Figure 1.

Anti-CD47 VHH-secreting A12 CAR T cells can be generated and function in vitro. A, Schematic of VHH-secreting CAR T cell. B, Design of various VHH-secreting CAR constructs. Constructs consist of fluorescent protein tracers and a CAR separated by a P2A sequence. The secreted VHH is encoded either multi-cistronically, with a P2A sequence or IRES sequence, or with a separate CMV promoter. TM, transmembrane. C, Transduction of constructs traced by a fluorescent reporter protein. D, Flow cytometry–based assay to detect expression of A4 VHH. Populations analyzed included transduced and untransduced cells and were stained with a fluorescent anti-CD47 mAb that competes for binding with A4. A4-secreting CAR T cells showed blockade of anti-CD47 binding. E, Anti-HA immunoblot on lysate of A12 P2A A4 CAR T cells shows production of A4 VHH (left). Anti-HA IP on supernatant of A12 A4-secreting CAR T cells shows secretion of A4 (right). F, B16F10 killing is measured with a CellTiter Glo–based assay (left). CAR T-cell activation is measured by IFNγ ELISA (right). E:T, effector-to-target ratio.