Engineering ErbB-targeted CAR T-cells to improve efficacy in treatment of solid tumours

Abstract

T-cells engineered with a second generation CD19 targeted chimeric antigen receptors (CARs) have recently shown great promise for the treatment of haematological malignancies, with three therapies now approved for the treatment of acute lymphoblastic leukaemia and diffuse large B-cell lymphoma. Despite the success of these and other CARs in haematological malignancies, CAR therapy for solid tumours has encountered numerous obstacles.
Even with a number of trials ongoing in solid tumours using second-generation CAR T-cells, it is apparent that these CARs are generally not effective in achieving an anti-tumour response. This indicates that there is a need for further development of CARs to improve their efficacy in the treatment of solid tumours. One way to achieve this is to re-engineer these receptors to deliver increased costimulation. This may help to increase the persistence, survival and anti-tumour activity of therapeutically infused CAR T-cells.
In this thesis, I set out to deliver enhanced costimulation using a novel CAR system known as a parallel CAR (pCAR). Parallel CARs comprise the co-expression of a second-generation CAR and a chimeric co-stimulatory receptor (CCR) which delivers a different co-stimulatory signal. To direct cells against multiple solid tumour types, pCAR T-cells were targeted against the pan-ErbB family. CAR and CCR specificity was directed against pan-ErbB using the EGF-TGFα chimeric protein, T1E, as the targeting moiety. Using mutated versions of T1E with reduced affinity for the epidermal growth factor receptor (EGFR; ErbB1), I investigated the relationship between the affinity of CAR and CCR for their respective target antigens. Decreasing the affinity of T1E in the CAR position decreased the anti-tumour activity of the CAR T-cells. Competition for the target antigen was ruled out by replacing the T1E CCR with a peptide targeted against a second tumour-associated antigen, αvβ6 integrin. When tested in vitro, pCARs with wildtype 3 T1E in both CAR and CCR or an αvβ6-targeted peptide in the CCR position showed better activity compared to a second-generation CAR. However, in vivo experiments were less conclusive, suggesting that further experimentation is warranted.
To test the pCAR format in a different ErbB-expressing tumour model, an scFv derived from the EGFR-specific antibody ICR62 was cloned into the pCAR format with T1E as a CCR. This was evaluated in models of glioblastoma since EGFR was identified as a valid target in this disease. In vitro testing again showed superiority of the pCAR over an equivalent second-generation CAR, however, again, further experimentation is required to confirm if there is any superiority of the pCAR in an in vivo model of glioblastoma.
In summary, pCARs show potential as a further evolution of CAR T-cells, providing greater persistence and cytokine support in vitro with potentially better persistence in vivo, however individual optimisation for each target is required.

Date of Award	1 Jul 2021
Original language	English
Awarding Institution	King's College London
Supervisor	John Maher (Supervisor) & Sophie Papa (Supervisor)