RevCAR-expressing immune effector cells for targeting of Fn14-positive glioblastoma.

In recent studies, we have established the unique adapter chimeric antigen receptor (CAR) platform RevCAR which uses, as an extracellular CAR domain, a peptide epitope instead of an antibody domain. RevCAR adapters (termed RevCAR target modules, RevTMs) are bispecific antibodies that enable the reversible ON/OFF switch of the RevCAR system, improving the safety compared to conventional CARs. Here, we describe for the first time its use for retargeting of both T and NK-92 cells. In addition, we describe the development and preclinical validation of a novel RevTM for targeting of the fibroblast growth factor-inducible 14 (Fn14) surface receptor which is overexpressed on Glioblastoma (GBM) cells, and therefore serves as a promising target for the treatment of GBM. The novel RevTM efficiently redirects RevCAR modified T and NK-92 cells and leads to the killing of GBM cells both in vitro and in vivo. Tumor cell killing is associated with increased IL-2, TNF-α and/or IFN-γ secretion. Hence, these findings give an insight into the complementary potential of both RevCAR T and NK-92 systems as a safe and specific immunotherapeutic approach against GBM.

Targeting colorectal cancer cells using AND-gated adaptor RevCAR T-cells.

Despite the success of chimeric antigen receptor (CAR) T-cells especially for treating hematological malignancies, critical drawbacks, such as "on-target, off-tumor" toxicities, need to be addressed to improve safety in translating to clinical application. This is especially true, when targeting tumor-associated antigens (TAAs) that are not exclusively expressed by solid tumors but also on hea9lthy tissues. To improve the safety profile, we developed switchable adaptor CAR systems including the RevCAR system. RevCAR T-cells are activated by cross-linking of bifunctional adaptor molecules termed target modules (RevTM). In a further development, we established a Dual-RevCAR system for an AND-gated combinatorial targeting by splitting the stimulatory and co-stimulatory signals of the RevCAR T-cells on two individual CARs. Examples of common markers for colorectal cancer (CRC) are the carcinoembryonic antigen (CEA) and the epithelial cell adhesion molecule (EpCAM), while these antigens are also expressed by healthy cells. Here we describe four novel structurally different RevTMs for targeting of CEA and EpCAM. All anti-CEA and anti-EpCAM RevTMs were validated and the simultaneous targeting of CEA+ and EpCAM+ cancer cells redirected specific in vitro and in vivo killing by Dual-RevCAR T-cells. In summary, we describe the development of CEA and EpCAM specific adaptor RevTMs for monospecific and AND-gated targeting of CRC cells via the RevCAR platform as an improved approach to increase tumor specificity and safety of CAR T-cell therapies.