Abstract
The use of engineered cell therapies for the treatment of certain malignancies is a rapidly advancing field. Second-generation chimeric antigen receptor (CAR) T cell therapies have now become a standard of care for relapsed or refractory large B-cell lymphomas. However, while initial response rates are high, long-term survival rates and frequency of severe toxicities could be improved upon. As such, there is a pressing need for the development of CAR T-cells that combine high efficacy with low toxicity.
Toll-like receptor 2 (TLR2) is expressed by certain T-cell subsets and can enhance expansion, effector function, and memory formation when activated1. When integrated into a CAR, the intracellular signalling portion of TLR2 has been shown to be an effective co-stimulatory molecule, enhancing overall CAR activity2. To build on this initial work performed by our collaborators at the Guangzhou Institutes of Biomedicine and Health (GIBH), we have investigated the function and signalling of a third-generation anti-CD19 CAR T-cell construct, incorporating an intracellular TLR2-derived signalling domain between membrane-proximal CD28 and cytoplasmic tail CD3ζ domains (1928T2z).
In vitro, 1928T2z CAR T-cells were able to upregulate the expression of surface activation markers, produce pro-inflammatory cytokines and specifically lyse antigen expressing K562-CD19 target cells. Through addition of a truncated EGFR surface tag (EGFRt), highly pure populations of CAR T-cells were generated for use in in vitro assays. Full activity of 1928T2z-EGFRt CAR T-cells required both dimerization of the TLR2 domain and recruitment of the adaptor protein MyD88, as demonstrated using a small molecule inhibitor of TLR2 signalling, ortho-vanillin, and by selective mutation of key TLR2 residues within the CAR. Furthermore, in comparison to a construct with a mutated TLR2 domain, 1928T2z-EGFRt CAR T-cells upregulated transcription of certain TLR-, APC- and Th2-associated genes.
Upon exposure to CD19, 1928T2z-EGFRt CAR T-cells demonstrated comparable activation to a second-generation CD28-containing CAR (1928z-EGFRt) and a third-generation CD28/4-1BB CAR (1928BBz-EGFRt). In a serial cytotoxicity assay, 1928T2z-EGFRt CAR T-cells displayed similar or superior activity compared to 1928z CAR T-cells. Surprisingly, however, production of inflammatory cytokines by 1928T2z-EGFRt was significantly lower than that by 1928z-EGFRt or 1928BBz-EGFRt CAR T-cells. Specifically, upon co-culture with CD19+ target cells and autologous monocytes, which have been shown to drive CAR T-cell-associated toxicities, 1928T2z CAR T-cells elicited lower levels of IFN-γ and GM-CSF, both of which are implicated in CAR T-cell neurotoxicity.
This thesis describes, for the first time, the characteristics of 1928T2z CAR T-cells, which include cytotoxicity against CD19 target cells in combination with reduced production of certain pro-inflammatory cytokines. I show that activity is dependent on TLR2 dimerization and MyD88 recruitment, and find that the TLR2 domain results in a distinct pattern of CAR T-cell gene expression and protein phosphorylation upon exposure to CD19. These findings correlate well with preliminary phase 1 clinical trial results using 1928T2z-EGFRt CAR T-cells against B-cell lymphomas (ENABLE, NCT04049513), and will inform the future development of CAR T-cells employing TLR domains.