Comparison of two structurally different RevTMs for the RevCAR system to specifically target CEA expressing cells

Despite the fact that chimeric antigen receptor (CAR) engineered T cells have shown encouraging therapeutic effects in hematological setups, the targeting of solid tumor-related antigens still represents a challenge. One main issue is that the expression of this type of antigens is not restricted to cancer cells, but normal tissues express them as well to some degree. In order to avoid strong side-effects caused by on-target/off-tumor effects, more controllable and specific CAR T cell-derived technologies need to be developed. In this line of thought, we developed the switchable, flexible and programmable Reverse (Rev) CAR platform. This system is based on engineered T cells expressing RevCAR molecules, which have extracellular short peptide epitopes incapable of recognizing surface antigens. Thus, the RevCAR T cells are per se inert and will only recognize the target cell through the interaction with an antigen-specific target module, named RevTM. RevTMs are bispecific antibodies designed to bind simultaneously to the target antigen and the short epitopes on the RevCAR engineered T cells. This interaction triggers a specific activation and cytotoxic activity of the RevCAR T cells redirecting them to eradicate the cancer cells.
Here, we adapted our RevCAR technology to target the carcinoembryonic antigen (CEA), which is a significant tumor marker for colorectal cancers and other carcinomas. Moreover, we developed two RevTMs with different structures: scFv- and IgG4-based. The first one is built by linking a scFv against CEA to a scFv that recognizes the RevCAR epitope through glycine and serine residues, resulting in a small sized molecule (<60 kDa) with two binding sites. The IgG4-based RevTM connects the same scFv-structures but in this case through the hinge and constant Fc regions (CH2 and CH3) of an IgG4 antibody. The formation of disulfide bridges on the hinge portion causes the formation of homodimers, resulting in a molecule with increased molecular weight (160 kDa) with a total of four binding sites. The generation of diverse RevTMs formats is of interest because their structures influence their half-life, biodistribution and killing efficiency, which are important features when it comes to adapting and customizing patient therapy using the RevCAR system.
We herein demonstrate that both scFv- and IgG4-based RevTMs can bind, on the one hand to CEA expressing cells, and on the other hand to engineered T cells expressing the RevCAR epitopes. Similarly, we confirmed that the simultaneous binding of the RevTMs to the RevCAR T cells and the CEA expressing cells promotes a specific lysis of the target cells in vitro, together with the secretion of pro-inflammatory cytokines. Additionally, we validated that CEA expressing cells are effectively eradicated by RevCAR T cells on the presence of RevTMs in vivo. Hereby, we proved that the RevCAR system can be directed to target CEA expressing cells using RevTMs with different formats, which encourages its further development as a future treatment option for solid tumors.