Design and binding specificity of the CD1d-CD19 fusion protein. (A) Schematic depicting the genetic fusion of human β2m with the sCD1d and the single chain of the human anti-CD19 antibody (CD19 scFV). DNA fragments were produced by polymerase chain reaction, including sequences for flexible glycine-serine rich linker (G₁₀S₃). A 6-histidine tag was added at the C terminus for Ni-NTA purification. (B) Illustration of the antigen-loaded fusion protein. (C) Depiction of how the CD1d-CD19 fusion “links” iNKT cells to tumor cells in a CD19-specific, yet CD1d-independent, manner (right). Once loaded with agonistic iNKT-directed lipids such as αGC, it is anticipated that the fusion will engage the iNKT TCR, induce iNKT activation, and promote lysis of CD19⁺CD1d⁻ tumor cells (see visual abstract). (D) CD1d expression on the cell lines used in this study, as determined by staining target cells with a phycoerythrin-labeled isotype, control and anti-CD1d antibody. (E) Binding of the CD1d-CD19 fusion protein to CD1d⁻ cell lines used in this study and detection of the fusion by a phycoerythrin-labeled anti-CD1d antibody. Data from >4 independent experiments are shown. Ab, antibody.