Crucial Role of Aspartic Acid at Position 265 in the CH2 Domain for Murine IgG2a and IgG2b Fc-Associated Effector Functions

Abstract

Replacement of aspartic acid by alanine at position 265 (D265A) in mouse IgG1 results in a complete loss of interaction between this isotype and low-affinity IgG Fc receptors (FcγRIIB and FcγRIII). However, it has not yet been defined whether the D265A substitution could exhibit similar effects on the interaction with two other FcγR (FcγRI and FcγRIV) and on the activation of complement. To address this question, 34-3C anti-RBC IgG2a and IgG2b switch variants bearing the D265A mutation were generated, and their effector functions and in vivo pathogenicity were compared with those of the respective wild-type Abs. The introduction of the D265A mutation almost completely abolished the binding of 34-3C IgG2a and IgG2b to all four classes of FcγR and the activation of complement. Consequently, these mutants were hardly pathogenic. Although oligosaccharide side chains of these mutants were found to contain higher levels of sialic acids than those of wild-type Abs, the analysis of enzymatically desialylated D265A variants ruled out the possibility that very poor Fc-associated effector functions of the D265A mutants were due to an increased level of the mutant Fc sialylation. Thus, our results demonstrate that aspartic acid at position 265 is a residue critically implicated in triggering the Fc-associated effector functions of IgG, probably by defining a crucial three-dimensional structure of the Fc region.

Antigen specificity can be irrelevant to immunocytokine efficacy and biodistribution

“…This unexpected observation shows that tumor targeting by the antibody variable region is dispensable for synergy with TA99. A single point mutation (D265A) in the antibody Fc region ablates interactions with FcγRs and complement, abolishing IgG effector functions (18). We introduced the D265A mutation to TA99-IL2 and sm3E-IL2 and found that the D265A immunocytokines did not perform significantly differently from their parent immunocytokines ( Fig.…”

Lack of Galactosylation Enhances the Pathogenic Activity of IgG1 but Not IgG2a Anti-Erythrocyte Autoantibodies

“…We have previously shown that the 34-3C IgG2a F243A mutant, which contained ∼20% sialylated glycovariants, efficiently interacted with FcgRs and activated complement, and was as pathogenic as barely sialylated WT Ab (28). However, sialylated 6A6 IgG1 and IgG2b antiplatelet glycovariants enriched through SNA lectin affinity column chromatography were shown to be less pathogenic as compared with their poorly sialylated counterparts (25).…”

“…Indeed, 34-3C IgG2a F243A displayed a markedly increased content of sialylated glycoforms, which accounted for ∼20% of total oligosaccharides, which contrasted with essentially absence of sialylated glycoforms (,1%) in WT 34-3C IgG2a mAb (28). Despite these remarkable differences in the content of sialylated glycoforms, the pathogenic activity of 34-3C IgG2a F243A mutant was comparable with that of WT 34-3C IgG2a.…”

“…Alternatively, because of a decreased interaction of highly sialylated IgG with FcgRs (25), it has been proposed that the enhanced pathogenicity of agalactosylated IgG could be attributed to their inability to undergo terminal sialylation, which is dependent on upstream galactosylation, rather than the lack of galactosylation (23,26). However, it should be noted that the anti-inflammatory properties of highly sialylated IgG remain a controversial issue (19,(27)(28)(29)(30). Notably, a more recent study reported that IgG galactosylation, but not sialylation, was associated with the improvement of disease activity during pregnancy in patients with rheumatoid arthritis (16).…”

The antitumor activity of the human FOLR1-specific monoclonal antibody, farletuzumab, in an ovarian cancer mouse model is mediated by antibody-dependent cellular cytotoxicity

“…The N297Q and the D265A mutations in the CH2 domain alter the oligosaccharide composition necessary for optimal recognition of IgG by human Fc gamma receptors, the former mutation by removing the site for N-linked carbohydrates 25 and the latter mutation by increasing galactosylation and sialylation. 26 In this respect, the L234A/L235A/K322A mutant is more similar to the G236R/L328R mutant, 21 in that they both target the key binding motif for C1q (K322A or L328R) as well as the motif involved in the high-affinity interaction with Fc gamma receptors, 27 likely without altering of the oligosaccharide composition.…”