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

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 1...

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Bibliographic Details
Published in:Cancer research (Chicago, Ill.) Ill.), 1999-11, Vol.59 (22), p.5758-5767
Main Authors: WILLUDA, J, HONEGGER, A, WAIBEL, R, SCHUBIGER, P. A, STAHEL, R, ZANGEMEISTER-WITTKE, U, PLÜCKTHUN, A
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Antibodies, Neoplasm - chemistry
Antibodies, Neoplasm - isolation & purification
Antibodies, Neoplasm - metabolism
Antibody Specificity
Antigens, Neoplasm - immunology
Antigens, Neoplasm - metabolism
Antineoplastic agents
Biological and medical sciences
Cell Adhesion Molecules - immunology
Cell Adhesion Molecules - metabolism
Epithelial Cell Adhesion Molecule
epithelial glycoprotein 2
Female
Hot Temperature
Humans
Immunoglobulin Fragments - chemistry
Immunoglobulin Fragments - isolation & purification
Immunoglobulin Fragments - metabolism
Immunoglobulin Variable Region - chemistry
Immunoglobulin Variable Region - isolation & purification
Immunoglobulin Variable Region - metabolism
Immunotherapy
Isotope Labeling
Medical sciences
Mice
Mice, Inbred BALB C
Mice, Nude
Models, Chemical
Molecular Sequence Data
Pharmacology. Drug treatments
Protein Structure, Secondary
Receptor, ErbB-2 - immunology
Receptor, ErbB-2 - metabolism
Sequence Alignment
Technetium
Tumor Cells, Cultured
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Summary: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.
ISSN:0008-5472
1538-7445