As recombinant antibodies (Abs) are increasingly used as therapeutic agents for the treatment of infectious disease and malignancy, it becomes critically important that we understand the mechanisms through which antibodies provide protection. Armed with this information, we will be able to produce the most effective Abs. It is also important that we are able to produce recombinant Abs at a cost that will make them broadly available. In the proposed experiments the investigators will address these issues. They will attempt to produce IgA with improved functional properties. IgA is the principal Ab providing protection in the hostile environment of the mucosal surface where it is susceptible to proteolytic attack and one goal will be to identify and alter the positions on sIgA that are most susceptible to proteolysis. These experiments should both yield a more stable sIgA and also give insights into its structure. The biologic properties of IgA suggest that it may be the ideal antibody for some parenteral applications. However, serum IgA is rapidly cleared. They will attempt to increase the serum half-life of IgA by providing it with an FcRn binding site and by identifying and removing the N-carbohydrate moiety responsible for the rapid clearance of IgA2 by the asialoglycoprotein receptor. These studies will provide IgA with a longer half-life and give additional insights into the mechanisms and pathways of Ab clearance. Complement activation can lead to the elimination of pathogens by both cytolysis and opsonization. They will attempt to produce IgA with the ability to activate complement. These experiments will provide an IgA with the ability to activate the inflammatory cascade and will also further our understanding of the sequences on the Ab that mediate interaction with the complement proteins. They will attempt to define the mechanisms of Ab-mediated protection using the suckling mouse model for enterotoxigenic Escherichia coli (ETEC) and monoclonal antibodies against the F41 antigen. In vitro activity in assays such as adherence, agglutination, complement-mediated cytotoxicity and stability will be compared with the ability to provide in vivo protection. As one step to our long-term goal of using the chicken as a bioreactor for Ab production, they will further define the fine specificity of the receptor responsible for Ab transport with the ultimate goal of cloning it. Definition of the sequences required for transport receptor recognition should make it possible to design Abs that are effectively transported. It also may be possible to use this sequence to target other proteins to the chicken egg. Cloning of the receptor will define its structure and allow us to fully characterize this novel transport system.
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