A fully human anti-Ep-CAM scFv-beta-glucuronidase fusion protein for selective chemotherapy with a glucuronide prodrug.
Monoclonal antibodies against tumour-associated antigens could be useful to deliver enzymes selectively to the site of a tumour for activation of a non-toxic prodrug. A completely human fusion protein may be advantageous for repeated administration, as host immune responses may be avoided. We have constructed a fusion protein consisting of a human single chain Fv antibody, C28, against the epithelial cell adhesion molecule and the human enzyme beta-glucuronidase. The sequences encoding C28 and human enzyme beta-glucuronidase were joined by a sequence encoding a flexible linker, and were preceded by the IgGkappa signal sequence for secretion of the fusion protein. A CHO cell line was engineered to secrete C28-beta-glucuronidase fusion protein. Antibody specificity and enzyme activity were retained in the secreted fusion protein that had an apparent molecular mass of 100 kDa under denaturing conditions. The fusion protein was able to convert a non-toxic prodrug of doxorubicin, N-[4-doxorubicin-N-carbonyl(oxymethyl)phenyl]-O-beta-glucuronyl carbamate to doxorubicin, resulting in cytotoxicity. A bystander effect was demonstrated, as doxorubicin was detected in all cells after N-[4-doxorubicin-N-carbonyl(oxymethyl)phenyl]-O-beta-glucuronyl carbamate administration when only 10% of the cells expressed the fusion protein. This is the first fully human and functional fusion protein consisting of an scFv against epithelial cell adhesion molecule and human enzyme beta-glucuronidase for future use in tumour-specific activation of a non-toxic glucuronide prodrug.
de Graaf
M
M
Department of Medical Oncology, Division of Gene Therapy, Vrije Universiteit Medical Centre, PO Box 7057, 1007 MB Amsterdam, The Netherlands. M.de_Graaf.oncol@med.vu.nl
Boven
E
E
Oosterhoff
D
D
van der Meulen-Muileman
I H
IH
Huls
G A
GA
Gerritsen
W R
WR
Haisma
H J
HJ
Pinedo
H M
HM
D000818:Animals; D000951:Antigens, Neoplasm / Q000235:genetics / Q000276:immunology / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology; D015153:Blotting, Western; D024201:Bystander Effect; D016466:CHO Cells; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000276:immunology / Q000494:pharmacology*; D002455:Cell Division / Q000187:drug effects; D006224:Cricetinae; D004317:Doxorubicin / Q000494:pharmacology; D000071858:Epithelial Cell Adhesion Molecule; D005786:Gene Expression Regulation; D005966:Glucuronidase / Q000235:genetics / Q000276:immunology / Q000494:pharmacology*; D006801:Humans; D008222:Lymphokines / Q000235:genetics / Q000276:immunology / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D011355:Prodrugs; D011786:Quality Control; D011993:Recombinant Fusion Proteins / Q000494:pharmacology*; D012795:Sialoglycoproteins / Q000235:genetics / Q000276:immunology / Q000494:pharmacology*
D000951:Antigens, Neoplasm; D000970:Antineoplastic Agents; D015815:Cell Adhesion Molecules; D000071858:Epithelial Cell Adhesion Molecule; C089422:Fv protein, human; D008222:Lymphokines; D011355:Prodrugs; D011993:Recombinant Fusion Proteins; D012795:Sialoglycoproteins; D004317:Doxorubicin; D005966:Glucuronidase