Fig. 3.

Generation of 4H11 CAR T cells secreting ESK1-BiTE. A Design of the study of a dual targeting model. 4H11 CAR T cells recognizing Muc16 surface antigen on ovarian cancer cells also secrete ESK1-BiTE which binds and kill the cancer cells expressing WT1 oncoprotein. ESK1-BiTE can also recruit non-CAR T cells to the targets, thereby adding orthogonal secondary effector cells (BioRender). B Schematic of retroviral vectors encoding the 4H11BBz CAR alone (top) and ESK1-4H11BBz CAR constructs encoding a secreted ESK1 bite (bottom). A 6xHis-Tag was added at the C-terminal of ESK1 bite for easy detection and affinity purification. A P2A element sequence was used to link ESK1 bite with MUC16 CAR sequences. C The (His)6-tag ESK1 bites were pulled down from the medium supernatants of WT, 4H11BBζ, and ESK1-4H11BBζ producer cells using Ni-NTA agarose beads, separated using SDS-PAGE, and analyzed by immunoblotting using an anti-His-Tag antibody. The ESK1 bite appeared at the predicted molecular weight (∽58 kDa) in the conditioned media of ESK1-4H11BBζ Galv9 producer cells