Tyrosine Sulfation at Antibody Light Chain CDR-1 Increases Binding Affinity and Neutralization Potency to Interleukine-4.

Abstract

Structure and function of therapeutic antibodies can be modulated by a variety of post-translational modifications (PTM). Tyrosine (Tyr) sulfation is a type of negatively charged PTM that occurs during protein trafficking through the Golgi. In this study, we discovered that an anti-interleukin (IL)-4 human IgG1, produced by transiently transfected HEK293 cells, contained a fraction of unusual negatively charged species. Interestingly, the isolated acidic species exhibited a two-fold higher affinity to IL-4 and a nearly four-fold higher potency compared to the main species. Mass spectrometry (MS) showed the isolated acidic species possessed an +80-Dalton from the expected mass, suggesting an occurrence of Tyr sulfation. Consistent with this hypothesis, we show the ability to control the acidic species during transient expression with the addition of Tyr sulfation inhibitor sodium chlorate or, conversely, enriched the acidic species from 30% to 92% of the total antibody protein when the IL-4 IgG was co-transfected with tyrosylprotein sulfotransferase genes. Further MS and mutagenesis analysis identified a Tyr residue at the light chain complementarity-determining region-1 (CDRL-1), which was sulfated specifically. These results together have demonstrated for the first time that Tyr sulfation at CDRL-1 could modulate antibody binding affinity and potency to a human immune cytokine.