Medical Biology - Theses
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ItemInvestigating the Role of Oligomeric State in Chimeric Antigen Receptor Function Using de novo Designed Transmembrane Structures( 2021)Chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of B cell malignancies by redirecting patient T cells to destroy cancer cells using engineered receptors. While CAR T cell therapies hold enormous potential as treatments in a wide range of tumour settings, treatments for non-B cell cancers have largely failed to significantly improve patient outcomes thus far. Furthermore, CAR therapies carry significant risk of inducing cytokine release syndrome (CRS), a potentially deadly toxicity caused by excessive release of inflammatory cytokines. The ability to minimize toxicity whilst maintaining adequate tumour cell-killing is therefore vital to the continued improvement of CAR therapies. We aimed to investigate the currently ill-defined relationship between CAR oligomeric state and potency using a novel protein engineering approach, with the aim of leveraging this knowledge to predictably modulate CAR activity. With de-novo protein design collaborators we identified synthetic transmembrane domain (TM) sequences that predictably formed defined homo-oligomeric structures. In addition to a previously validated trimeric TM sequence, I used X-ray protein crystallography to determine the structure of a dimeric TM peptide that agreed closely with its predicted structure. I inserted these novel oligomeric TM sequences into a well-established anti-HER2 CAR construct (comprising an anti-HER2 scFv attached via stalk/TM to costimulatory and stimulatory tail sequences) and validated their oligomeric state and signalling capacity in a mouse T cell line. When expressed in primary mouse T cells and incubated with HER2+ target cells, dimeric and trimeric CARs exhibited enhanced target cell killing compared to a reference anti-HER2 CAR. Using an in vivo mouse tumour model it was subsequently demonstrated that CAR oligomeric state correlates positively with CAR T cell anti-tumour efficacy. CARs encoding synthetic oligomeric TM’s also demonstrated a dramatic reduction in the release of inflammatory, CRS-associated cytokines within in vitro experiments. Using rational TM sequence mutations I identified lateral interactions between CARs and the endogenous T cell costimulatory molecule CD28 in primary mouse T cells as the key determinant of CAR cytokine release. These findings present an opportunity to improve efficacy and safety of CAR T cell therapies and warrant further validation in other clinically relevant CAR T cell disease models.