Cell surface display of a lysosomal enzyme for extracellular gene-directed enzyme prodrug therapy

Heine, Daniel; Müller, Rolf; Brüsselbach, Sabine

doi:10.1038/sj.gt.3301474

Cited by 31 publications

(27 citation statements)

References 24 publications

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“…34 Similarly, Brusselbach et al demonstrated that cell surface display of hbG for extracellular gene-directed enzyme prodrug therapy can produce strong bystander killing and potent antitumor activity. 36 We recently demonstrated that mbG expressed on tumor cells effectively activated a glucuronide prodrug and produced tumor regressions in a mouse model. 37 bG is therefore an attractive enzyme for specific conversion of glucuronide prodrugs for cancer therapy.…”

Section: Discussionmentioning

confidence: 99%

Gene expression imaging by enzymatic catalysis of a fluorescent probe via membrane-anchored β-glucuronidase

Chuang

Wang

et al. 2007

Gene Ther

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Development of nonimmunogenic and specific reporter genes to monitor gene expression in vivo is important for the optimization of gene therapy protocols. We developed a membrane-anchored form of mouse b-glucuronidase (mbG) as a reporter gene to hydrolyze a nonfluorescent glucuronide probe (fluorescein di-b-D-glucuronide, (FDGlcU) to a highly fluorescent reporter to assess the location and persistence of gene expression. A functional b-glucuronidase (bG) was stably expressed on the surface of murine CT26 colon adenocarcinoma cells where it selectively hydrolyzed the cell-impermeable FDGlcU probe. FDGlcU was also preferentially converted to fluorescent probe by (bG) on CT26 tumors. The fluorescent intensity in bG-expressing CT26 tumors was 240 times greater than the intensity in control tumors. Selective imaging of gene expression was also observed after intratumoral injection of adenoviral bG vector into carcinoma xenografts. Importantly, mbG did not induce an antibody response after hydrodynamic plasmid immunization of Balb/c mice, indicating that the reporter gene product displayed low immunogenicity. A membraneanchored form of human bG also allowed in vivo imaging, demonstrating that human bG can be employed for imaging. This imaging system therefore, displays good selectivity with low immunogenicity and may help assess the location, magnitude and duration of gene expression in living animals and humans.

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Section: Discussionmentioning

confidence: 99%

Gene expression imaging by enzymatic catalysis of a fluorescent probe via membrane-anchored β-glucuronidase

Chuang

Wang

et al. 2007

Gene Ther

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“…However, secreted enzymes might leak away from the site of the tumour. Therefore, cell surface-tethered forms of prodrugconverting enzymes, such as b-glucuronidase or carboxypeptidase G2, were developed to prevent leakage of untargeted enzyme from the tumour, while prodrug activation is retained (Spooner et al, 2000;Heine et al, 2001). Another way to prevent diffusion of the enzyme from the tumour is secretion by transduced tumour cells of a fusion protein consisting of an scFv antibody and a prodrugconverting enzyme, which can subsequently bind to tumour cells Oosterhoff et al, 2002).…”

Section: Discussionmentioning

confidence: 99%

“…Given the fact that current gene transfer technologies do not allow transduction of all tumour cells, a bystander effect is warranted to achieve effective kill of untransduced tumour cells. To improve the bystander effect of adenoviral vector-mediated GDEPT approaches, secreted and surface-tethered prodrug-converting enzymes have been investigated (Spooner et al, 2000;Weyel et al, 2000;Heine et al, 2001;Oosterhoff et al, 2003). We envisioned that a targeted, secreted form of CE2, consisting of the secreted form of CE2 (sCE2) fused to a tumour-specific scFv antibody would provide an enlarged bystander effect and would furthermore theoretically prevent leakage of the protein into the circulation, thereby reducing systemic side effects.…”

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confidence: 99%

Adenoviral vector-mediated expression of a gene encoding secreted, EpCAM-targeted carboxylesterase-2 sensitises colon cancer spheroids to CPT-11

et al. 2005

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CPT-11 (irinotecan or 7-ethyl-10[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin) is an anticancer agent in use for the treatment of colon cancer. In order to be fully active, CPT-11 needs to be converted into by the enzyme carboxylesterase (CE). In humans, only a minority of CPT-11 is converted to SN-38. To increase the antitumour effect of CPT-11 by gene-directed enzyme prodrug therapy, we constructed a replication-deficient adenoviral vector Ad.C28-sCE2 containing a fusion gene encoding a secreted form of human liver CE2 targeted to the surface antigen epithelial cell adhesion molecule (EpCAM) that is highly expressed on most colon carcinoma cells. By targeting CE2 to EpCAM, the enzyme should accumulate specifically in tumours and leakage into the circulation should be minimised. Ad.C28-sCE2-transduced colon carcinoma cells expressed and secreted active CE that bound specifically to EpCAM-expressing cells. In sections of three-dimensional colon carcinoma spheroids transduced with Ad.C28-sCE2, it was shown that C28-sCE2 was capable of binding untransduced cells. Most importantly, treatment of these spheroids with nontoxic concentrations of CPT-11 resulted in growth inhibition comparable to treatment with SN-38. Therefore, Ad.C28-sCE2 holds promise in gene therapy approaches for the treatment of colon carcinoma.

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“…The solution to this problem could be secretion of a targeted form or a surface tethered form of GUSh by transduced tumour cells, as it might result in retention of the enzyme within the tumour. Recently, a surface tethered form of GUSh has been constructed by fusion of the transmembrane domain of the human PDGF receptor to a C-terminally truncated form of GUSh (Heine et al, 2001). This GDEPT system has been shown to produce a potent anti-tumour effect in vivo.…”

Section: Experimental Therapeuticsmentioning

confidence: 99%

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

et al. 2002

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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 b-glucuronidase. The sequences encoding C28 and human enzyme b-glucuronidase were joined by a sequence encoding a flexible linker, and were preceded by the IgGk signal sequence for secretion of the fusion protein. A CHO cell line was engineered to secrete C28-bglucuronidase 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-

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Cell surface display of a lysosomal enzyme for extracellular gene-directed enzyme prodrug therapy

Cited by 31 publications

References 24 publications

Gene expression imaging by enzymatic catalysis of a fluorescent probe via membrane-anchored β-glucuronidase

Gene expression imaging by enzymatic catalysis of a fluorescent probe via membrane-anchored β-glucuronidase

Adenoviral vector-mediated expression of a gene encoding secreted, EpCAM-targeted carboxylesterase-2 sensitises colon cancer spheroids to CPT-11

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

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