STRUCTURE AND FUNCTION OF MUTANT MONOCLONAL ANTIBODIES PRODUCED BY HYBRID CELL LINES

Abstract

The central aim of the work presented in this thesis was to generate a series of cell lines producing structurally altered immunoglobulins from a hybridoma line secreting an antigen-binding mouse IgG(,2b) antibody. The goals of this project were: (1) to show whether the instability of immunoglobulin genes observed in several mouse myeloma cell lines was also a property of hybrid cell lines, (2) To use structurally altered antigen-binding immunoglobulins in studying antibody effector functions in ways that had not been previously possible with nonantigen-binding myeloma proteins and (3) to investigate the potentials for generating new kinds of monoclonal antibody reagents that are potentially more suitable for medical use as well as more convenient reagents in the research laboratory.;A hybridoma cell line secreting an IgG(,2b) immunoglobulin binding the hapten p-azophenylarsonate was derived by cell fusion of a non-immunoglobulin-producing mouse myeloma cell line to splenic lymphocytes from an immunized mouse. From this line a series of lines with mutations affecting the constant region of the (gamma)2b gene was derived by methods used previously to select Ig mutants in the MPC-11 myeloma line. These mutants appear to contain deletions in the constant region. The molecular weights and covalent and non-covalent assembly patterns were determined for each mutant. It was observed that some mutants appear to synthesize multiple sizes of mutant H-chains. In some cases this was shown to be due to glycosylation differences between the H-chains, but other lines appear to synthesize more than one H-chain polypeptide. To determine what portion of the IgG(,2b) molecule had been altered a set of rat monoclonal anti-IgG(,2b) antibodies were generated. It was possible to identify roughly where the antigenic determinants recognized by these antibodies were on the mouse IgG(,2b) molecule using mutant immunglobulins from MPC-11 the location of whose deletions are known. The reactivity of these rat anti-mouse IgG(,2b) antibodies with the set of antigen-binding mutants allowed the approximate locations of their deletions to be determined. Each antigen-binding mutant was examined for its ability to exhibit three activities carried out by normal mouse IgG(,2b): complement fixation, binding to mouse Fc receptors and binding to protein A from Staphlococcus aureus. The information obtained to date with these antigen-binding mutants, whose immune complexes are more similar to natural antigen-antibody complexes, is by and large consistent with that obtained with chemically-aggregated Ig fragments. This approach offers the potential for obtaining a finer resolution of the functional sites of the immunoglobulin constant regions. The properties of the mutant monoclonal antibodies may prove superior to native Ig in many clinical and research settings.