Production and characterization of a set of mouse-human chimeric immunoglobulin G (IgG) subclass and IgA monoclonal antibodies with identical variable regions specific for Pseudomonas aeruginosa serogroup O6 lipopolysaccharide.
The heavy- and light-chain variable regions from a murine monoclonal antibody that recognize Pseudomonas aeruginosa serogroup O6 lipopolysaccharide (LPS) were used to generate a series of chimeric mouse-human monoclonal antibodies with identical variable regions. The murine variable-region gene segments were cloned into an immunoglobulin (Ig) cDNA expression vector that contained the human kappa light-chain and IgG1 constant regions. The IgG1 heavy-chain constant region was then replaced with the human IgG2, IgG3, IgG4, or IgA1 heavy-chain constant region. The five different expression vectors were transfected into Chinese hamster ovary cells for antibody production. The chimeric antibodies exhibited immunoreactivity and affinity similar to that of the parental murine IgG antibody toward whole cells of a serogroup O6 strain. In vitro complement deposition assays demonstrated that the chimeric IgG4 and IgA antibodies did not mediate the deposition of complement component C3 onto the surface of either purified LPS or whole bacteria. The chimeric IgG1 and IgG3 antibodies were similar in their ability to deposit C3 onto the surface of both bacteria and LPS, while IgG2 antibody was more effective at depositing C3 onto the surface of bacteria than onto purified LPS. The pattern of opsonophagocytic activity of the chimeric monoclonal antibodies was similar to that of complement deposition onto bacterial cells in that the chimeric IgG1 and IgG3 had the highest opsonic activity. Although IgG2 deposited more C3 onto the bacterial surface than did IgG4 or IgA, all three of these isotypes had low opsonic activity against the serogroup O6 target strain. This series of related antibodies will help reveal functional differences in efficacy among protective antibodies to P. aeruginosa and will be critical for defining the optimal formulation of either a vaccine for active immunization or a polyclonal intravenous IgG or monoclonal antibody cocktail for passive immunotherapy.
Preston
M J
MJ
Channing Laboratory, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA. mpreston@channing.harvard.edu
Gerçeker
A A
AA
Reff
M E
ME
Pier
G B
GB
D000818:Animals; D000907:Antibodies, Bacterial / Q000235:genetics / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology; D016466:CHO Cells; D003165:Complement System Proteins / Q000276:immunology; D006224:Cricetinae; D006801:Humans; D007070:Immunoglobulin A / Q000276:immunology*; D007127:Immunoglobulin Constant Regions / Q000235:genetics / Q000276:immunology; D007074:Immunoglobulin G / Q000235:genetics / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology*; D007145:Immunoglobulin kappa-Chains / Q000235:genetics / Q000276:immunology; D008070:Lipopolysaccharides / Q000276:immunology*; D051379:Mice; D011550:Pseudomonas aeruginosa / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D007070:Immunoglobulin A; D007127:Immunoglobulin Constant Regions; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains; D008070:Lipopolysaccharides; D011993:Recombinant Fusion Proteins; D003165:Complement System Proteins