Oral Presentation Asia-Pacific Vaccine and Immunotherapy Congress 2023

Identifying optimal gene promoters for CRISPR-HDR generated armoured CAR T cells (#42)

Kah Min Yap 1 2 , Amanda Chen 1 2 , Imran House 1 2 , Phil Darcy 1 2 , Paul 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

Chimeric antigen receptor (CAR) T cell therapy has shown tremendous success in haematological malignancies but limited efficacy in solid tumours, owing to immunosuppressive tumour microenvironment, tumour heterogeneity and inefficient trafficking to tumours. One promising attempt to overcoming these includes the development of armoured CAR T cells that co-express CAR with a therapeutic transgene. We have 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, artificial promoter systems have demonstrated insufficiencies in achieving site-specific transgene expression, which can cause systemic toxicities that had resulted in the 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 have previously shown that CRISPR knock-out (KO) of immunosuppressive genes like adenosine A2A receptor enhanced CAR T cell function3. Now, we aim to exploit CRISPR-homology directed repair (HDR) technology to knock-in (KI) transgenes into tumour-specific gene loci for tighter regulation of transgene expression. We performed genome-wide RNA-Sequencing on CAR T cells and identified a list of genes with high and 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, target genes that did not adversely impact CAR T cell function/phenotype following KO had a reporter gene knocked in. Our investigations have revealed novel gene promoters that upon KI elicit enhanced transgene expression in tumours but low transgene expression at non-tumour sites relative to the prototypic PD-1 promoter. This is anticipated to ultimately enable the generation of armoured CAR T cells with higher cytokine-secreting capacity specifically in tumours and thus improved safety and efficacy profiles.

  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