A novel CD34-derived hinge for rapid and efficient detection and enrichment of CAR T cells.

Immunotherapy including chimeric antigen receptor (CAR) T cell therapy has revolutionized modern cancer therapy and has achieved remarkable remission and survival rates for several malignancies with historically dismal outcomes. The hinge of the CAR connects the antigen binding to the transmembrane domain and can be exploited to confer features to CAR T cells including additional stimulation, targeted elimination or detection and enrichment of the genetically modified cells. For establishing a novel hinge derived from human CD34, we systematically tested CD34 fragments of different lengths, all containing the binding site of the QBend-10 monoclonal antibody, in a FMC63-based CD19 CAR lentiviral construct. A final construct of 99 amino acids called C6 proved to be the best candidate for flow cytometry-based detection of CAR T cells and >95% enrichment of genetically modified T cells on MACS columns. The C6 hinge was functionally indistinguishable from the commonly used CD8α hinge in vitro as well as in in vivo experiments in NSG mice. We also showed that the C6 hinge can be used for a variety of different CARs and mediates high killing efficacy without unspecific activation by target antigen-negative cells, thus making C6 ideally suited as a universal hinge for CARs for clinical applications.

CD44v6-targeted CAR T-cells specifically eliminate CD44 isoform 6 expressing head/neck squamous cell carcinoma cells.

Immune checkpoint blockade can cause regression of recurrent and/or refractory head and neck squamous cell carcinoma (HNSCC). As a second type of immunotherapy, adoptive cellular therapy with genetically modified patient's T-cells redirected against the autologous malignant cells by expressing chimeric antigen receptors (CARs) recognizing tumor-associated antigens has been established as highly efficient personalized treatment for hematological malignancies. In solid cancers however, the application of these genetically modified immune effector cells still lacks equal response rates. CD44v6 is an isoform of the hyaluronic receptor CD44 that is almost exclusively expressed at high levels on solid cancers and has been associated with tumorigenesis, tumor cell invasion and metastasis. Here, we established a highly specific CAR against CD44v6 on HNSCC cells that can be expressed on normal T-cells with lentiviral vectors. Using primary human HNSCC cells in combination with CRISPR/Cas9 and overexpression approaches allowed us to confirm the high specificity of our CAR construct for the tumor-associated CD44v6 as target antigen and to demonstrate a direct correlation between CD44v6 expression levels and cytotoxicity of the CAR T-cells. Importantly, the design of our clinically applicable lentiviral vector facilitates to co-express a second transgene for in vivo control of CAR T-cells, if undesired side-effects or toxicities occur.