Chimeric Antigen Receptor (CAR) T cell therapy, albeit a promising treatment option for cancer, still faces a variety of challenges that hinder its efficacy. One factor that seems to affect CAR-T cell performance is cell phenotype: for example, a high proportion of exhausted or terminally differentiated effector cells in the CAR product is associated with a less satisfactory performance, while a high proportion of memory or naïve-like T cells is correlated with a better response to the therapy with higher rates of complete remission [1]. Cell phenotype is modulated through a complex interaction of several factors including culture conditions such as culture duration and media, added cytokines and pharmaceuticals, etc. In particular, there seems to be a relation between cell phenotype and the cells’ intrinsic pathways and metabolism; for instance, an increase in glycolysis and related pathways, such as the PI3K/Akt/mTOR pathway, have been associated with unfavorable T cell differentiation into effector or exhausted cells, resulting in a mediocre CAR-T performance [2]. Conversely, increased oxidative phosphorylation and fatty acid oxidation have been linked to memory/naïve CAR-T cells and a better overall therapeutic effect. In this context, we postulated that CAR-T performance can
be boosted by inhibiting factors that push T cell differentiation into less desirable phenotypes. We opted to transiently inhibit gene expression of target genes (such as Akt) via antisense oligonucleotides (ASOs). In this study, CAR-T cells were treated with ASOs targeting genes of interest (e.g. Akt) during the ex-vivo manufacturing process, and their phenotype and anti-tumor effect were examined. By starting the ASO treatment early in the process, we expect the cells to maintain their naïve-like/memory phenotypes, and drift less towards terminally differentiated and exhausted phenotypes, ultimately leading to a higher tumor killing efficiency.

Acknowledgement: This work was supported by the National Research Foundation of Korea (NRF) grants funded by the Korea government (MIST), (No.RS-2024-00347668) (No.RS-2024-00405360).

References
[1] J. A. Fraietta et al., “Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia,” Nat Med, vol. 24, no. 5, pp. 563–571, May 2018, doi: 10.1038/s41591-018-0010-1.
[2] M. Z. Madden and J. C. Rathmell, “The Complex Integration of T-cell Metabolism and Immunotherapy,” Cancer Discovery, vol. 11, no. 7, pp. 1636–1643, Jul. 2021, doi: 10.1158/2159-8290.CD-20-0569.