Figure 2. In vitro cell binding of radiolabeled conjugates.

(A) Representative LigandTracer measurements of real-time binding of CD44v6-scFv-A11 labeled with 125I using either Iodogen (top curve) or Chloramine T (bottom curve, dotted line) on A431 cells. Binding traces using three subsequent concentrations (10, 20, 60 nM) were obtained for at least 1 h per concentration (marked by vertical dotted lines), followed by a dissociation measurement for at least 10 h. CPS = Counts per second. (B) Representative LigandTracer measurements of real-time binding of CD44v6-scFv-A11 (filled lines) and CD44v6-scFv-H12 (dotted lines) on A431 cells, normalized to percentage of maximum binding. Binders were labeled with either ¹¹¹In (grey lines) or ¹²⁵I (black lines). Binding traces using three subsequent concentrations (1, 3, 9 nM) were obtained for at least 1 h per concentration (marked by vertical dotted lines), followed by a dissociation measurement for at least 10 h. (C) Signal intensities during equilibrated conditions after 2 h of incubation with 9 nM of conjugate using LigandTracer. Incubation was preceded by 2 + 2 h of uptake measurements at 1 and 3 nM. Measurements were performed using ¹²⁵I-A11(Iodogen) (52.5 ng, 4 kBq, black bars), ¹²⁵I-H12(Iodogen) (52.5 ng, 3 kBq, black striped bars), ¹¹¹In-DTPA-A11 (52.5 ng, 8.8 kBq, grey bars) and ¹¹¹In-DTPA-H12 (52.5 ng, 8.8 kBq, grey striped bars) on A431 cells of high CD44v6 density and UM-SCC-74B cells of low CD44v6 density. Signal from target area (cells) were subtracted by signal from the background area. CPS = Counts per second. Error bars show standard deviation (N=5). (D) Blocking assays for 125I-A11(Iodogen), 125I-H12(Iodogen), 111In-DTPA-A11 and 111In-DTPA-H12 on A431 cells. Radiolabeled conjugate was added at a concentration of 10 nM. In blocked samples, 100-times molar excess of non-labeled binder was also added to block specific binding of labeled conjugate. The samples were allowed to incubate for 4 hours at 37 °C, 5% CO2. Differences between unblocked and blocked samples were found to be significant for all conjugates according to student’s t test (P<0.05). Error bars show standard deviation (N>5).