Dirk Chelius

Isomerization of a Single Aspartyl Residue of Anti-Epidermal Growth Factor Receptor Immunoglobulin γ2 Antibody Highlights the Role Avidity Plays in Antibody Activity

A new isoform of the light chain of a fully human monoclonal immunoglobulin gamma2 (IgG2) antibody panitumumab against human epidermal growth factor receptor (EGFR) was generated by in vitro aging. The isoform was attributed to the isomerization of aspartate 92 located between phenylalanine 91 and histidine 93 residues in the antigen-binding region. The isomerization rate increased with increased temperature and decreased pH. A size-exclusion chromatography binding assay was used to show that one antibody molecule was able to bind two soluble extracellular EGFR molecules in solution, and isomerization of one or both Asp-92 residues deactivated one or both antigen-binding regions, respectively. In addition, isomerization of Asp-92 showed a decrease in in vitro potency as measured by a cell proliferation assay with a 32D cell line that expressed the full-length human EGFR. The data indicate that antibodies containing either one or two isomerized residues were not effective in inhibiting EGFR-mediated cell proliferation, and that two unmodified antigen binding regions were needed to achieve full efficacy. For comparison, the potency of an intact IgG1 antibody cetuximab against the same receptor was correlated with the bioactivity of its individual antigen-binding fragments. The intact IgG1 antibody with two antigen-binding fragments was also much more active in suppressing cell proliferation than the individual fragments, similar to the IgG2 results. These results indicated that avidity played a key role in the inhibition of cell proliferation by these antibodies against the human EGFR, suggesting that their mechanisms of action are similar.

Identification of N-linked oligosaccharides of rat insulin-like growth factor binding protein-4.

Insulin-like growth factor binding protein-4 (IGFBP-4) is, like the other five IGFBPs, a critical regulator of the activity of insulin-like growth factor (IGF)-I and IGF-II. Whereas IGFBP-1 and IGFBP-2 are not glycosylated, IGFBP-3 and IGFBP-4 are N-glycosylated and IGFBP-5 and IGFBP-6 are O-glycosylated. In this study we identified the glycosylation of IGFBP-4 using a nanoflow LC/MS/MS techniques. Although N-linked oligosaccharides are structurally diverse, their variants are well reported in the literature. Based on the molecular weight of the possible oligosaccharide moieties, we identified five different glycosylation isoforms of the protein. Identified glycans were biantennary and differ in the number of sialic acid terminal residues and/or core modification with fucose.