Poly(ethylene glycol) modification of β-glucuronidase-antibody conjugates for solid-tumor therapy by targeted activation of glucuronide prodrugs

Cheng, Tian‐Lu; Chen, Bing‐Mae; Chan, Lai-Yee; Wu, Pin-Yi; Chern, Ji‐Wang; Roffler, Steve R.

doi:10.1007/s002620050387

Cited by 46 publications

(58 citation statements)

References 28 publications

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“…15 Recombinant Escherichia coli-derived bG was produced as described. 16 Succinimidyl succinate poly(ethylene glycol) (PEG) (5 kDa) was purchased from Sigma Chemical Company, St Louis, MO. phOx and dansyl were linked to fluorescein via a diaminopentane spacer to generate phOx-FITC and dansyl-FITC as described.…”

Section: Reagentsmentioning

confidence: 99%

“…bG activity was measured as described. 16 The number of phOx groups introduced into bG was estimated as described. 19 bG and phOx-bG were modified with succinimidyl succinate PEG at a weight ratio of 2.5:1 in 0.1 M borate buffer, pH 8.0 at room temperature for 2 hours as described.…”

Section: Generation Of -Glucuronidase Derivatized With Phox and Peg (mentioning

confidence: 99%

“…18 The combined binding and enzymatic activities of phOx-bG-PEG conjugates were assayed as described. 16 Figure 1 Hapten-directed enzyme prodrug therapy. phOx-bG-PEG can bind to phOx scFv receptors on cells.…”

Section: Generation Of -Glucuronidase Derivatized With Phox and Peg (mentioning

confidence: 99%

“…16 Triplicate wells were also washed with PBS and incubated with phOx-bG-PEG (5 mg/ml) for 1 hour. The cells were washed twice with PBS and then 50 mM HAMG or pHAM was added to the cells for 24 hours.…”

Section: In Vitro Bystander Killing Effectmentioning

confidence: 99%

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Hapten-directed targeting to single-chain antibody receptors

et al. 2004

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Artificial recombinant receptors may be useful for selectively targeting imaging and therapeutic agents to sites of gene expression. To evaluate this approach, we developed transgenes to express highly on cells a single-chain antibody (scFv) against the hapten 4-ethoxymethylene-2-phenyl-2-oxazoline-5-one (phOx). A phOx enzyme conjugate was created by covalently attaching phOx molecules to polyethylene glycol (PEG)-modified b-glucuronidase. Cells expressing phOx scFv but not control scFv receptors were selectively killed after exposure to -glucuronidase derivatized with phOx and PEG (phOx-bG-PEG) and a glucuronide prodrug (phydroxy aniline mustard b-D-glucuronide, HAMG) of p-hydroxyaniline mustard. Targeted activation of HAMG produced bystander killing of receptor-negative cells in mixed populations containing as few as 10% phOx-receptor-positive cells. Functional phOx scFv receptors were stably expressed on B16-F1 melanoma tumors in vivo. Treatment of mice bearing established phOx-receptorpositive tumors with phOx-bG-PEG and HAMG significantly (Pp.0005) suppressed tumor growth as compared with treatment with bG-PEG and HAMG or prodrug alone. phOx was unstable in the serum, suggesting alternative haptens may be more suitable for in vivo applications. Our results show that therapeutic agents can be targeted to artificial hapten receptors in vitro and in vivo. The expression of artificial receptors on target cells may allow preferential delivery of therapeutic or imaging molecules to sites of transgene expression.

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Section: Reagentsmentioning

confidence: 99%

Section: Generation Of -Glucuronidase Derivatized With Phox and Peg (mentioning

confidence: 99%

Section: Generation Of -Glucuronidase Derivatized With Phox and Peg (mentioning

confidence: 99%

Section: In Vitro Bystander Killing Effectmentioning

confidence: 99%

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Hapten-directed targeting to single-chain antibody receptors

et al. 2004

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“…The combined antigenbinding and enzymic activity of L6-hβG was determined as described in [5]. Activity of sCD13 was determined by adding 20 µl of culture medium or purified sCD13 to 160 µl of PBS containing 0.5 % BSA.…”

Section: Activity Assaysmentioning

confidence: 99%

A simple method for the production of recombinant proteins from mammalian cells

Balasubramanian

et al. 2004

Biotech and App Biochem

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Expression of recombinant proteins in mammalian cells is useful for obtaining products with normal posttranslational modifications. We describe a simple and economical method for the production of milligram levels of proteins in murine fibroblasts. Retroviral or LIPOFECTAMINE TM (Gibco Laboratories) transduction was employed to generate stable murine-fibroblast producer cells. Confluent cultures of stable fibroblast clones were maintained for up to 1 month in 0.5 % serum. Culture medium was collected every 2-3 days and polyhistidine-tagged proteins were purified by ammonium sulphate precipitation and Ni 2+ -nitrilotriacetic acid affinity chromatography. Highly pure, active, glycosylated recombinant proteins, including human β-glucuronidase, mouse β-glucuronidase, aminopeptidase N (CD13) and a single-chain antibody-enzyme fusion protein, were obtained with yields of 3-6 mg/l of culture medium. Fc-tagged proteins were also produced and purified in a single step by Protein A affinity chromatography with yields of 6-12 mg/l. The techniques described here allow simple and economical production of recombinant mammalian proteins with post-translational modifications.

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Cure of malignant ascites and generation of protective immunity by monoclonal antibody–targeted activation of a glucuronide prodrug in rats

et al. 1997

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We examined the in vivo efficacy of targeting ␤-glucuronidase (␤G) to activate a glucuronide prodrug (BHAMG) of p-hydroxyaniline mustard (pHAM) at hepatoma ascites in Sprague-Dawley rats. Injection i.p. of 500 g RH1-␤G, a conjugate formed between recombinant ␤G and monoclonal antibody RH1 with specificity for an antigen expressed on AS-30D rat hepatoma cells, into rats bearing AS-30D ascites resulted in the accumulation of 54 g conjugate per 10 9 tumor cells after 2 hr. Ascites fluid and serum contained 0.53 and 0 g/ml, respectively, RH1-␤G 2 hr after injection of the conjugate. Conjugate binding to AS-30D cells was heterogeneous and non-saturated, as determined by flow cytometry. BHAMG was less toxic than pHAM to SD rats based on measures of animal mortality, weight loss and hematological toxicity. Treatment of rats bearing established hepatoma ascites with 500 g RH1-␤G followed 2 hr later with a single i.p. injection of 30 mg/kg BHAMG or 3 i.p. injections of 10 mg/kg BHAMG 2, 3 and 4 hr later resulted in the cure of 6/8 and 8/8 animals, respectively. Treatment with BHAMG or pHAM alone did not produce cures, whereas treatment with a control antibody-␤G conjugate and BHAMG produced significantly greater hematological toxicity compared to treatment with RH1-␤G and BHAMG. All cured rats were completely protected from rechallenge with 2 ؋ 10 7 AS-30D cells, indicating that successful treatment of animals induced protective immunity.

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Poly(ethylene glycol) modification of β-glucuronidase-antibody conjugates for solid-tumor therapy by targeted activation of glucuronide prodrugs

Cited by 46 publications

References 28 publications

Hapten-directed targeting to single-chain antibody receptors

Hapten-directed targeting to single-chain antibody receptors

A simple method for the production of recombinant proteins from mammalian cells

Cure of malignant ascites and generation of protective immunity by monoclonal antibody–targeted activation of a glucuronide prodrug in rats

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