Pfuse2ss clig hk

The anti-murine CD45 and anti-Y-DOTA bispecific Ab was produced starting with two Pfuse plasmid constructs (Invitrogen, Grand Island, NY), pFUSE2ss-CHIg-hG1 and pFUSE2ss-CLIg-hK, carrying the heavy and light chain genes, respectively, of the anti-murine CD45 30F11 Ab (19 (link)). The light chain construct also has DNA encoding for the variable heavy and light chains of the C825 scFv radio metal trap cloned downstream of the anti-murine CD45 light chain. Additional bispecific Ab expression, production and purification details are described in Supplemental Methods (available online). Control bispecific Ab LDL-Fc (targeting LDL but without the C825 scFv radiometal trap for Y-DOTA) and CC49-Fc-C825 (targeting the irrelevant adenocarcinoma antigen TAG-72 and Y-DOTA) were generated as described elsewhere (20 (link)).

For antibody expression, the heavy chain (342 bp) and light chain (324 bp) variable domains of the B12 sequence were cloned separately into pFUSEss human IgG1 expression vectors (pFUSEss-CHIg-hG1 and pFUSE2ss-CLIg-hk, Invitrogen) and co-transfected into HEK293T cells. The serum was collected after 72 hours, filtered through a 0.45 μm filter, and purified using a 5 mL HiTrap Protein A HP column (GE Healthcare). The column was equilibrated with 20 mM sodium phosphate at pH 6.8. The serum was loaded onto the column, washed with equilibration buffer for 10 column volumes, and bound protein was eluted with 0.1M citric acid at pH 3.5. Eluted IgG was collected, concentrated using a 50 kDa centrifugal filter (Millipore), and buffer exchanged into PBS using a Sephadex G-25 PD-10 desalting column (GE Healthcare). The purity of the final B12 IgG was analyzed by reduced and nonreduced SDS-PAGE, and protein concentration was measured based on absorbance at 280 nm using a NanoDrop One UV – Vis Spectrophotometer (Thermo-Fisher).