The Origins, Specificity, and Potential Biological Relevance of Human Anti-IgG Hinge Autoantibodies

Human anti-IgG hinge (HAH) autoantibodies constitute a class of immunoglobulins that recognize cryptic epitopes in the hinge region of antibodies exposed after proteolytic cleavage, but do not bind to the intact IgG counterpart. Detailed molecular characterizations of HAH autoantibodies suggest that they are, in some cases, distinct from natural autoantibodies that arise independent of antigenic challenge. Multiple studies have attempted to define the specificity of HAH autoantibodies, which were originally detected as binding to fragments possessing C-terminal amino acid residues exposed in either the upper or lower hinge regions of IgGs. Numerous investigators have provided information on the isotype profiles of the HAH autoantibodies, as well as correlations among protease cleavage patterns and HAH autoantibody reactivity. Several biological functions have been attributed to HAH autoantibodies, ranging from house-cleaning functions to an immunosuppressive role to restoring function to cleaved IgGs. In this review, we discuss both the historic and current literature regarding HAH autoantibodies in terms of their origins, specificity, and proposed biological relevance

Proteolytic cleavage and loss of function of biologic agents that neutralize tumor necrosis factor in the mucosa of patients with inflammatory bowel disease

BACKGROUND & AIMS: Many patients with inflammatory bowel disease (IBD) fail to respond to anti–tumor necrosis factor (TNF) agents such as infliximab and adalimumab, and etanercept is not effective for treatment of Crohn’s disease. Activated matrix metalloproteinase 3 (MMP3) and MMP12, which are increased in inflamed mucosa of patients with IBD, have a wide range of substrates, including IgG1. TNFneutralizing agents act in inflamed tissues; we investigated the effects of MMP3, MMP12, and mucosal proteins from IBD patients on these drugs. METHODS: Biopsy specimens from inflamed colon of 8 patients with Crohn’s disease and 8 patients with ulcerative colitis, and from normal colon of 8 healthy individuals (controls), were analyzed histologically, or homogenized and proteins were extracted. We also analyzed sera from 29 patients with active Crohn’s disease and 33 patients with active ulcerative colitis who were candidates to receive infliximab treatment. Infliximab, adalimumab, and etanercept were incubated with mucosal homogenates from patients with IBD or activated recombinant human MMP3 or MMP12 and analyzed on immunoblots or in luciferase reporter assays designed to measure TNF activity. IgG cleaved by MMP3 or MMP12 and antihinge autoantibodies against neo-epitopes on cleaved IgG were measured in sera from IBD patients who subsequently responded (clinical remission and complete mucosal healing) or did not respond to infliximab. RESULTS: MMP3 and MMP12 cleaved infliximab, adalimumab, and etanercept, releasing a 32-kilodalton Fc monomer. After MMP degradation, infliximab and adalimumab functioned as F(ab’)2 fragments, whereas cleaved etanercept lost its ability to neutralize TNF. Proteins from the mucosa of patients with IBD reduced the integrity and function of infliximab, adalimumab, and etanercept. TNF-neutralizing function was restored after incubation of the drugs with MMP inhibitors. Serum levels of endogenous IgG cleaved by MMP3 and MMP12, and antihinge autoantibodies against neo-epitopes of cleaved IgG, were higher in patients who did not respond to treatment vs responders. CONCLUSIONS: Proteolytic degradation may contribute to the nonresponsiveness of patients with IBD to anti-TNF agents

Membrane cholesterol content accounts for developmental differences in surface B cell receptor compartmentalization, actin reorganization and signaling

B cells undergo different antigen receptor induced signaling responses and cell fate decisions depending on their stage of development. Transitional immature B cells contain less cholesterol than mature B cells, and antigen receptor stimulated transitional immature B cells fail to compartmentalize their B cell receptor (BCR) with cholesterol-enriched plasma membrane. Cholesterol is a key component in the formation of the membrane microdomain termed the lipid raft, and lipid rafts have been proposed to function as platforms for antigen receptor signal transduction. Augmentation of cholesterol levels in transitional immature B cells such that the plasma membrane has equal levels of cholesterol compared to mature B cells results in antigen receptor stimulated association of the BCR with cholesterol-enriched plasma membrane. Association of the BCR with lipid rafts correlates with sustained signaling through the PLCγ2/NFκB/c-myc pathway. In addition, mature B cells undergo directed actin polymerization following BCR stimulation, while transitional immature B cells are deficient in this response. Interestingly, Vav phosphorylation in mature B cells is stronger than transitional immature B cells, and this correlates with increased GTP-loading of Rac1. Also, BCR stimulation of mature B cells leads to membrane ruffling, a Rac1-dependent process. Transitional immature B cells do not undergo membrane ruffling following BCR stimulation consistent with the defect in Rac1-GTP loading. Cholesterol addition to transitional immature B cells rescues Rac1-GTP loading and directed actin polymerization and membrane ruffling. These studies provide a link between B cell cholesterol levels and downstream cellular signaling processes

IgG Fc engineering to modulate antibody effector functions

ABSTRACT Therapeutic monoclonal antibodies are among the most effective biotherapeutics to date. An important aspect of antibodies is their ability to bind antigen while at the same time recruit immune effector functions. The majority of approved recombinant monoclonal antibody therapies are of the human IgG1 subclass, which can engage both humoral and cellular components of the immune system. The wealth of information generated about antibodies has afforded investigators the ability to molecularly engineer antibodies to modulate effector functions. Here, we review various antibody engineering efforts intended to improve efficacy and safety relative to the human IgG isotype. Further, we will discuss proposed mechanisms by which engineering approaches led to modified interactions with immune components and provide examples of clinical studies using next generation antibodies