Tumor cell killing by in vitro affinity-matured recombinant human monoclonal antibodies.
We have developed a method that allows the rapid improvement of the affinity of phage-displayed antibody fragments by selection on intact eukaryotic cells. A single chain Fv fragment, specific for the tumor-associated Ep-Cam molecule, was mutagenized by shuffling of the immunoglobulin light chain variable region and DNA shuffling of both heavy and light chain variable regions. Higher-affinity mutants were selected from small phage display libraries by cell panning under stringent conditions. When converted to an intact fully human antibody, the mutagenized anti-tumor monoclonal antibody displayed an affinity of 0.4 nM, a 15-fold improvement over the affinity of the original antibody. Compared to previously reported affinity maturation schemes, panning on intact cells does not require purified targets for selection and may be particularly useful when the target molecule can not be expressed as a recombinant molecule or easily purified without disrupting its native configuration. In vitro tumor cell killing assays demonstrated an improved performance of the higher-affinity antibody in complement-mediated tumor cell killing. In contrast, the lower-affinity antibody performed somewhat better in antibody-dependent cellular cytotoxicity assays and penetrated better in multicell spheroids of tumor cells, an in vitro model for the tumor penetration capacity of antibodies.
Huls
G
G
Crucell and Department of Immunology HP F.03.821, University Hospital Utrecht, Heidelberglaan 100, 3508 GA Utrecht, The Netherlands.
Gestel
D
D
van der Linden
J
J
Moret
E
E
Logtenberg
T
T
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000627:therapeutic use*; D000951:Antigens, Neoplasm / Q000276:immunology; D002460:Cell Line; D003110:Colonic Neoplasms / Q000378:metabolism; D003165:Complement System Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005057:Eukaryotic Cells / Q000378:metabolism; D006801:Humans; D007147:Immunoglobulin Light Chains / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D018613:Microscopy, Confocal; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019151:Peptide Library; D017386:Sequence Homology, Amino Acid; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D007147:Immunoglobulin Light Chains; D019151:Peptide Library; D003165:Complement System Proteins