ABSTRACT
Purpose
A new universal tool for specific, non-covalent and non-destructive attachment of a recombinant antibody fragment to a polymer-modified adenovirus has been utilised to regulate the tropism of adenoviral gene delivery vector.
Methods
We have prepared a multivalent reactive N-(2-hydroxypropyl)methacrylamide-based copolymer (PHPMA) bearing an α-bungarotoxin-binding peptide (BTXbp). The copolymer was used for covalent surface modification of adenoviral vectors (Ad). The α-bungarotoxin protein (BTX) has a nanomolar binding affinity for BTXbp, allowing non-covalent linkage of BTX fusion proteins. A single chain variable fragment of anti-PSMA antibody bearing BTX (scFv-BTX) binding to the prostate-specific membrane antigen (PSMA) was conjugated with the copolymer-coated adenovirus to enable specific infection of prostate cancer cells via PSMA receptors.
Results
As shown by ELISA, the copolymer-coated virus exhibited much reduced binding to anti-Ad antibodies. Infection of PC-3 and LNCaP prostate cancer cells was ∼100-fold less efficient with copolymer-coated Ad than with un-modified Ad. Conjugation of scFv-BTX with Ad-PHPMA-BTXbp led to 5–10-fold restoration of infection in PSMA-positive LNCaP cells. In PSMA-negative PC-3 cells, the conjugation of scFv-BTX with Ad-PHPMA-BTXbp gave no enhancement of infection.
Conclusions
We have shown that the presented Ad-PHPMA-BTXbp/scFv-BTX system can be used as a universal tool for a receptor-specific virotherapy.
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Abbreviations
- Ad:
-
adenovirus
- BCA:
-
bicinchoninic acid
- BTX:
-
α-bungarotoxin
- BTXbp:
-
α-bungarotoxin-binding peptide
- HEPES:
-
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- HINGS:
-
heat-inactivated normal goat serum
- HPMA:
-
N-(2-hydroxypropyl)methacrylamide
- IPTG:
-
isopropyl β-D-1-thiogalactopyranoside
- Ma-GG-OH:
-
N-methacryloylglycylglycine
- Ma-GG-TT:
-
3-(N-methacryloylglycylglycyl) thiazolidine-2-thione
- PEG:
-
poly(ethylene glycol)
- PHPMA:
-
N-(2-hydroxypropyl)methacrylamide-based copolymer
- PBS:
-
phosphate buffered saline
- PSMA:
-
prostate specific membrane antigen
- scFv-BTX:
-
single chain variable fragment of anti-PSMA antibody bearing BTX
- TMB:
-
3,3′,5,5′-tetramethylbenzidine
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ACKNOWLEDGEMENTS
Financial support of this project by EU grant GIANT No. 512087, the Grant Agency of the Czech Republic, grant No. 203/08/0543 and the Academy of Sciences of the Czech Republic, grant No. 200200651 is gratefully acknowledged.
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Ralph A. Willemsen, Michal Pechar and Robert C. Carlisle contributed equally to the manuscript
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Willemsen, R.A., Pechar, M., Carlisle, R.C. et al. Multi-component Polymeric System for Tumour Cell-Specific Gene Delivery Using a Universal Bungarotoxin Linker. Pharm Res 27, 2274–2282 (2010). https://doi.org/10.1007/s11095-010-0088-8
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DOI: https://doi.org/10.1007/s11095-010-0088-8