Figure 2A. Schematic of the 2H7-Fc-C825 (anti-CD20 x anti-Y-DOTA) bispecific Fc fusion antibody gene. An anti-human CD20 2H7 scFv gene and an yttrium-DOTA capturing C825 disulfide-stabilized scFv (ds-scFv) gene were fused to the human IgG1 Fc fragment at the amino and carboxyl ends, respectively. An N-linked glycosylation containing linker (NLG) was incorporated between the Fc and C825 ds-scFv domains, as shown.
Figure 2B. SDS-PAGE analysis of the 2H7-Fc-C825 FP. Bispecific 2H7-Fc-C825 fusion polypeptides expressed in CHO-DG44 cells spontaneously formed dimers via the hinge regions. Two batches of 2H7-Fc-C825 FP (5μg) were analyzed by electrophoresis on a 4–20% MES SDS PAGE gel (Invitrogen). Lanes 1 and 5: Seeblue marker proteins; Lanes 2 and 6 show the non-reduced 2H7-Fc-C825 FP (samples boiled); Lanes 3 and 7 show the monomeric 2H7-Fc-C825 FP (samples boiled and reduced with 2-mercaptoethanol); Lane 4 is empty. The gel was stained with Coomassie blue.
Figure 2C. Flow Cytometric Analysis of Binding of Purified 2H7-Fc-C825 FP to cells transduced to express hCD20 (EL4-CD20, [black]) and to untransduced (control) EL4 cells (red). EL4 or EL4-CD20 cells (0.5x106 each) were incubated in 100μl of HBSS buffer containing 2% FBS and treated with 1.8μg of the 2H7-Fc-C825 FP for 30 min at 4°C. After washing, the cells were mixed with 2 μl of PE-anti-human Fc antibody in 40μl of HBSS-2% FBS buffer for 30 min at 4°C. After washing 3x,cells were re-suspended in 400μl of PBS buffer containing 1% of formaldehyde and analyzed on a Guava cytometer.
Figure 2D. Sandwich ELISA assay demonstrating concentration-dependent binding of the 2H7-Fc-C825 FP to microtiter wells coated with the Y-DOTA ligand. A 96-well plate was coated with 70μl of the BSA-Y-DOTA conjugate (1μg/ml in PBS) and then blocked with 200μl of 2% BSA in PBS buffer. After washing, the wells were treated with 100μl of fusion proteins at a concentration of 16μg/ml followed by serial dilution as indicated. The plate was further treated with HRP-anti-human Fc antibody followed by TMB. A control FP shows no binding to Y-DOTA.