Conferring a chimeric antigen receptor (CAR) to T cells supercharges their functionalities against cancerous tumors. However, enhancing their therapeutic potency also requires proper regulation to prevent any adverse side effects.1 To reshape the tumor microenvironment in a customized, regulated, and tumor-dependent manner, 3 genes of the T cell activation pathway (namely TCR, CD25 and PD1) were specifically repurposed in this study to allow the conditional secretion of IL-12P70 (a pro-inflammatory cytokine) by CAR T cells.2 For this, Cellectis exploited their proprietary gene editing TALEN® technology as well as HDR strategy to deliver a CAR into the TCRα gene (TRACCAR) and to insert IL-12P70 into either IL2Rα or PDCD1 genes.2 This publication provides a detailed account of their “smart” CAR T-cells’ activity.2
Cellectis picks TLA to confirm targeted insertion in their engineered T cells
For the in-depth genetic QC of their engineered T cells, the researchers leveraged TLA technology. Our data confirmed targeted insertions and our analysis also suggested possible concatemers or homology-independent integrations.2
Ultimately, the performed targeted modifications generated T cells that (1) lacked TCR and PD1, (2) were endowed with CAR expression, and (3) preserved the expression of CD25 (an important component of the IL-2 receptor). Following T-cell customization, IL-12P70 was seen to be transiently secreted upon engaging a tumor target, which in turn stimulated the build-up of TRACCAR T cells. Consequently, the tailored T cells displayed a significantly improved antitumor activity.
However, it is important to realize that the powerful anticancer agent IL-12 brings about pleiotropic effects and that its systemic infusion in patients is highly toxic.3 For this reason, Valton (Vice President of Gene Therapy at Cellectis) warns that the “immunostimulatory agent must be precisely delivered at the tumor site at an appropriate dose to prevent potential toxic side effects - while avoiding the toxicity of a systemic injection of IL-12.”3
Next-generation CAR T-cells for the treatment of different malignancies
In summary, through an ingenious engineering strategy, the Cellectis team was able to successfully rewire the natural TCR regulatory pathway such that, upon tumor sensing and subsequent (tumor) engagement by the CAR, IL-12 would be secreted.2 In other words, the scientists were able to manipulate T cells such that they could regulate themselves and adapt to the tumor environment accordingly, for better therapeutic outcome. Valton notes that “these highly intelligent CAR T-cells can sense and remodel their microenvironment in a tailored, highly regulated, and antigen-specific manner, allowing us to have more control over increasingly potent treatments and less risk of general secretion into healthy tissues. This engineering strategy could bring smarter, safer, and more effective treatments to the forefront for patients in need."4
Complete genetic characterization of engineered immune cells
TLA enables the complete targeted sequencing of any regions of interest. With a single primer pair, we will be able to uncover any (trans)gene(s) and will also sequence their neighboring regions (up to hundreds of kilobases in size). For this reason, TLA analysis is often used for the unbiased mapping of transgene insertion and will help you and your team QC your genetic engineering. More precisely, TLA can identify the genomic positions of integration sites as well as assess the integrity of the integrated vector sequence in a hypothesis-neutral manner. Therefore, our TLA-based solutions have great relevance in the complete genetic characterization of transgene insertion for producer cell banks and immunotherapy products, within the cell and gene therapy manufacturing space.
We have recently been interviewed by Genetic Engineering & Biotechnology News (GEN), who has recognized our proprietary technology as an improved analytical genetic characterization and QC method for cell and gene therapy products. Check out the article and/or watch our latest webinar, here below, to learn more about our TLA capabilities!
GEN article: Gene Therapy Adopts New Tools to Guarantee Quality
References
[1] Leonard, J. P. et al. Effects of single-dose interleukin-12 exposure on interleukin-12-associated toxicity and interferon-gamma production. Blood 90, 2541–2548 (1997).
[2] Sachdeva, M., Busser, B.W., Temburni, S. et al. Repurposing endogenous immune pathways to tailor and control chimeric antigen receptor T cell functionality. Nat Commun 10, 5100 (2019). https://doi.org/10.1038/s41467-019-13088-3
[3] Philippidis, A. (2019, November 13). Cellectis details proof-of-concept for “smart” CAR T-cells. Genetic Engineering & Biotechnology News. https://www.genengnews.com/news/cellectis-details-proof-of-concept-for-smart-car-t-cells/
[4] Cellectis. (2019, November 13). Cellectis publishes creation of “smart CAR T-cells” for potentially safer, more effective treatments for cancer in Nature Communications. Cellectis. https://www.cellectis.com/en/press/cellectis-publishes-creation-of-smart-car-t-cells-for-potentially-safer-more-effective-treatments-for-cancer-in-nature-communications
