{
  "abstract": "Background The tremendous success of chimeric antigen receptor (CAR) T cell therapy in haematological malignancies has not been recapitulated in the solid tumour setting, owing to tumour-induced immunosuppression, tumour antigen heterogeneity and inefficient tumour trafficking of CAR T cells. One promising solution includes ‘armouring’ CAR T cells with therapeutic transgenes such as pro-inflammatory cytokines. Indeed, we demonstrated that CAR T cells engineered to express dendritic cell growth factor Flt3L could effectively engage host anti-tumour immunity crucial for overcoming antigen-negative relapse. 1 However, synthetic promoters have demonstrated insufficiencies in driving tumour-restricted cytokine expression, which had caused systemic toxicities and trial termination.2 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/Cas9-mediated knock-out (KO) of immunosuppressive gene A2AR enhanced CAR T cell function.3 Now, we aim to exploit a CRISPR/Cas9-mediated knock-in (KI) strategy to leverage endogenous gene regulatory elements to restrict transgene expression to the tumour site (figure 1), and hypothesise that this would enable the generation of armoured CAR T cells with improved safety and efficacy (Accepted, Nature).Methods Genome-wide RNA sequencing was performed on human anti-Lewis Y and murine anti-Her2 CAR T cells isolated from tumours and spleens of xenogeneic and syngeneic mouse models, respectively. 27 genes upregulated in intratumoural relative to splenic CAR T cells were identified as potential KI sites. As KI disrupts target gene expression, the impact of knocking out each gene on CAR T cell function or phenotype was first assessed. 8 genes that did not show adverse impact following KO had GFP knocked in.Results NR4A2 and RGS16 emerged as novel tumour-specific genes upon GFP KI. While NR4A2 was highly tumour-restricted and could deliver highly potent cytokines (e.g., IL-12) without inducing toxicities in mice (figure 2A), RGS16 had high intratumoural expression and could mediate the efficacy of less potent cytokines (e.g., IL-2) (figure 2B).Conclusions Endogenous tumour-specific promoters enabled the generation of IL-12- and IL-2-armoured CAR T cells with enhanced efficacy in both xenogeneic and syngeneic mouse models. These improvements were associated with increased CAR T cell proliferative capacity and polyfunctionality, along with activation of endogenous anti-tumour immune responses, all in the absence of systemic toxicities. Notably, this CRISPR-KI strategy demonstrated superior tumour specificity compared to conventional synthetic promoter systems (e.g., NFAT-inducible promoter), and was successfully applied to patient-derived CAR T cells, demonstrating its translational potential.References Lai J, 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;21(8):914-926. doi:10.1038/s41590-020-0676-7Zhang L, Morgan RA, et al. Tumor-infiltrating lymphocytes genetically engineered with an inducible gene encoding interleukin-12 for the immunotherapy of metastatic melanoma. Clin Cancer Res. 2015;21(10):2278-2288. doi:10.1158/1078-0432.Ccr-14-2085Giuffrida L, Sek K, et al. CRISPR/Cas9 mediated deletion of the adenosine A2A receptor enhances CAR T cell efficacy. Nat Commun. 2021;12(1):3236. doi:10.1038/s41467-021-23331-5Ethics Approval All murine experiments were performed in accordance with the Peter MacCallum Cancer Centre (PMCC) Animal Experimentation Ethics Committee under projects #E582, #E664, #E671 and #E693. Frozen apheresis samples were collected from patients in accordance with the PMCC Human Research and Ethics Committee under HREC/74245/PMCC.Abstract 297 Figure 1CRISPR-KI strategy for generating next-generation armoured CAR T cells with enhanced safety and efficacy. Targeted insertion of an armouring gene into an endogenous gene locus, which is specifically expressed by CAR T cells at the tumour site and encodes for a protein inhibitory to CAR T cell functionAbstract 297 Figure 2Identification of NR4A2 and RGS16 as optimal tumour-specific genes for generating cytokine-armoured CAR T cells with enhanced safety and efficacy.Human anti-Lewis Y (LeY) CAR T cells CRISPR-engineered to express IL-12 or IL-2 from the NR4A2 or RGS16 locus were adoptively transferred into mice bearing subcutaneous OVCAR-3 tumours, following which tumour progression and body weight were monitored",
  "authors": [
    {
      "affiliations": [
        "Peter MacCallum Cancer Centre, Carlton, VIC, Australia",
        "The University of Melbourne, Parkville, VIC, Australia"
      ],
      "name": "Kah Min Yap"
    },
    {
      "affiliations": [
        "The University of Melbourne, Parkville, VIC, Australia",
        "Peter MacCallum Cancer Centre, Melbourne, VIC, Australia"
      ],
      "name": "Amanda XY Chen"
    },
    {
      "affiliations": [
        "The University of Melbourne, Parkville, VIC, Australia",
        "Peter MacCallum Cancer Centre, Melbourne, VIC, Australia"
      ],
      "name": "Imran G House"
    },
    {
      "affiliations": [
        "The University of Melbourne, Parkville, VIC, Australia",
        "Peter MacCallum Cancer Centre, Melbourne, VIC, Australia"
      ],
      "name": "Phillip K Darcy"
    },
    {
      "affiliations": [
        "The University of Melbourne, Parkville, VIC, Australia",
        "Peter MacCallum Cancer Centre, Melbourne, VIC, Australia"
      ],
      "name": "Paul A Beavis"
    }
  ],
  "title": "297 Identifying optimal tumour-specific genes for CRISPR/Cas9 engineering of armoured CAR T cells with enhanced safety and efficacy",
  "uid": "ed8356d7-b420-596b-a4a7-de07a1d2cc0a"
}