IgG3 anti‐Kell allotypic variation results in differential antigen binding and phagocytosis

Abstract

BACKGROUND Human immunoglobulin G (hIgG) includes four different subtypes (IgG1, IgG2, IgG3, and IgG4). Due to genetic variations, each IgG subtype contains different isoallotypes. It was previously shown that a Food and Drug Administration–approved monoclonal anti‐IgG failed to recognize 2 of 15 recombinant, human IgG3 anti‐Kell (K1) isoallotypes (rIgG3‐03 and rIgG3‐13) by indirect antiglobulin test (IAT). STUDY DESIGN AND METHODS We expressed and purified 15 recombinant human rIgG3 anti‐K1 isoallotypes and investigated their antigen binding and ability to induce phagocytosis using homozygous (KK) and heterozygous (Kk) K1‐positive red blood cells (RBCs) by gel IAT, flow cytometry, and a monocyte monolayer assay (MMA) with peripheral blood monocytes and cultured inflammatory (M1) and anti‐inflammatory (M2) macrophages. RESULTS MMA results showed that differences in the Fc region of rIgG3 anti‐K1 led to distinctive phagocytic activity with both monocytes and M1 macrophages. rIgG3‐18 and rIgG3‐19 showed an enhanced ability to induce phagocytosis. Differences in Fc regions also led to variations in the number of antibodies bound to KK RBCs. Despite the differences in phagocytic activity, all 15 rIgG3 clones are predicted to induce clinically significant hemolysis if K1‐positive blood was transfused into patients. CONCLUSION These results argue that antiglobulin reagents that fail to detect isoallotype rIgG3‐03 or rIgG3‐13 could present a transfusion risk or lack of detection of a potentially clinically significant anti‐K1 in hemolytic disease of the fetus and newborn.

Three non-classical mechanisms for anemic disease of the fetus and newborn, based on maternal anti-Kell, anti-Ge3, anti-M, and anti-Jra cases

“…Maternal alloantibody against Ge3 antigen of the Gerbich blood group system expressed on glycophorin C, which is tied to the cytoskeleton and is essential in maintaining red cell shape and membrane functions, is supposed to suppress erythroid proliferation via non-classical apoptosis. Further, the lowest level of IgG class anti-M as causative in very severe ADFN should be elucidated, including from the standpoint of allotypic variations, and Fc fucosylation and galactosylation in IgG, as suggested by allotypic variations of the IgG Fc region manifesting different abilities to induce phagocytosis [37,38].…”

Characterization and refinement of monoclonal anti‐human globulins that lack reactivity with human IgG4

“…The frequency with which individuals that are homozygous or compound heterozygotes for IgG3‐03 and/or IgG3‐13 is unclear; however, such individuals have been documented to exist and at a substantial frequency, albeit through the study of a very small sample size (i.e., 10 individuals) . Moreover, IgG3‐03 and IgG3‐13 have recently been reported to have hemolytic activity in the monocyte monolayer assay . Thus, in our view, the risk of missing a hemolytic antibody and causing a potentially lethal hemolytic transfusion reaction is nontrivial and is in need of addressing.…”

Beyond Allotypes: The Influence of Allelic Diversity in Antibody Constant Domains

“…It is less intuitive to note mutation in the CH2/CH3 domains can also propagate allosteric changes to modulate antigen binding (54). Differences in antigen binding have been found for different alleles with the same variable region, although without high resolution analysis to determine exactly which mutations are contributing to the effect (55).…”