Genetic engineering, expression, and activity of a chimeric monoclonal antibody-avidin fusion protein for receptor-mediated delivery of biotinylated drugs in humans.
The genetic engineering, expression, and validation of a fusion protein of avidin (AV) and a chimeric monoclonal antibody (mAb) to the human insulin receptor (HIR) is described. The 15 kDa avidin monomer was fused to the carboxyl terminus of the heavy chain of the HIRMAb. The fusion protein heavy chain reacted with antibodies specific for human IgG and avidin, and had the same affinity for binding to the HIR extracellular domain as the original chimeric HIRMAb. The fusion protein qualitatively bound biotinylated ligands, but was secreted fully saturated with biotin by COS cells, owing to the high level of biotin in tissue culture medium. Chinese hamster ovary (CHO) cells were permanently transfected with a tandem vector expressing the fusion protein genes, and high expressing cell lines were isolated by methotrexate amplification and dilutional cloning. The product expressed by CHO cells had high binding to the HIR, and migrated as a homogeneous species in size exclusion HPLC and native polyacrylamide gel electrophoresis. The CHO cells were adapted to a 4 week culture in biotin depleted medium, and the HIRMAb-AV fusion protein expressed under these conditions had 1 unoccupied biotin binding site per molecule, based on a [3H]-biotin ultrafiltration assay. The HIRMAb-AV increased biotin uptake by human cells >15-fold, and mediated the endocytosis of fluorescein-biotin, as demonstrated by confocal microscopy. In summary, the HIRMAb-AV fusion protein is a new drug targeting system for humans that can be adapted to monobiotinylated drugs or nucleic acids.
Boado
Ruben J
RJ
ArmaGen Technologies, Inc, Santa Monica, California 90401, USA.
Zhang
Yufeng
Y
Zhang
Yun
Y
Xia
Chun-fang
CF
Wang
Yuntao
Y
Pardridge
William M
WM
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics* / Q000494:pharmacology*; D001360:Avidin / Q000737:chemistry; D001710:Biotin / Q000737:chemistry* / Q000493:pharmacokinetics; D015153:Blotting, Western; D016466:CHO Cells; D019556:COS Cells; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003412:Cricetulus; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D003956:Dialysis; D016503:Drug Delivery Systems; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005818:Genetic Engineering; D006801:Humans; D018613:Microscopy, Confocal; D050506:Mutant Chimeric Proteins / Q000235:genetics* / Q000494:pharmacology*; D011955:Receptors, Drug / Q000187:drug effects*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D012710:Serum Albumin, Bovine / Q000737:chemistry; D014462:Ultrafiltration
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D050506:Mutant Chimeric Proteins; D011955:Receptors, Drug; D011993:Recombinant Fusion Proteins; D001360:Avidin; D012710:Serum Albumin, Bovine; D001710:Biotin