Boosting CAR T cell anti-tumor potency through endomembrane engineering

Background.

CAR T cells represent a highly promising type of cancer immunotherapy. They have yielded impressive clinical response rates in the treatment of hematologic cancers, but for the treatment of solid tumors, their potency, tumor specificity and safety need to be further improved (Hou, 2021; Cappell, 2023). For that purpose, complex proteins including CARs, components of logic gated signaling circuits, drug-regulated protein switches, etc. have to be expressed (Zajc, 2020). Unfortunately, many of these complex proteins show poor expression in T cells, thus calling for improvements of their protein expression machinery. Moreover, the secretory capacity of the CAR T cell is also decisive for its very basic anti-tumor effector functions, which mostly rely on surface-expressed or secreted proteins (CARs, cytokines, perforin, granzymes, etc.) (Hou, 2021).
To improve the secretory capacity of CAR T cells and thereby their anti-tumor potency, we will use endomembrane engineering to adapt the size and/or morphology of the endoplasmic reticulum (ER) in CAR T cells. Such a morphologic change and/or expansion of the ER has been associated with increased protein secretion in various expression systems.

Aims and Methods.

Our extensive expertise with endomembrane engineering in other cell types (plants, yeast) - combined with our CAR T cell knowhow - will serve as a foundation for endomembrane engineering in human CAR T cells. CAR T cell engineering will be conducted by using lentiviruses and CRISPR/Cas9 technology. Expression of complex CAR molecules (e.g. bispecific CARs that prevent tumor resistance mechanisms), cytokine secretion and cytotoxic activity in response to cancer cells will be assessed using established assay systems (Salzer, 2020; Zajc, 2020). The top candidates will be analyzed in more detail, e.g. their effects on ER size and morphology within CAR T cells (confocal and 2D/3D electron microscopy).
This project will (1) improve our understanding of the molecular processes regulating protein expression in human T cells and (2) establish strategies to elevate their secretion capacity, ultimately enabling the design of next generation CAR T cells with improved anti-tumor potency.

REFERENCES
1. Cappell, K. M. and J. N. Kochenderfer (2023) Long-term outcomes following CAR T cell therapy: what we know so far. Nat Rev Clin Oncol 20, 359-371. doi: 10.1038/s41571-023-00754-1
2. Hou, A. J., L. C. Chen and Y. Y. Chen (2021) Navigating CAR-T cells through the solid-tumour microenvironment. Nat Rev Drug Discov 20, 531-550. doi: 10.1038/s41573-021-00189-2
3. Salzer, B., C. M. Schueller, C. U. Zajc, T. Peters, M. A. Schoeber, B. Kovacic, M. C. Buri, E. Lobner, O. Dushek, J. B. Huppa, C. Obinger, E. M. Putz, W. Holter, M. W. Traxlmayr and M. Lehner (2020) Engineering AvidCARs for combinatorial antigen recognition and reversible control of CAR function. Nat Commun 11, 4166. doi: 10.1038/s41467-020-17970-3
4. Zajc, C. U., M. Dobersberger, I. Schaffner, G. Mlynek, D. Puhringer, B. Salzer, K. Djinovic-Carugo, P. Steinberger, A. De Sousa Linhares, N. J. Yang, C. Obinger, W. Holter, M. W. Traxlmayr and M. Lehner (2020) A conformation-specific ON-switch for controlling CAR T cells with an orally available drug. Proc Natl Acad Sci U S A 117, 14926-14935. doi: 10.1073/pnas.1911154117