Figure 3
Recovery of binding activities of the recombinant antibodies after removal of inhibitory domains by MMP-2. CBa-anti-EGFR antibody (A), C2b-anti-EGFR antibody (B) or LAP-anti-EGFR antibody (C) that were digested by MMP-2 for indicated time were added to microtiter plates pre-coated with MDA-MB-468 cells. Degree of antibody binding to the cells was compared to the binding of the cells by the unmodified anti-EGFR antibody (set as 100%). The LAP ___domain reduced antibody binding activity by 53.8%. The CBa and C2b domains masked antibody binding sites ineffectively, only marginally reducing the antibody binding activity by 9.3% and 21%, respectively. Note that after MMP-2 digestion of the LAP-anti-EGFR antibody, the binding activity rose progressively from 46.2% to 100%. (D) The binding activities of MMP-2 substrate peptide-linked anti-EGFR antibody (GPLGVR-anti-EGFR Ab) and MMP-2 substrate peptide-removed anti-EGFR antibody (GPLGVR-anti-EGFR Ab + MMP) showed the MMP-2 substrate peptide does not affect the binding activity of the antibody. (E) MMP-2 digested antibodies were resolved by reducing SDS-PAGE and analyzed by western blot using HRP-conjugated goat anti human-IgG Fcγ antibodies. GPLGVR peptide was indeed removed from the heavy chains of anti-EGFR antibodies by MMP-2, as indicated by reduction of molecular size. GPLGVR-HC: MMP-2 substrate peptide-linked heavy chain. HC: heavy chain. The original blot image for panel E is presented in Supplementary Figure S6.
