UniCAR T-Cell Potency-A Matter of Affinity between Adaptor Molecules and Adaptor CAR T-Cells?

Although Chimeric Antigen Receptor (CAR) T-cells have shown high efficacy in hematologic malignancies, they can cause severe to life-threatening side effects. To address these safety concerns, we have developed adaptor CAR platforms, like the UniCAR system. The redirection of UniCAR T-cells to target cells relies on a Target Module (TM), containing the E5B9 epitope and a tumor-specific binding moiety. Appropriate UniCAR-T activation thus involves two interactions: between the TM and the CAR T-cell, and the TM and the target cell. Here, we investigate if and how alterations of the amino acid sequence of the E5B9 UniCAR epitope impact the interaction between TMs and the UniCAR. We identify the new epitope E5B9L, for which the monoclonal antibody 5B9 has the greatest affinity. We then integrate the E5B9L peptide in previously established TMs directed to Fibroblast Activation Protein (FAP) and assess if such changes in the UniCAR epitope of the TMs affect UniCAR T-cell potency. Binding properties of the newly generated anti-FAP-E5B9L TMs to UniCAR and their ability to redirect UniCAR T-cells were compared side-by-side with the ones of anti-FAP-E5B9 TMs. Despite a substantial variation in the affinity of the different TMs to the UniCAR, no significant differences were observed in the cytotoxic and cytokine-release profiles of the redirected T-cells. Overall, our work indicates that increasing affinity of the UniCAR to the TM does not play a crucial role in such adaptor CAR system, as it does not significantly impact the potency of the UniCAR T-cells.

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.