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

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.

Genetic Engineering and Enrichment of Human NK Cells for CAR-Enhanced Immunotherapy of Hematological Malignancies

“…We previously established a novel element for CARs, the CD34-derived C6 hinge, that facilitates detection and enrichment of CAR T cells in clinical processes and does not negatively influence the functional characteristics of genetically transduced and enriched T cells in vitro and in vivo in mice ( 28 ). In the present study, we demonstrated that C6 as a hinge in CAR constructs expressed on human NK cells also allows to easily detect transduced NK cells in vitro or the peripheral blood of mice using the QBend-10 antibody by flow cytometry.…”

“…All CAR constructs were optimized for human codon usage and synthesized by GeneArt (Thermo Fisher Scientific) or BioCat (Heidelberg, Germany). The CARs were co-expressed via a T2A site with the monomeric tag blue fluorescent protein (BFP from Evrogen, Moscow, Russia) under the control of the myeloproliferative sarcoma virus (MPSV) promoter ( 27 ) and equipped with our CD34-derived hinge C6 ( 28 ), CD28 transmembrane, and co-stimulatory domains as well as the CD3 zeta-chain unit ( 27 ). The CD19, CD33, CD123, and epidermal growth factor receptor (EGFR) (clone cetuximab/C225, from now on referred to as Cetux) CARs were previously described ( 20 , 27 – 29 ).…”

“…Three to four days after transduction, CAR NK cells were enriched on an OctoMACS™ Separator using MS columns or on a QuadroMACS™ Separator using LS columns after staining with CD34 microbeads (clone QBend-10) according to the manufacturer’s instructions (Miltenyi Biotec) and similarly as described previously for CAR T cells ( 28 ). In order to investigate the MACS enrichment efficiency, the CAR NK cell purity and yield, and the expression of the different CAR constructs on the transduced NK cells, the three cell fractions—before MACS, flowthrough, and after MACS—were collected and analyzed by flow cytometry for BFP expression and/or CD34 positivity.…”

“…Each group consisted of 3 animals. Peripheral blood was analyzed at days 5, 10, 20, and 27 after lysis of erythrocytes with BD Pharm Lyse (BD Biosciences, San Jose, CA, USA) on a MACSQuant™ Analyzer 10 for murine CD18, human CD45, CD56, and EGFP-expressing cells (all antibodies were REA clones from Miltenyi Biotec) as described previously ( 28 ).…”

“…For the engraftment of REH cells expressing a firefly luciferase/EGFP fusion protein (REH LucEG ) ( 28 ), 7- to 10-week-old NSG mice were treated by i.p. injection of 25 mg/kg of busulfan and the next day intravenously transplanted with 3.5 × 10 6 REH LucEG cells via tail vein injections.…”