Poster Presentation Melbourne Immunotherapy Network Winter Symposium 2021

Using CRISPR/Cas9 to engineer next-generation armoured CAR T cells with improved efficacy against solid tumours (#104)

Amanda X. Y. Chen 1 2 , Matthew H Porteus 3 4 , Phillip 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
  3. Department of Pediatrics , Stanford University School of Medicine, Stanford, California, USA
  4. Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, California, USA

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-cancer immunity. 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 cells. We hypothesised that engineering CAR T cells to express cytokines under the transcriptional control of tumour-specific promoters would enhance the safety and efficacy of armoured CAR T cells.

METHODS: Genome-wide RNA-sequencing was used to identify genes in CAR T cells with tumour-specific expression. PD-1 and NR4A2 were key candidates due to their inhibitory role in T cells. A novel CRISPR/Cas9-mediated homology directed repair (CRISPR HDR) strategy was then employed to knock in (KI) proinflammatory cytokines into these gene loci. This enabled the simultaneous deletion of an inhibitory gene while achieving tumour-specific control of cytokine expression.

RESULTS: CRISPR-engineered primary murine T cells exhibited gene KI efficiency of ~50%. Integrating TNF into either PD-1 or NR4A2, which are upregulated upon TCR or CAR stimulation, led to antigen-specific induction of transgene expression in tumour cocultures. In vivo, PD-1 or NR4A2 knockout CAR T cells exhibited superior anti-tumour function and integrating TNF into either PD-1 or NR4A2 led to tumour-specific TNF expression. Ongoing studies are assessing the therapeutic potential of this approach.

 CONCLUSION: CRISPR HDR enables the generation of armoured CAR T cells with tumour-restricted cytokine secretion. This is superior to analogous strategies currently used clinically such as artificial NFAT-based promoter systems.