Radiolabelling of hu3S193 antibodies

Hu3S193 antibodies were radiolabelled with four isotopes: ¹²⁵I, ¹³¹I, ¹¹¹In and ¹⁷⁷Lu. Iodine-125 and lutetium-177 were obtained from PerkinElmer (PerkinElmer Life and Analytical Sciences, Waltham, MA), iodine-131 was obtained from ANSTO (ANSTO, Menai, Australia) and indium-111 was obtained from MDS Nordion (Canada).

Radioiodination was performed using pH-neutralised isotopes, catalysed by iodogen-coated glass beads as previously published [12, 33]. After a 10-min incubation period, the reaction mixture was purified through a Sephadex G50 desalting column (Sigma-Aldrich, Sydney, Australia) equilibrated with 0.9 % NaCl containing 0.05 % human serum albumin.

Radiolabelling of the hu3S193 antibody constructs with indium-111 and lutetium-177 was done by using the bifunctional metal ion chelate C-functionalized trans-cyclohexyl diethylenetriaminepentaacetic acid (CHX-A″ DTPA) [34, 35]. A chelate to antibody ratio of 3:1 was employed, using 0.1 M sodium bicarbonate buffer (pH 8.6) containing 0.9 % NaCl. Incubation was allowed for 16 h at room temperature. Under these conditions, one to two chelates are expected per antibody molecule. The radiolabelled mixture was purified through a Sephadex G50 desalting column equilibrated with 0.9 % NaCl containing 0.05 % human serum albumin.

Radiolabelling was performed on the day of injection into mice. Prior to injection, the percentage of unbound radionuclide content was determined by ITLC as previously described [36]. Determination of the immunoreactivity of radiolabelled hu3S193 antibody constructs was performed by a single-point binding assay, where 10 × 10⁶ Le^y-positive A431 cells were incubated with 20 ng of radiolabelled antibody constructs for 45 min at room temperature with continuous mixing throughout to keep the cells in suspension. Cells were washed three times, and pellets were measured in a gamma counter (Cobra II, Model 5002, Packard Instruments, Canberra, Australia). Three samples of radiolabelled antibody at the same concentration as that initially added to the cells were measured at the same time of the cell pellets, and immunoreactivity was calculated: (cpm cell pellet/mean cpm radioactive antibody standards) × 100. Serum stability was analysed by determination of immunoreactivity on the day of injection, at 48 h, and 7 days after 20 ng of radiolabelled antibody was incubated in human serum at 37 °C.