Immunotheranostic target modules suitable for imaging and navigation of UniCAR T-cells to strike FAP-expressing solid tumours and their microenvironment

Chimeric antigen receptor (CAR) T-cells are unquestionably considered one of the most promising approaches in cancer immunotherapy. Nonetheless, mild to severe toxicities are associated with this approach which include e.g. on-target/off-tumour toxicities and cytokine release syndrome. Aiming for increased clinical safety, a modular universal CAR (UniCAR) platform was developed by our group in which UniCAR T-cells are exclusively activated in the presence of a target module (TM) that specifically establishes the cross-link between target cells and UniCAR T-cells. Fibroblast activation protein (FAP) is highly expressed on cancer-associated fibroblasts (CAFs) present in the tumour stroma and also found to be overexpressed in tumour cells. This protein plays an important role in promoting tumour growth, metastasis, and immunosuppression and has therefore been studied as a target for cancer diagnosis and treatment. Given this and the demonstrated efficacy, flexibility and switchability of the UniCAR system, currently demonstrated in phase I clinical trials, we hereby aimed to develop TMs targeting FAP that can be used for both immunotherapeutic and theranostic approaches. For that, the single-chain variable fragment (scFv) of an anti-human FAP mAb was fused to the peptide epitope E5B9 that is recognized by the UniCAR T-cells, creating low molecular weight TMs that are rapidly eliminated allowing a specific and recurrent on/off switch of UniCAR T-cell activity via TM dosing. Additionally, extended half-life anti-FAP TMs based on the human IgG4 Fc-domain, including a mutated version, were created intending to strengthen anti-tumour responses and to ease the clinical TM administration at later stages of tumour therapy. All TMs were tested in vitro based on naturally and artificially overexpressing 2D and 3D models, and proven to specifically redirect UniCAR T-cells to FAP-expressing target cells. Positron emission tomography (PET) using 64Cu radiolabelled anti-FAP IgG4 TMs demonstrated a FAP specific enrichment of these TMs at the tumour site of FAP overexpressing HT1080 xenografts resulting in a tumour SUV of 50 at 48h p.i. with almost no background. Single-photon emission tomography (SPECT) using 177Lu radiolabelled anti-FAP IgG4 TMs furthermore confirmed a high FAP-dependent tumour uptake. In conclusion, we hereby designed novel TMs targeting FAP with different formats that can be used for both endoradionuclide therapy and immunotherapeutic approaches using UniCAR T-cells, proving to be promising and innovative immunotheranostic tools to foster cancer treatment allowing a more convenient, individualized, and safe treatment of cancer patients.