Short-lived and extended half-life target modules for redirecting UniCAR T-cells against sialyl-Tn expressing cancer cells

Background
The development of chimeric antigen receptors (CARs) has rapidly emerged as a promising approach in cancer immunotherapy. Nonetheless, drawbacks associated with CAR T cell therapies include on-target/off-tumor effects and cytokine release syndrome. Aiming an increased clinical safety while preserving the efficacy of such therapy, we developed a novel modular universal CAR platform termed UniCAR. UniCAR T-cells are exclusively activated in the presence of a target module (TM), which establishes the cross-link between antigen-specific cancer cells and UniCAR T-cells in an individualized time- and target-dependent manner. The carbohydrate antigen sialyl-Tn (STn) is a particularly interesting target due to its expression in several types of cancer and absence in normal healthy tissues. Given the small size of such TMs, they are rapidly eliminated and thus, possible side effects and activation of UniCAR T-cells can be easily controlled by TM dosing. In late phases of treatment, TMs with extended half-life may play an important role by improving the eradication of residual tumor cells.
Methods
In this work, a novel longer-lasting TM against STn was developed, characterized and compared to the previously developed short-lived anti-STn TM. Short-lived TMs are composed of a tumor-specific binding moiety fused to the La peptide epitope (E5B9) which is recognized by UniCAR T-cells. In extended half-life TMs, these two components are fused via an Fc domain derived from the human IgG4 molecule. Functional and pharmacokinetic properties were assessed using in vitro and in vivo assays.
Results
The developed anti-STn IgG4-based TM efficiently activates and redirects UniCAR T-cells to STn-expressing tumors in a highly efficient target-specific and target-dependent manner, promoting the secretion of pro-inflammatory cytokines, tumor cell lysis of breast and bladder cancer cells in vitro and of breast cancer cells in experimental mice. A comparable or increased killing efficiency was obtained at a lower concentration range in comparison to the results obtained for the anti-STn scFv-based TM. Additionally, PET studies demonstrate the specific enrichment of the anti-STn IgG4-based TM at the tumor site presenting a prolonged serum half-life compared to the scFv short-lived TM.
Conclusions
Taken together, these data demonstrate the effective and potential application of this CAR T cell-derived modular system to target STn in different types of cancer using different TM formats. The use and combination of such molecules with different formats and half-lives provides highly promising and customized tools for retargeting of UniCAR T-cells in a flexible, individualized and safe manner at different stages of treatment.