Figure 1.

RevCAR platform targeting CD33 and CD123. (a) Schematic overview of the RevCAR system and (b) design of the RevTMs. (a) RevCARs consist of the extracellular peptide epitope E5B9 or E7B6 and CD28 (28) hinge domain (HiD), CD28 transmembrane domain (TMD), the intracellular CD28 costimulatory (CSD) and CD3 zeta (3z) activating signaling domain (ASD). RevCAR-E5B9-28/3z or RevCAR-E7B6-28/3z modified T-cells are redirected towards CD33 or CD123 expressed on AML blasts via adaptor target modules, named RevTMs. RevTMs are bispecific antibodies (bsAbs) consisting of two different single-chain fragments variable (scFvs) binding on the one hand to E5B9 or E7B6 of the RevCAR and on the other hand to CD33 or CD123 on the surface of AML blasts. (b) In detail, the scFvs of the RevTMs are constructed with the variable heavy (V_H) and light chain (V_L) domains derived from the monoclonal antibodies (mAbs) CD33, CD123, 5B9, or 7B6 connected via glycine (G)-serine (S) linkers. RevTMs are expressed in eukaryotic cell lines and secreted into the cell culture supernatant mediated by the Ig kappa leader sequence (Igk). After purification via histidine tag (His), RevTMs were separated by SDS-PAGE and analyzed using Coomassie staining (c) and immunodetection after blotting on nitrocellulose membrane via anti-His Ab and AP-conjugated anti-mouse Ab (d). The whole western blot figures can be found in Figure S3.