Background CAR T-cell therapy has revolutionized cancer treatment, especially for hematological
diseases, and is also a focus of research for solid tumors. In CAR T-cell therapy,
the extracellular variable single chain fragment of a monoclonal antibody is connected
via a hinge domain to a transmembrane and intracellular T-cell activation motifs, thereby combining
antigen recognition and T-cell activation in a single molecule. The aim of the work
is to use the hinge to improve CAR T-cell stimulation or enable their targeted elimination or detection
and enrichment.
Methods We established six hinge domains based on the truncated form of CD34, which contained
the epitope for the CD34 antibody (Qbend-10). The fragments were cloned into lentiviral
CAR constructs and the CAR-positive T-cells were enriched with CD34-specific microbeads
via the MACS system (Miltenyi). After in vitro functionality measurements (FACS, cytotoxicity),
the CARs with C6 hinge were tested in vivo in NOD-SCID-gamma mice.
Results A final construct of 99 amino acids (C6) was the best candidate for an efficient>
95% enrichment using the MACS system and additinally allows the direct detection of
the CARs on the T-cells. The C6 hinge was functionally indistinguishable from the
approved CD8α hinge both in vitro and in vivo.
Conclusion We have shown that our CD34 C6 hinge domain can be used for a wide variety of CARs
and malignancies. It shows a high killing efficiency without mediating unspecific
activation by target antigen-negative cells. This makes the new C6 hinge ideal for
CARs in clinical applications, including head and neck cancer.
Düsseldorf School of Oncology
