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A fully human anti-Ep-CAM scFv-beta-glucuronidase fusion protein for selective chemotherapy with a glucuronide prodrug.

de Graaf M, Boven E, Oosterhoff D, van der Meulen-Muileman IH, Huls GA, Gerritsen WR, Haisma HJ, Pinedo HM - Br. J. Cancer (2002)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 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

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Related in: MedlinePlus

Schematic representation of the C28-GUSh expression cassette. The structural elements include the cytomegalovirus (CMV) promotor, IgG kappa leader sequence (L), and a C-terminal myc- and 6His-tag (mycHis) for easy detection and purification. The anti-Ep-CAM scFv C28 is inserted as an SfiI/NotI fragment. The gene encoding GUSh is inserted as a NotI/NotI fragment, but not before the (Gly4Ser)2 linker is inserted in the NotI and ApaI restriction sites. The primers encoding the linker were designed in such a way that the NotI site at the 3′ end of the scFv disappeared upon downstream insertion, and a new one is introduced at the 3′ end of the linker.
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fig1: Schematic representation of the C28-GUSh expression cassette. The structural elements include the cytomegalovirus (CMV) promotor, IgG kappa leader sequence (L), and a C-terminal myc- and 6His-tag (mycHis) for easy detection and purification. The anti-Ep-CAM scFv C28 is inserted as an SfiI/NotI fragment. The gene encoding GUSh is inserted as a NotI/NotI fragment, but not before the (Gly4Ser)2 linker is inserted in the NotI and ApaI restriction sites. The primers encoding the linker were designed in such a way that the NotI site at the 3′ end of the scFv disappeared upon downstream insertion, and a new one is introduced at the 3′ end of the linker.

Mentions: The plasmid pC28-GUSh was constructed as shown in Figure 1Figure 1


A fully human anti-Ep-CAM scFv-beta-glucuronidase fusion protein for selective chemotherapy with a glucuronide prodrug.

de Graaf M, Boven E, Oosterhoff D, van der Meulen-Muileman IH, Huls GA, Gerritsen WR, Haisma HJ, Pinedo HM - Br. J. Cancer (2002)

Schematic representation of the C28-GUSh expression cassette. The structural elements include the cytomegalovirus (CMV) promotor, IgG kappa leader sequence (L), and a C-terminal myc- and 6His-tag (mycHis) for easy detection and purification. The anti-Ep-CAM scFv C28 is inserted as an SfiI/NotI fragment. The gene encoding GUSh is inserted as a NotI/NotI fragment, but not before the (Gly4Ser)2 linker is inserted in the NotI and ApaI restriction sites. The primers encoding the linker were designed in such a way that the NotI site at the 3′ end of the scFv disappeared upon downstream insertion, and a new one is introduced at the 3′ end of the linker.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2375299&req=5

fig1: Schematic representation of the C28-GUSh expression cassette. The structural elements include the cytomegalovirus (CMV) promotor, IgG kappa leader sequence (L), and a C-terminal myc- and 6His-tag (mycHis) for easy detection and purification. The anti-Ep-CAM scFv C28 is inserted as an SfiI/NotI fragment. The gene encoding GUSh is inserted as a NotI/NotI fragment, but not before the (Gly4Ser)2 linker is inserted in the NotI and ApaI restriction sites. The primers encoding the linker were designed in such a way that the NotI site at the 3′ end of the scFv disappeared upon downstream insertion, and a new one is introduced at the 3′ end of the linker.
Mentions: The plasmid pC28-GUSh was constructed as shown in Figure 1Figure 1

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 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

Show MeSH
Related in: MedlinePlus