Oral Presentation 49th Annual Scientific Meeting of the Australian and New Zealand Society for Immunology 2021

Using CRISPR/Cas9 to engineer next-generation armoured CAR T cells with improved efficacy against solid tumours (#18)

Amanda X. Y. Chen 1 2 , Phil K Darcy 1 2 , Imran G House 1 2 , Paul A Beavis 1 2
  1. Cancer Immunology Program, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia
  2. Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Victoria, Australia

INTRODUCTION: Chimeric antigen receptor (CAR) T cell therapy, while highly efficacious for the treatment of certain haematological malignancies, remains ineffective in solid cancers. This is largely due to the immunosuppressive nature of the tumour microenvironment (TME). “Armouring” CAR T cells to express proinflammatory cytokines is a promising strategy to modulate the TME and engage endogenous anti-tumour immunity, which we have demonstrated to be critical for the treatment of heterogeneous solid cancers1. However, the use of a synthetic promoter to drive cytokine expression leads to unrestricted cytokine production, and can cause severe clinical toxicities. Recent advances in CRISPR/Cas9 gene editing for primary T lymphocytes have presented new avenues for the precise engineering of armoured CAR T cells2. We hypothesised that engineering CAR T cells to express cytokines under the control of endogenous gene promoters with appropriate regulation would enhance the efficacy and safety of CAR T cells.

METHODS: Genome-wide RNA-sequencing was used to identify candidate gene loci. A novel CRISPR/Cas9-mediated homology directed repair (CRISPR HDR) strategy was then employed to knock in (KI) proinflammatory cytokines into these gene loci to produce CRISPR-engineered "armoured" T cells. This represents the first study to successfully knock-in genes to primary murine T cells. This approach was applied to syngeneic CAR T and OT-I mouse models that enable simultaneous assessment of efficacy and safety1,2.

RESULTS: CRISPR-engineered primary murine T cells exhibited gene KI efficiency of ~50% with enhanced cytokine production in vitro. In vivo, CRISPR-engineered T cells improved anti-tumour efficacy in both the anti-Her2 CAR T cell and OT-I models. Importantly, this approach led to undetectable systemic expression of cytokines and a favourable safety profile. 

CONCLUSION: CRISPR HDR enables the generation of armoured CAR T cells with improved safety and efficacy. This is superior to analogous strategies currently used clinically such as artificial NFAT-based promoter systems.

  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. 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.