The major objectives of this proposal are to clarify the molecular basis for, and the significance and generality of, Fc region-dependent, cooperative binding of antibodies to antigen. In addition, we will characterize IgG subclass-associated difference in other functional properties. The experimental approaches to be employed are delineated below. First, the functional affinities of IgG antibodies expressing different heavy chain constant domains will be compared with respect to different (particularly with regard to epitope spacing) forms of antigen under a variety of conditions (e.g., temperature or ionic strength). Monoclonal antibodies (mAbs) of different IgG subclasses, but expressing identical, or comparable, variable domains will be used for these studies. Second, the sites in the gamma 3 Fc region that are involved in cooperative binding will be mapped by serologic and electron microscopic methods. Third, the murine IgG subclasses will be compared with respect to clearance and protection in a murine model infection with Streptococcus pneumoniae. Forth, mAbs with novel binding phenotypes will be selected and characterized, and new strategies for endowing antibodies with the ability to bind cooperatively will be evaluated. These studies should contribute to an understanding of the functional differences among IgG subclasses, particularly in the contexts of anti- bacterial and anti-polysaccharide antibody responses. Further more, by increasing our understanding of the relationships between antibody constant domain structure and function, these studies should suggest novel ways to alter antibody structure to enhance desired functional properties.
