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

Arthur Bister; Tabea Ibach; Corinna Haist; Denise Smorra; Katharina Roellecke; Martin Wagenmann; Kathrin Scheckenbach; Norbert Gattermann; Constanze Wiek; Helmut Hanenberg

doi:10.1016/j.omto.2021.11.003

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

Mol Ther Oncolytics. 2021 Nov 11:23:534-546. doi: 10.1016/j.omto.2021.11.003. eCollection 2021 Dec 17.

Authors

Affiliations

¹ Department of Otorhinolaryngology, Head & Neck Surgery, Heinrich Heine University, 40225 Düsseldorf, Germany.
² Department of Pediatrics III, University Children's Hospital, University of Duisburg-Essen, Hufelandstrasse 55, 45147 Essen, Germany.
³ Department of Hematology, Heinrich Heine University, 40225 Düsseldorf, Germany.

Abstract

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.

Keywords: CAR; CAR T cells; CD34; CD34 hinge; MACS enrichment; chimeric antigen receptor; detection; hinge; immunotherapy; lentiviral vector; selection of genetically modified cells.