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

Abstract

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.

In vitro and in vivo modifications of recombinant and human IgG antibodies

“…76 Isomerization of Asp92 in the light chain CDR3 of a recombinant monoclonal IgG2 antibody deactivated its antigen binding capability. 83 As discussed in the previous section, isoAsp can be formed in vivo in monkey 72,73 and in human 74 in both recombinant mAbs and human endogenous IgG antibodies. 74 Therefore, isoAsp from isomerization is not foreign to the human immune system, indicating a lower risk of immunogenicity.…”

Stability of Protein Pharmaceuticals: An Update

“…Some of the same LC-MS methods used to identify deamidation in MAbs have been used to monitor Asp isomerization as well (27,55,116). Both degradation pathways have been observed in MAbs (43,55).…”

Structural and functional characterization of the trifunctional antibody catumaxomab

“…Information about modifications like oxidation, [73][74][75] deamidation, [76][77][78] isomerization, 79,80 C-and N-terminal variants, [81][82][83] as well as some unexpected modifications [84][85][86] would not have been possible without the development of new mass spectrometers with increased sensitivity, resolution, mass accuracy, and mass range. However, "older" techniques such as SDS-PAGE or SEC still provide useful information and are important for routine analysis in a Good Manufacturing Practice (GMP) environment.…”