Figure 2

A2/NY-ESO-1₁₅₇-specific BiTE engages peripheral blood T cells with target cells. (a) A2/NY-ESO-1₁₅₇-specific BiTE containing an A2/NY-ESO-1₁₅₇-specific scFv (clone 3M4E5) and a human CD3-specific scFv was generated. A hexahistidine (6xHis) tag was inserted at the C-terminus of the construct. Purified 1 μg control BiTE and A2/NY-ESO-1₁₅₇ BiTE were similarly detected by immunoblotting (IB) using anti-His mAb. The full-length blotting image is shown in Supplementary Fig. S4 (top). (b) 10 μg/mL A2/NY-ESO-1₁₅₇ peptide or HIV Gag₇₇ peptide was loaded onto T2 cells. After floating peptides had been removed, the cells were stained with 10 μg/mL A2/NY-ESO-1₁₅₇ BiTE or control BiTE (left). To see the binding of each BiTE to the CD3 molecule, CD3⁺ Jurkat cells and CD3⁻ Jurkat 76 cells were stained with each BiTE (right). PE-conjugated anti-His mAb was utilized to detect the binding of BiTEs. Dotted lines show the isotype control. (c) Freshly isolated peripheral blood T cells derived from 5 different donors were individually incubated with T2 cells pulsed with 10 μg/mL NY-ESO-1₁₅₇ peptide or HIV Gag₇₇ peptide in the presence of 1 μg/mL A2/NY-ESO-1₁₅₇ BiTE or control BiTE. Cytokine production by peripheral blood T cells was measured by intracellular cytokine staining via flow cytometry. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., not significant.