Investigating the Role of Oligomeric State in Chimeric Antigen Receptor Function Using de novo Designed Transmembrane Structures
AuthorChandler, Nicholas John
Document TypePhD thesis
Access StatusThis item is embargoed and will be available on 2023-09-08.
© 2021 Nicholas John Chandler
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.
KeywordsImmunotherapy; CAR T cell; Structural biology; De novo protein design; Immunology; Oligomeric proteins; Receptor biology
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