Objectives: One in two patients with stage 4 melanoma will not survive their first year after diagnosis. Although recent treatment advances have improved survival, there is an unmet need for alternative therapeutics. Chimeric antigen receptor (CAR) T cell therapy involves re-directing autologous T lymphocytes against tumour-associated antigens via a receptor which couples an antigen-binding domain to CD3-zeta and costimulatory signalling domains. Melanoma cell adhesion molecule (MCAM) is upregulated in melanoma, promotes invasive cell behaviour and angiogenesis, and predicts poor survival. CAR T cells redirected against this tumour-associated antigen could represent a novel treatment option in late-stage melanoma. Methods: 5 single chain variable fragments (scFvs), derived from MCAM-specific antibody sequences, were cloned into a retroviral second-generation, 4-1BB costimulated CAR construct with CD8 hinge and transmembrane regions. CARs were screened for their ability to activate the JRT3-T3.5 cell line in response to MCAM-expressing cells. Selected CAR constructs were then expressed in primary T cells, and T cell effector functions were assessed in response to target cells. CAR T cells were also cocultured with HUVEC cells in monolayers and networks. Results: M1.BBz and M40.BBz CARs drove modest activation of the JRT3-T3.5 cell line in response to target cells expressing the MCAM ectodomain. These CARs redirected primary T cells effector functions, causing MCAM-dependent pro-inflammatory cytokine release, upregulation of cell surface activation markers and tumour cell lysis. However, the CAR T cells also targeted HUVEC cells, causing significant destruction of capillary-like networks. Conclusions: Whilst this study demonstrates the feasibility of generating MCAM-specific CAR T cells, further work is needed to confirm their anti-tumour efficacy in vivo. Ultimately, the potential for on-target/off-tumour toxicity, particularly towards the vasculature, must be considered. Further engineering has the potential to increase their safety and specificity, but the risk of toxicities may ultimately preclude the safe targeting of MCAM in this context.
|Date of Award||1 Aug 2023|
- The University of Manchester
|Supervisor||Paul Lorigan (Supervisor) & Adam Hurlstone (Supervisor)|
- Chimeric antigen receptor