The effect of pH dependence of antibody-antigen interactions on subcellular trafficking dynamics.

A drawback of targeting soluble antigens such as cytokines or toxins with long-lived antibodies is that such antibodies can prolong the half-life of the target antigen by a "buffering" effect. This has motivated the design of antibodies that bind to target with higher affinity at near neutral pH relative to acidic endosomal pH (~pH 6.0). Such antibodies are expected to release antigen within endosomes following uptake into cells, whereas antibody will be recycled and exocytosed in FcRn-expressing cells. To understand how the pH dependence of antibody-antigen interactions affects intracellular trafficking, we generated three antibodies that bind IL-6 with different pH dependencies in the range pH 6.0-7.4. The behavior of antigen in the presence of these antibodies has been characterized using a combination of fixed and live cell fluorescence microscopy. As the affinity of the antibody:IL-6 interaction at pH 6.0 decreases, an increasing amount of antigen dissociates from FcRn-bound antibody in early and late endosomes, and then enters lysosomes. Segregation of antibody and FcRn from endosomes in tubulovesicular transport carriers (TCs) into the recycling pathway can also be observed in live cells, and the extent of IL-6 association with TCs correlates with increasing affinity of the antibody:IL-6 interaction at acidic pH. These analyses result in an understanding, in spatiotemporal terms, of the effect of pH dependence of antibody-antigen interactions on subcellular trafficking and inform the design of antibodies with optimized binding properties for antigen elimination.

Neonatal Fc receptor blockade by Fc engineering ameliorates arthritis in a murine model.

Multiple autoimmune diseases are characterized by the involvement of autoreactive Abs in pathogenesis. Problems associated with existing therapeutics such as the delivery of intravenous immunoglobulin have led to interest in developing alternative approaches using recombinant or synthetic methods. Toward this aim, in the current study, we demonstrate that the use of Fc-engineered Abs (Abs that enhance IgG degradation [Abdegs]) to block neonatal FcR (FcRn) through high-affinity, Fc region binding is an effective strategy for the treatment of Ab-mediated disease. Specifically, Abdegs can be used at low, single doses to treat disease in the K/B×N serum transfer model of arthritis using BALB/c mice as recipients. Similar therapeutic effects are induced by 25- to 50-fold higher doses of i.v. Ig. Importantly, we show that FcRn blockade is a primary contributing factor toward the observed reduction in disease severity. The levels of albumin, which is also recycled by FcRn, are not affected by Abdeg delivery. Consequently, Abdegs do not alter FcRn expression levels or subcellular trafficking behavior. The engineering of Ab Fc regions to generate potent FcRn blockers therefore holds promise for the therapy of Ab-mediated autoimmunity.