Invited Speaker Abstract MIN Spring Retreat 2023

CRISPR/Cas9 Engineering of Next-Generation Armoured CAR T Cells (#23)

Paul Beavis 1
  1. Peter MacCallum Cancer Centre, Melbourne, VIC, Australia

Chimeric antigen receptor (CAR) T cell therapy, while highly efficacious for the treatment of certain haematological malignancies, remains ineffective in solid cancers. This can be attributed to various factors, including immunosuppression in the tumour microenvironment (TME). “Armouring” CAR T cells to express anti-tumour factors such as proinflammatory cytokines and chemokines is a promising strategy to address their limitations in the solid tumour setting and engage endogenous anti-tumour immunity, which we have demonstrated to be critical for the treatment of heterogeneous solid cancers1. However, conventional armouring approaches utilise a synthetic promoter to drive cytokine expression, which leads to unrestricted cytokine production and can cause severe clinical toxicities2. Recent advances in CRISPR/Cas9 gene editing for primary T lymphocytes have presented new avenues for the precise engineering of armoured CAR T cells3. We aimed to engineer CAR T cells to express anti-tumour factors under the transcriptional control of tumour-specific promoters, and hypothesised that this would enhance the safety and efficacy of armoured CAR T cells.

Through a screen of promoters that are selectively active in CAR T cells isolated form the tumour site but not peripheral sites such as the spleen, we have identified two lead candidates that enable potent and highly restricted cytokine production. A novel CRISPR/Cas9-mediated homology directed repair (CRISPR HDR) strategy was employed to knock in (KI) anti-cancer factors into these gene loci and was applied to syngeneic CAR T and OT-I murine models that enable clinically relevant assessment of efficacy and safety1, 3.

Integrating proinflammatory cytokines and chemokines downstream of these identified promoters led to antigen-specific induction of transgene expression and tumour-restricted expression in vivo resulting in significantly enhanced efficacy. Importantly, these factors did not exhibit elevated systemic expression, and provided a favourable safety profile to conventional armoured CAR T cell approaches. 

  1. Lai, J. and Mardiana, S. et al. Adoptive cellular therapy with T cells expressing the dendritic cell growth factor Flt3L drives epitope spreading and antitumor immunity. Nat Immunol. 2020;10.1038/s41590-020-0676-7.
  2. Zhang, L. et al. Improving adoptive T cell therapy by targeting and controlling IL-12 expression to the tumor environment. Mol Ther. 2011 Apr;19(4):751-9. doi: 10.1038/mt.2010.313. Epub 2011 Feb 1. PMID: 21285960; PMCID: PMC3070103.
  3. Giuffrida, L. and Sek, K. et al. CRISPR/Cas9 mediated deletion of the adenosine A2A receptor enhances CAR T cell efficacy. Nat Commun. 2021 May 28;12(1):3236. doi: 10.1038/s41467-021-23331-5.