T cells, engineered to express chimeric antigen receptors (CAR), are capable to generate an extremely potent and highly specific immune response against defined surface-expressed antigens. The very same features however hinder a more general clinical application, first because on-target off-tumor toxicity can cause life-threatening damage to vitally important healthy tissue and second because heterogeneity as well as plasticity of tumor tissue circumvent elimination through modulation or loss of target epitopes.

One elegant way to achieve temporal (on/off) as well as qualitative (change and combine target antigens) control of CAR T cell function, is to split antigen recognition and effector cell activation using adapter molecules. Here, we report on the development of a novel adapter CAR (aCAR) system. By harnessing the unique properties of a novel single chain variable fragment (scFv), recognizing a neo-epitope comprising the endogenous vitamin biotin, conjugated to an adapter molecule e.g. a monoclonal antibody, in context of a special linker moiety, the technology can be used to specifically redirect CAR-T cells to a target structure referred to as linker-label epitope (LLE).

CAR design was empirically tested using five different extracellular spacer formats combined with a second generation 4-1BB-CD3-ζ signaling backbone. aCAR expression was found to transmit potent T cell activation, cytokine secretion and target cell lysis exclusively in presence of biotinylated antibodies (b-mAbs) or antibody fragments targeting specific antigens at concentrations spanning several log units (10 pg/ml - 100 µg/ml). Importantly, no impairment or inhibition of signaling was detected in human serum or at supra-physiological concentrations of free biotin, providing evidence for specificity against the LLE. Short spacers (IgG4 or CD8 hinge) performed best, independent of the utilized biotinylated antibody format (full-size monoclonal antibodies (mAb), Fab2 or Fab fragments). aCAR T cells were tested in vitro in combination with over 30 different b-mAbs directed against antigens expressed on tumor cells of various cancer entities. With all tested antibodies, aCAR T cells were highly cytotoxic in the presence of a single antibody against an expressed antigen, thusverifying the specificity and applicability of the aCAR system. Further, we were able to demonstrate the simultaneous targeting of multiple antigens can prevent selection of intrinsically resistant antigen-loss variants. Importantly, we found that combining adapters against different antigens expressed on the same target cell mediates aCAR activation at adapter concentrations below the activation threshold for single antigen targeting, allowing target cell recognition and elimination based on multifactorial expression profiles. Finally, in vivo efficacy was demonstrated, targeting CD19 in a NALM-6 NSG mouse xenograft model.

In conclusion, we demonstrate a novel aCAR system for maximal flexibility. aCAR T cell activation and cytotoxicity exclusively depends on the presence of biotinylated adapter molecules. Therefore, aCAR-T cell function can be stringently titrated by b-mAb infusion and withdrawal. This could add to an increased safety profile of CAR T cell therapy. Moreover, simultaneous targeting of multiple antigens can reduce selection pressure and the occurrence of resistance to therapy. Finally, since various mAbs have been FDA-approved or are close to enter the clinical setting and since the biotinylation of mAbs can be easily performed in a GMP-compliant manner, future clinical trials in leukemia and solid tumors can be envisioned based on our aCAR system.


Seitz: Miltenyi Biotec GmbH: Patents & Royalties, Research Funding. Schlegel: Miltenyi Biotec GmbH: Patents & Royalties, Research Funding. Mittelstaet: Miltenyi Biotec GmbH: Employment. Lock: Miltenyi Biotec GmbH: Employment. Kaiser: Miltenyi Biotec GmbH: Employment, Patents & Royalties. Lang: Miltenyi Biotec GmbH: Patents & Royalties, Research Funding. Handgretinger: Miltenyi Biotec GmbH: Patents & Royalties, Research Funding.

Author notes


Asterisk with author names denotes non-ASH members.