Engineering a human IgG2 antibody stable at low pH

Abstract

IgG2 subclass antibodies have unique properties that include low effector function and a rigid hinge region. Although some IgG2 subclasses have been clinically tested and approved for therapeutic use, they have a higher propensity than IgG1 for aggregation, which can curtail or abolish their biological activity and enhance their immunogenicity. In this regard, acid-induced aggregation of monoclonal antibodies during purification and virus inactivation must be prevented. In the present study, we replaced the constant domain of IgG2 with that of IgG1, using anti-2,4-dinitrophenol (DNP) IgG2 as a model antibody, and investigated whether that would confer greater stability. While the anti-DNP IgG2 antibody showed significant aggregation at low pH, this was reduced for the IgG2 antibody containing the IgG1 CH2 domain. Substituting three amino acids within the CH2 domain-namely, F300Y, V309L, and T339A (IgG2_YLA)-reduced aggregation at low pH and increased CH2 transition temperature, as determined by differential scanning calorimetric analysis. IgG2_YLA exhibited similar antigen-binding capacity to IgG2, low affinity for FcγRIIIa, and low binding ability to C1q. The same YLA substitution also reduced the aggregation of panitumumab, another IgG2 antibody, at low pH. Our engineered human IgG2 antibody showed reduced aggregation during bioprocessing and provides a basis for designing improved IgG2 antibodies for therapeutic applications. K E Y W O R D Sacid-induced aggregate, ADCC, aggregation, antibody, CDC, IgG2 subclass, subclass change

“IgG’s: contending with aggregating circumstances”

“…14 Parallel needs exist for tractable and informative approaches for assaying IgG aggregation, together with approaches for preparing aggregated IgG's as comparative controls. For the latter (and as an IgG aggregation model), transient exposure to low pH (as relevant to producer cell culture and IgG purification) is commonly and effectively employed to induce IgG aggregation [15][16][17][18] (with heat, agitation, shear force, and high pressure as additional options). Assays of aggregated IgG's include first those which can be essentially 2-step, and report on relative frequencies of aggregates.…”