title: High thermal stability is essential for tumor targeting of antibody fragments: engineering of a humanized anti-epithelial glycoprotein-2 (epithelial cell adhesion molecule) single-chain Fv fragment.
abstract: The epithelial glycoprotein-2 is abundantly expressed on many solid tumors and is a suitable target for antibody-based therapy. In the present study, an antiepithelial glycoprotein-2 single-chain Fv (scFv) was derived from the hybridoma MOC31 by phage display. Despite its high affinity (KD = 3.9 x 10(-9) M), however, this antibody fragment failed to significantly enrich at lung tumor xenografts in mice, mostly because of its insufficient thermal stability. To overcome this limitation, the antigen-binding residues of the MOC31 scFv fragment were grafted onto the framework of the highly stable and well-folding anti-c-erbB2 scFv 4D5. Further modification of the resulting 4D5 MOC-A, which was performed by transferring eight additional residues of the heavy chain variable domain core of the parent MOC31 antibody, produced 4D5 MOC-B, resulting in increased serum stability at 37 degrees C and also significantly improved expression behavior while retaining the antigen specificity and affinity of the parent MOC31 scFv. In mice, the scFv 4D5 MOC-B, which was radiolabeled with 99mtechnetium using a new histidine-tag specific labeling method (Waibel et al., Nature Biotechnol., 17: 897-901, 1999), showed favorable blood clearance and efficient enriches at lung tumor xenografts, with a tumor:blood ratio of 5.25 and a total dose of 1.47% injected dose per gram after 24 h. Biophysical properties such as high thermal stability are thus decisive for whether these molecules are useful in vivo, and our approach may provide a general strategy to solve this problem. This is also the first report of using a humanized anti-EGP-2 scFv in vivo for targeting solid tumors, which is a promising targeting moiety for the diagnostics and therapy of EGP-2-positive tumors in patients.
authors.affiliation: Department of Biochemistry, University of Zürich, Switzerland.
authors.forename: J
authors.initials: J
authors.lastname: Willuda
authors.forename: A
authors.initials: A
authors.lastname: Honegger
authors.forename: R
authors.initials: R
authors.lastname: Waibel
authors.forename: P A
authors.initials: PA
authors.lastname: Schubiger
authors.forename: R
authors.initials: R
authors.lastname: Stahel
authors.forename: U
authors.initials: U
authors.lastname: Zangemeister-Wittke
authors.forename: A
authors.initials: A
authors.lastname: Plückthun
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000912:Antibodies, Neoplasm / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D000918:Antibody Specificity; D000951:Antigens, Neoplasm / Q000276:immunology* / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000276:immunology* / Q000378:metabolism; D000071858:Epithelial Cell Adhesion Molecule; D005260:Female; D006358:Hot Temperature; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D007553:Isotope Labeling; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008956:Models, Chemical; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology* / Q000378:metabolism; D016415:Sequence Alignment; D013667:Technetium; D014407:Tumor Cells, Cultured
chemical_list: D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm; D015815:Cell Adhesion Molecules; D000071858:Epithelial Cell Adhesion Molecule; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D018719:Erb-b2 Receptor Tyrosine Kinases; D013667:Technetium