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. 2016 Jul 15;5(9):e1211220. doi: 10.1080/2162402X.2016.1211220

Figure 2.

Figure 2.

Specificity of ULBP2-aCD19, ULBP2-aCD33, ULBP2-aCD19-aCD33 and ULBP2-aCD19-aCD19 immunoligands to their respective target moieties. (A) CD19+ (MEC1) and CD19− (HL60) cell lines were incubated with 10 µg/mL of ULBP2-aCD19 or ULBP2-aCD19-aCD19 and binding was detected by either anti c-Myc tag antibody (upper panel—“c-Myc”) or recombinant human NKG2D receptor (lower panel—“NKG2D”) where the latter confirms that immunoligands can bind to CD19 and NKG2D simultaneously. Gray-filled area depicts the background staining. Pre-blocking of CD19 antigen by anti-CD19scFv (20 µg/mL) inhibited the binding of immunoligands (dashed line—“MEC1”). (B) CD19+CD33+ cell line BV173 was incubated with 10 µg/mL of ULBP2-aCD19, ULBP2-aCD33 and ULBP2-aCD19-aCD33 and binding was detected by recombinant human NKG2D receptor (“No blocking”). Pre-blocking of CD19 or CD33 antigen by respective blocking construct (20 µg/mL) prevented the binding of ULBP2-aCD19 and ULBP2-aCD33, respectively (“Block + Bispecific ILs”) but not of ULBP2-aCD19-aCD33 (“Block + ULBP2-aCD19-aCD33”). Only simultaneous blocking of both CD19 and CD33 antigens on BV173 cells could completely inhibit the binding of ULBP2-aCD19-aCD33 (“Block + ULBP2-aCD19-aCD33”).