Abstract
Vascular targeting agents (VTAs) can be produced by linking antibodies or antibody fragments directed against endothelial cell markers to effector moieties. So far, it has been necessary to produce the components of VTAs (antibody, antibody fragment, linker, and effector) separately and, subsequently, to conjugate them by biochemical reactions. We devised a cloning and expression system to allow rapid generation of recombinant VTAs from hybridoma cell lines. The VTAs consist of a single chain Fv antibody fragment as a targeting moiety and either truncated Pseudomonas exotoxin (resulting in immunotoxins) or truncated human tissue factor (resulting in coaguligands) as effectors. The system was applied to generate recombinant immunotoxins and coaguligands directed against endoglin, vascular endothelial growth factor (VEGF):VEGF receptor (VEGFR) complex and vascular cell adhesion molecule 1 (VCAM-1). The fusion proteins exhibited similar functional activity to analogous biochemical constructs. This is the first report to describe the generation and characterization of recombinant coaguligands.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Animals
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Antigens, CD
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Cell Division / drug effects
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Cell Line
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Cloning, Molecular
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Dose-Response Relationship, Drug
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Endoglin
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Endometrial Neoplasms / metabolism
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Endometrial Neoplasms / pathology
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Endothelial Growth Factors / immunology
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Endothelial Growth Factors / metabolism
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Endothelium, Vascular / cytology
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Endothelium, Vascular / drug effects
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Endothelium, Vascular / metabolism
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Factor Xa / drug effects
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Factor Xa / metabolism
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Female
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Humans
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Hybridomas
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Immunoglobulin Fragments / genetics
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Immunoglobulin Fragments / pharmacology*
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Immunohistochemistry
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Immunotoxins / genetics
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Immunotoxins / pharmacology*
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Lymphokines / immunology
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Lymphokines / metabolism
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Mice
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Neovascularization, Pathologic / prevention & control*
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Plasmids / genetics
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Protein Binding
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Receptor Protein-Tyrosine Kinases / immunology
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Receptor Protein-Tyrosine Kinases / metabolism
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Receptors, Cell Surface
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Receptors, Growth Factor / immunology
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Receptors, Growth Factor / metabolism
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Receptors, Vascular Endothelial Growth Factor
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Recombinant Fusion Proteins / isolation & purification
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Recombinant Fusion Proteins / metabolism
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Recombinant Fusion Proteins / pharmacology
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Thromboplastin / genetics
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Thromboplastin / pharmacology*
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Vascular Cell Adhesion Molecule-1 / immunology
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Vascular Cell Adhesion Molecule-1 / metabolism
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Vascular Endothelial Growth Factor A
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Vascular Endothelial Growth Factors
Substances
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Antigens, CD
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ENG protein, human
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Endoglin
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Endothelial Growth Factors
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Immunoglobulin Fragments
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Immunotoxins
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Lymphokines
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Receptors, Cell Surface
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Receptors, Growth Factor
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Recombinant Fusion Proteins
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Vascular Cell Adhesion Molecule-1
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Vascular Endothelial Growth Factor A
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Vascular Endothelial Growth Factors
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immunoglobulin Fv
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Thromboplastin
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Receptor Protein-Tyrosine Kinases
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Receptors, Vascular Endothelial Growth Factor
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Factor Xa