Poster Presentation Asia-Pacific Vaccine and Immunotherapy Congress 2024

Identifying Optimal Tumour-specific Promoters for CRISPR Knock-in Generated Armoured CAR T Cells (#122)

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

The tremendous success of chimeric antigen receptor (CAR) T cell therapy in haematological malignancies has yet to be recapitulated in the solid tumour setting, owing to immunosuppressive tumour microenvironment, tumour heterogeneity and inefficient tumour trafficking. One promising attempt to overcoming these barriers includes “armouring” CAR T cells with a therapeutic transgene. We previously demonstrated that CAR T cells engineered to secrete dendritic cell growth factor Flt3L could effectively engage the host immunity, which is critical in overcoming antigen-negative relapse1. However, synthetic promoters have demonstrated insufficiencies in achieving site-specific transgene expression, which had caused systemic toxicities and ultimately termination of a trial that expressed IL-12 using an NFAT-responsive promoter2. The advent of CRISPR/Cas9 gene editing tool has enabled the precise engineering of CAR T cells for safety and efficacy enhancements. We previously showed that CRISPR knock-out (KO) of immunosuppressive gene A2AR enhanced CAR T cell function3. Now, we aim to exploit a CRISPR knock-in (KI) strategy that leverages all endogenous regulatory elements of target genes to restrict transgene expression to the tumour site. We performed genome-wide RNA-Sequencing on CAR T cells and identified 27 genes with tumour-specific expression as potential KI sites. As target gene expression is disrupted during KI, we first assessed the impact of each gene KO on CAR T cell function/phenotype. Subsequently, 7 genes that did not adversely impact function/phenotype following KO had the reporter gene GFP knocked in. RGS16 and NR4A2 emerged as novel promoters that upon KI elicit higher transgene expression in tumours and lower transgene expression at non-tumour sites relative to the prototypic PD-1 promoter. This enabled the generation of armoured CAR T cells that secrete proinflammatory cytokines such as IL-12 and IL-2 specifically at the tumour site, leading to enhanced safety and efficacy in both syngeneic and xenogeneic models that was concomitant with improved CAR T cell polyfunctionality and proliferative capacity as well as activation of the host anti-tumour immunity. Notably, we also showed that this CRISPR KI strategy was applicable using patient-derived CAR T cells, further demonstrating the clinical translatability of this approach.

  1. Lai, J., Mardiana, S., House, I. G., Sek, K., Henderson, M. A., Giuffrida, L., . . . Beavis, P. A. (2020). Adoptive cellular therapy with T cells expressing the dendritic cell growth factor Flt3L drives epitope spreading and antitumor immunity. Nature Immunology, 21(8), 914-926. doi:10.1038/s41590-020-0676-7
  2. Zhang, L., Morgan, R. A., Beane, J. D., Zheng, Z., Dudley, M. E., Kassim, S. H., . . . Rosenberg, S. A. (2015). Tumor-Infiltrating Lymphocytes Genetically Engineered with an Inducible Gene Encoding Interleukin-12 for the Immunotherapy of Metastatic Melanoma. Clinical Cancer Research, 21(10), 2278-2288. doi:10.1158/1078-0432.Ccr-14-2085
  3. Giuffrida, L., Sek, K., Henderson, M. A., Lai, J., Chen, A. X. Y., Meyran, D., . . . Beavis, P. A. (2021). CRISPR/Cas9 mediated deletion of the adenosine A2A receptor enhances CAR T cell efficacy. Nat Commun, 12(1), 3236. doi:10.1038/s41467-021-23331-5