lines. The anti-5T4 CAR T-cells resulted in minimal activity against the AML cell lines, suggesting resistance that could be, in part, due to the expression of immune checkpoint receptors. Summary: This study shows the significance of the length/nature of hinge and TM regions for optimal CAR expression and T-cell function, this importance varies, depending on the co-stimulation receptor. The study also demonstrates the presence of 5T4 antigen expression in certain AML subtypes, with the potential need for combination therapy for better CAR T-cell outcomes.en