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Monoclonal antibodies targeting the extracellular region of the human IgE heavy chain membrane-tethering domain have been proposed for treating allergies caused by hyperproliferative monoclonal expansion of IgE-producing B cells. Antibodies against this target are expected to deplete membrane IgE (mIgE) displaying B cells and leave B cells of other immunoglobulin isotypes intact. Because of alternative splicing, the mIgE heavy chain has two isoforms that differ in their membrane-proximal segment. In the long isoform, the CH4 domain is followed by a 67-amino acid-long extracellular portion. Out of these 67 amino acids, the first 52 amino acids following the CH4 domain constitute the CɛmX segment while the rest of the 15 amino acids immediately adjacent to the membrane constitute the ɛ-migis. In the short isoform the CɛmX segment is absent and the CH4 domain is followed only by the 15-amino acid-long ɛ-migis segment. Using antibodies derived from a phage display library, we investigated: (1) ɛ-migis and (2) the junction of CɛmX and ɛ-migis (CɛmX.migis), as potential therapeutic antibody targets. Our results indicate that antibodies obtained from our phage library that target ɛ-migis bind to a variety of human cells irrespective of mIgE expression, possibly due to homology between ɛ-migis and a region of phosphoinositide-binding protein (ARAP3). In contrast, antibodies specific for the CɛmX.migis junctional region, bound specifically to transfected and primary B cells expressing human mIgE and elicited antibody-dependent cellular cytotoxicity and reduction in IgE production. These antibodies did not bind secreted IgE or the mIgE isoform in which CɛmX is absent. These results suggest that CɛmX.migis junctional region is a promising antibody target and the human antibodies we describe warrant further evaluation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The first three-dimensional structure of a human Fc fragment genetically engineered for improved pharmacokinetics properties is reported. When introduced into the C(H)2 domain of human immunoglobulin G (IgG) molecules, the triple mutation M252Y/S254T/T256E ('YTE') causes an about 10-fold increase in their binding to the human neonatal Fc receptor (FcRn). This translates into an almost 4-fold increase in the serum half-life of YTE-containing human IgGs in cynomolgus monkeys. A recombinantly produced human Fc/YTE fragment was crystallized and its structure solved at a resolution of 2.5A using molecular replacement. This revealed that Fc/YTE three-dimensional structure is very similar to that of other human Fc fragments in the experimentally visible region spanning residues 236-444. We propose that the enhanced interaction between Fc/YTE and human FcRn is likely mediated by local effects at the substitutions sites. Molecular modeling suggested that potential favorable hydrogen bonds along with an increase in the surface of contact between the two partners may account in part for the corresponding increase in affinity.