Monoclonal antibodies are used to treat various human diseases by binding and inhibiting disease associated antigens, but many antibodies with proven in vitro activity fail to exhibit efficacy in vivo. Some of the most difficult targets to inhibit in vivo include the molecules that have a high rate of synthesis, which require high doses of antibody for treatment. Because antibody binding stabilizes the antigen through endosomal recycling, a high affinity antibody tends to increase the target antigen concentration compared to the baseline (i.e. no antibody), making it progressively more difficult to neutralize the antigen. In this regard, an antibody that can inhibit an antigen without increasing its serum stability may exhibit improved in vivo efficacy. In particular, reducing the serum concentration of a pathogenic antigen using an engineered antibody should provide therapeutic benefits. We propose to engineer such antigen clearing antibody by introducing mutations for pH dependent (PHD) antigen binding. A PHD antibody with high affinity at neutral pH and weaker affinity at acidic pH can eliminate an antigen by forming a stable antibody-antigen complex in the serum that dissociates in the endosome after endocytosis. Dissociated antigen is sorted to the lysosome for degradation, while the antibody is recycled by binding to endosomal neonatal Fc receptor (FcRn). The pH dependent interaction is used by some natural receptor-ligand complexes to remove circulating ligands, supporting the generality of this strategy for removing circulating antigens. We will further engineer the antibody to bind FcRn with increased affinity at pH 7.4 to improve the efficiency of antibody capture at the cell surface. Increasing the FcRn affinity is predicted to overcome a kinetic bottleneck due to fluid-based endocytosis and accelerate the rate of antigen endocytosis and degradation, further potentiating the antigen clearing capability of PHD antibody. In this proposal, we will evaluate the use of an engineered PHD antibody in the treatment of Staphylococcal enterotoxin B (SEB) intoxication. SEB is a virulence factor secreted by certain strains of Staphylococcus aureus (SA) that cause infection in crowded communities and hospitals. Since SEB can cause a toxic shock at a low concentration, treatment of SEB intoxication should include inhibition and elimination of circulating SEB. Current treatment options for SEB intoxication include antibiotics to inhibit bacterial growth and synthesis of SEB, but antibody-based immunotoxicotherapy against the toxin is not clinically available. Considering that SEB causes food poisoning and is a known bioterrorism agent, there is an urgent need for effective treatment against SEB intoxication. We will engineer and test if PHD anti-SEB antibody can lower the SEB concentration in mice under transient and steady-state infection conditions. We will also measure the secretion of SEB-induced inflammatory cytokines and the survival rate of mice after SEB injection to determine the efficacy of the antibody. The proposed study is the first of its kind in which an engineered PHD antibody is used to treat a toxin-induced disease, and it is expected to generate important molecular insights to guide future development of novel antibody therapeutics.
Antibody therapy is difficult to develop against targets with a high turnover rate because they require high doses of antibody for treatment. In this regard, eliminating disease-associated antigens with pH dependent (PHD) antibody is a promising and achievable therapeutic strategy. We propose to engineer antibody with PHD binding of Staphylococcal enterotoxin B (SEB) and evaluate its effect on the SEB pharmacokinetics in mice and therapeutic efficacy against SEB intoxication.
