Anderson, G. S. | Search bioRxiv

bioRxiv2023

Targeting Myeloma Essential Genes using NOT Gated CAR- T Cells, a computational approach.

Sensitive cell surface proteomics studies have shown that the number of completely tumour-specific targets for adoptive cellular immunotherapy is extremely low. Even approved CAR T-cell targets appear to have expression in the central nervous system, leading to long-term neurological complications. We propose that this toxicity could be significantly improved by adoption of NOT-gates, which have been shown to limit CAR T-cell activity against healthy tissue expressing a second target that is absent on the tumour. Furthermore, the approach could also target essential, but non-specific proteins on tumour cells. The use of a NOT gate confers the specificity, whilst targeting the essential protein limits antigen escape. Here we explore the feasibility of such an approach for CAR T-cell targeting of primary myeloma. We show that none of the 45 most essential proteins are unique to the myeloma cell. However, whilst widely expressed, one of the most important proteins for myeloma cell survival, the transferrin receptor, could safely be targeted by a NOT-gate approach. Exploring co-expression patterns demonstrate 26 proteins that are not expressed on myeloma cells, but which are coexpressed with the transferrin receptor in all healthy tissues. We also describe a web app, NOTATER, which can be used by scientists with no bioinformatic capabilities to explore potential NOT-gate combinations in myeloma.

cancer biology↗

bioRxiv2023

Cell surface proteomics reveal a strategy for targeting myeloma through combinatorial targeting of TNFRSF8 and TMPRSS11E

Chimeric antigen receptor (CAR) T-cell therapy is a highly effective novel treatment in haematological malignancies that has shown promise as a therapeutic option in multiple myeloma. However, widespread adoption of CAR T-cell therapy in myeloma has been hindered by the challenge of unbiased target antigen identification and selection. As activation of CAR T-cells requires minimal antigen on the cell surface, a major risk of toxicity is destruction of healthy tissue expressing the target protein, i.e. on-target, off-tumour toxicity. Indeed, examination of the myeloma surface proteome demonstrated that there was no single target that was completely unique to myeloma cells. One approach to achieve target specificity is to require simultaneous expression of two proteins on the target cells, so-called AND-gate targeting. To identify potential AND-gate combinations for myeloma, we devised an algorithm to prioritise pairings that exhibited pan-myeloma expression and no overlapping expression in vital healthy tissue, as predicted by proteomics. Through this approach, we identified over 600 combinations. To minimise the risk of exhaustion or priming by CAR T-cells, any combination whereby one of the two antigens was expressed in T-cells was also excluded, leading to the prioritisation of 144 candidate pairings. This demonstrates the potential for AND-gating to expand the repertoire of CAR T-cell targets for myeloma. We evaluated one of these candidate pairings, TMPRSS11E and TNFRSF8, in vitro. Activation in the Jurkat cell line co-expressing a suboptimal CAR against TNFRSF8 and a chimeric costimulatory receptor (CCR) against TMPRSS11E was markedly enhanced following co-culture with a dual-target positive myeloma cell line compared with single-target positive K562, demonstrating improved discrimination between tumour and non-tumour cells.

cancer biology↗