title: Light-chain shuffling results in successful phage display selection of functional prokaryotic-expressed antibody fragments to N-glycolyl GM3 ganglioside.
abstract: Phage display technology makes it possible to introduce and rapidly screen diversity in antibody binding sites. Chain shuffling has been successfully used to humanize murine antibody fragments and also to obtain affinity matured variants. Here we report a different application of this method: the use of chain shuffling to overcome improper prokaryotic expression behavior of a hybridoma-derived single-chain antibody fragment. Construction and expression of such recombinant antibody fragments remain as empirical entities, hampered by the inability to express some antibody genes coming from eukaryotic cells in bacterial expression systems. Such problems are different for each combination of variable regions and can be serious enough to preclude the use of some hybridomas as sources of V regions to obtain recombinant antibody fragments. The particular binding properties and potential usefulness of some monoclonal antibodies make it highly desirable to bypass these technical limitations in order to develop smaller size therapeutic agents in the form of antibody fragments. The 14F7 mouse monoclonal antibody is one such attractive candidate due to its high specificity for the N-glycolyl GM3 ganglioside overexpressed in tumor cells and its ability to distinguish this antigen from closely related gangliosides like N-acetyl GM3. Our goal was to construct a phage-displayed single-chain Fv antibody fragment derived from 14F7. After cloning the original variable regions from the 14F7 hybridoma in a phagemid vector, we were unable to detect either binding activity or even expression of antibody fragments in bacteria, despite repetitive efforts. We constructed light-chain shuffling libraries, from which functional antibody fragments were readily selected. These combined the original 14F7 heavy chain variable region with a wide variety of unrelated murine and human light-chain variable regions. New antibody fragments retained the valuable properties of the monoclonal antibody in terms of fine specificity, affinity and tumor recognition. They were readily produced by bacteria, either in phage-displayed form or as soluble molecules, and provided a panel of potentially useful variants for cancer diagnosis and immunotherapy. Chain shuffling and phage display were found to be useful strategies for selecting antibody fragments on the basis of both prokaryotic expression and antigen binding criteria.
authors.affiliation: Recombinant Antibodies Laboratory, Pharmaceuticals Division, Center for Genetic Engineering and Biotechnologym, P.O. Box 6162, Ave 31 e/ 158 y 190, Cubanacán, Playa, La Habana 10600, Cuba. gertrudis.rojas@cigb.edu.cu
authors.forename: Gertrudis
authors.initials: G
authors.lastname: Rojas
authors.forename: Ariel
authors.initials: A
authors.lastname: Talavera
authors.forename: Yasmiana
authors.initials: Y
authors.lastname: Munoz
authors.forename: Enrique
authors.initials: E
authors.lastname: Rengifo
authors.forename: Ute
authors.initials: U
authors.lastname: Krengel
authors.forename: Jonas
authors.initials: J
authors.lastname: Angström
authors.forename: Jorge
authors.initials: J
authors.lastname: Gavilondo
authors.forename: Ernesto
authors.initials: E
authors.lastname: Moreno
mesh_terms: D000818:Animals; D004926:Escherichia coli / Q000276:immunology*; D005679:G(M3) Ganglioside / Q000031:analogs & derivatives* / Q000276:immunology; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000235:genetics* / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D051379:Mice; D019151:Peptide Library
chemical_list: D005679:G(M3) Ganglioside; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D019151:Peptide Library