Identification of residues in the monoclonal antitumor antibody L6 important for binding to its tumor antigen.
L6 is a monoclonal antitumor antibody which recognizes an epitope located in a 42-residue extracellular domain of a tumor-associated approximately 22 kDa glycoprotein antigen. The L6 mAb localizes to solid tumors in vivo and triggers complement activation and antibody-dependent cellular cytotoxicity. It has been the subject of phase I clinical trials. Previously, we had reported the derivation and analysis of a three-dimensional model of the L6 Fv. The model suggests that L6 displays a generally aromatic CDR surface. We aim at improving the affinity for tumor antigen of L6 by in vitro mutagenesis. As the first step toward this end, we have attempted to identify residues critical for the binding of L6 to tumor antigen. On the basis of the model, seven residues were selected which we thought may be critical for L6 antigen binding. Criteria for the selection of these residues were their accessibility and central position on the CDR surface and the residue character. Large polar or charged residues such as arginine, asparagine, and tyrosine were preferred. Nine site-specific single and double mutants were generated using oligonucleotide-directed mutagenesis in an M13 expression vector encoding the L6 Fab. The binding of these mutant Fabs to the L6 tumor antigen and a set of three anti-idiotypic antibodies was quantified in an ELISA. In eight out of nine mutants, binding to L6 tumor antigen was either abolished or substantially reduced. In contrast, the binding of the mutants to the anti-idiotypic antibodies was largely unaffected, suggesting that no significant structural perturbations were introduced as a consequence of these mutations.
Stenzel-Johnson
P R
PR
Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121.
Yelton
D
D
Bajorath
J
J
D000595:Amino Acid Sequence; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000648:ultrastructure; D000912:Antibodies, Neoplasm / Q000737:chemistry* / Q000648:ultrastructure; D000937:Antigen-Antibody Reactions; D000954:Antigens, Surface / Q000378:metabolism*; D001483:Base Sequence; D001666:Binding Sites, Antibody; D017931:DNA Primers / Q000737:chemistry; D000939:Epitopes; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry; D066298:In Vitro Techniques; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013329:Structure-Activity Relationship
D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D000954:Antigens, Surface; D017931:DNA Primers; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D009363:Neoplasm Proteins; C074075:TM4SF1 protein, human