Verbesserte Therapieeffizienz durch duales Targeting mit CAR-T-Zellen und BiTEs gegen EpCAM bei Kopf- und Halstumoren

Background:The success of CAR T-cell therapy against B-cell malignancies could so far not be translated to the treatment of solid tumors due to a heterogeneous expression of target antigens, a hostile tumor microenvironment and on-target off-tumor effects. We investigated if CAR T-cell therapy can be enhanced by additionally releasing bi-specific T-cell engagers (BiTEs) recognizing a 2nd tumor antigen. The transmembrane protein EpCAM is a prognostic biomarker for HNSCC and a promising candidate for targeted immunotherapy.

Methods:EpCAM expression was analyzed on 26 HNSCC cell lines by FACS. The cytotoxicity and specificity of human T-cells expressing EpCAM CAR constructs was evaluated against HNSCC cells in cytotoxic assays. EpCAM knockout (KO) cells were created by CRISPR/Cas9 to detect off-target effects of EpCAM CARs. EpCAM BiTE constructs were generated by connecting scFvs via a linker to scFv of the CD3 antibody. The BiTE-mediated cytotoxicity was measured and compared to CAR-associated cytotoxicity of T-cells.

Results:80% of all HNSCC cells expressed EpCAM and were efficiently and specifically killed by EpCAM CAR T-cells, regardless of antigen density. The EpCAM-specific BiTEs demonstrating comparable or enhanced cytotoxicity of unmodified T-cells plus BiTEs compared to their respective CAR counterparts expressed on T-cells. For dual targeting, CD44v6 CAR-mediated lysis was combined with EpCAM BiTE secretion. The efficacy was demonstrated by cytotoxicity assays using wild-type or EpCAM/CD44v6 KO HNSCC cells.

Conclusion: The combination of CAR T-cell therapy with the secretion of BiTEs will facilitate to increase the effectiveness of CAR T-cells by recruiting residual T-cells in the tumor and by reducing the antigen escape due to dual targeting possibilities.

nein