Fig. 3. Influence of extra- and intracellular CAR domains.

a, b Clustering of a hHER2-specific scFv. scFv-containing CARs were either based on a BBz and Ser-BBz-backbone, as shown in (a) (SEQ-IDs 5 and 6, respectively). b The figure shows the cytolytic and secretory activity of mock-T cells (no CAR) and T cells expressing the BBz- or Ser-BBz-CAR in co-cultures with hHER2t^pos Jurkat cells [flow cytometry-based assay; three independent experiments with different donors; *p < 0.05, calculated by two-tailed paired t-test (left panel) or by two-tailed ratio-paired t-test (right panel)]. c Schematic of diabody formation of two scFvs by intermolecular heterodimerization of VHs and VLs. d, e Prevention of diabody formation enables defined CAR dimerization. d Schematic of a CAR with VH and VL on separate polypeptide chains for preventing diabody formation (SEQ-IDs 7 and 8). Both constructs are shown in the heterodimerized state together forming a hHER2-specific Fv-based CAR. e Cytolytic activity of mock-T cells (no CAR) or T cells expressing either the hHER2-specific scFv-CAR [shown in (a)] or the VH and VL constructs [shown in (d)] in co-cultures with hHER2t^pos Jurkat cells. AP20187 was added for homodimerization of the VH-construct as indicated (filled red boxes). [flow cytometry-based assay; two independent experiments with three different donors; *p < 0.05, calculated by two-tailed paired t-test and by two-tailed unpaired t-test (scFv Ser-BBz vs. VH + VL chain)]. f, g Influence of co-stimulatory domains. f Schematic of hEGFR-specific rcSso7d-based low-affinity CARs which contain different co-stimulatory domains. The 4-1BB domain of the AvidCAR was substituted by the endodomain of CD28, ICOS, OX40, or CD2 (SEQ-IDs 9, 10, 11, 12, and 13, respectively). The FKBP12-F36V domain was integrated for conditional homodimerization. g rcSso7d-based low-affinity CARs with different co-stimulatory domains were expressed in T cells and co-cultured with hEGFRt^pos Jurkat cells. The figure shows the cytolytic activity of the T cells and its dependence on AP20187-mediated CAR dimerization (flow cytometry-based assay; two independent experiments with three different donors; *p < 0.05, calculated by two-tailed paired t-test). Exact p values are given in Supplementary Data 2. Source data are provided as a Source data file.