Light-chain shuffling results in successful phage display selection of functional prokaryotic - expressed antibody fragments to N-glycolyl GM3 ganglioside
Artikel i vetenskaplig tidskrift, 2004

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-di splayed 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. © 2004 Elsevier B.V. All rights reserved.

escherichia-coli

monoclonal-antibody

recombinant antibodies

affinity

specificity

generation

variable-region

pseudomonas exotoxin

v-gene libraries

complementarity-determining regions

Författare

Gertrudis Rojas

Ctr. Genetic Eng. Biotechnologym

Ariel Talavera

Center of Molecular Immunology Havana

Yasmiana Munoz

Ctr. Genetic Eng. Biotechnologym

Enrique Rengifo

Center of Molecular Immunology Havana

Ute Krengel

Chalmers, Institutionen för kemi och biovetenskap

Jonas Ångström

Göteborgs universitet

Jorge Gavilondo

Ctr. Genetic Eng. Biotechnologym

Ernesto Moreno

Center of Molecular Immunology Havana

Journal of Immunological Methods

0022-1759 (ISSN)

Vol. 293 71-83

Ämneskategorier

Immunologi inom det medicinska området

Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci)

DOI

10.1016/j.jim.2004.07.002