Targeted tumor therapy with a fusion protein of an antiangiogenic human recombinant scFv and yeast cytosine deaminase.
In adults, endothelial cell division occurs only in wound healing, during menstruation, or in diseases such as wet age-related macular degeneration or development of benign or malignant tissues. Angiogenesis is one of the major requirements to supply the fast developing tumor tissue with oxygen and nutrients, and enables it to spread into other tissues far from its origin. We selected the extradomain B (ED-B), a splice variant of fibronectin, which is exclusively expressed in ovaries, uterus, during wound healing, and in tumor tissues, as a target for the development of an innovative antiangiogenic, prodrug-based targeted tumor therapy approach. We designed a fusion protein termed L19CDy-His, consisting of the antibody single chain fragment L19 for targeting ED-B and yeast cytosine deaminase for the conversion of 5-fluorocytosine into cytotoxic 5-fluorouracil. We purified high amounts of the fusion protein from Pichia pastoris that is stable, enzymatically active, and retains 75% of its activity after incubation with human plasma for up to 72 hours. The binding of L19CDy-His to ED-B was confirmed by an enzyme-linked immunosorbent assay and quantified by surface plasmon resonance spectroscopy determining a KD value of 81±7 nM. L19CDy-His successfully decreased cell survival of the murine ED-B-expressing teratocarcinoma cell line F9 upon addition of the prodrug 5-fluorocytosine. Our data demonstrate the suitability of targeting ED-B by L19CDy-His for effective prodrug-based tumor therapy.
Institut für Laboratoriumsmedizin, Klinische Chemie und Pathobiochemie, Charité-Universitätsmedizin Berlin, Berlin, Germany.
Nicole
N
Schellmann
Hossein
H
Panjideh
Patricia
P
Fasold
Diana
D
Bachran
Christopher
C
Bachran
Peter M
PM
Deckert
Hendrik
H
Fuchs
D020533:Angiogenesis Inhibitors / Q000627:therapeutic use*; D000818:Animals; D000951:Antigens, Neoplasm / Q000276:immunology / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D043525:Cytosine Deaminase / Q000627:therapeutic use*; D005353:Fibronectins / Q000037:antagonists & inhibitors*; D005437:Flucytosine / Q000627:therapeutic use; D005656:Fungal Proteins / Q000627:therapeutic use*; D051379:Mice; D058990:Molecular Targeted Therapy; D010843:Pichia; D011993:Recombinant Fusion Proteins / Q000627:therapeutic use*; D057127:Single-Chain Antibodies / Q000235:genetics / Q000378:metabolism*; D018243:Teratocarcinoma / Q000628:therapy*
D020533:Angiogenesis Inhibitors; D000951:Antigens, Neoplasm; D005353:Fibronectins; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D005437:Flucytosine; D043525:Cytosine Deaminase