The ability to produce antibody molecules by genetic engineering and gene transfection has revolutionized the study of structure/function relationships within the antibody molecule. Since human constant regions can be used chimeric antibodies provide for the first time a continuous source of antigen specific antibodies of all human isotypes. Increased understanding of the functional properties of antibodies should give insights into the basis for antibody caused diseases and should enable us to produce antibodies with the optimal combination of effector functions for immunotherapy. Our studies to date have focused on human IgG constant regions and have investigated the functional role of the hinge region and the constant region carbohydrate and more completely defined the residues recognized by FcgammaRI. We propose to expand our studies of IgG and will focus our attention on several questions. We will attempt to identify the residues which determine the differential ability of the IgG isotypes to activate complement. We will determine why IgG4 cannot activate C', why IgG2 is less effective than IgG1 and IgG3, and why IgG3 is better at binding C1q while IgG1 has been reported to be more efficient at C4 activation. With this information in hand we will attempt to produce an IgG more effective at C' activation both by in vitro mutagenesis and by making a polymeric IgG molecule. We will now shift our attention from FcgammaRI to FcgammaRII and FcgammaRIII and we determine their isotype specific binding patterns and the residues on IgG they recognize. Experiments have begun to characterize the specific residues recognized by rheumatoid factors (RFs) and will continue focusing on defining the epitopes on IgG3 recognized by monoclonal RFs. We have shown that chimeric IgG1 and IgG3 exhibit different serum half-lives in mice and will now attempt to identify the amino acid sequence determining serum half-life with the objective of producing antibodies with the optimal serum half-life in humans. We will also determine if human IgG2 non-covalently polymerizes. We will extend our studies to additional human isotypes focusing on IgD and IgA. A vector directing the synthesis of secreted chimeric IgD has been prepared and we will begin an analysis of the functional relationship within that molecule focusing on the role of carbohydrate and on defining the sequence determining binding to cellular receptors. An expression vector for human IgA1 has also been constructed. It is not known if the isotype of the light chain plays a role in the functional properties of the antibody molecule. We will now produce a chimeric light chain with human lambda constant region and compare the functional properties of the resulting antibodies with those with kappa light chains.
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