Genetic Engineering, Expression, and Activity of a Chimeric Monoclonal Antibody−Avidin Fusion Protein for Receptor-Mediated Delivery of Biotinylated Drugs in Humans

Abstract

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.

High-level expression of tamavidin 2 in human cells by codon-usage optimization

“…The cells of mammals such as human, mouse, rat, and Chinese hamster serve as hosts for the expression of fusion proteins of biotin-binding proteins and antibodies or other pharmaceutical proteins. [11][12][13][14][15][16][17][18] Given that the codon preferences among these mammals are similar, there is a good chance that mam-tam2A and mam-tam2B would also be highly expressed in the cells of these other mammals. One or two amino acids of mam-tam2A and mam-tam2B could be changed to create derivatives with a lower isoelectric point, 7) reversible biotin-binding capability, 8) or extremely high thermal stability.…”

“…In many cases, the fusion proteins were expressed in mammalian cells so that the characteristics of the fusion partners, which are quite often mammalian proteins, would be fully retained. [11][12][13][14][15][16][17][18] Monoclonal antibodies against targets such as receptors and surface proteins of cancer cells are popular fusion partners, because these fusion proteins can attach to the cancer cells to deliver the biotinylated therapeutics.…”

Tamavidin, a versatile affinity tag for protein purification and immobilization

“…Avidin and streptavidin have been suggested as potential affinity tags for protein purification (Airenne et al, 1999a;Sano and Cantor, 2000) and for protein immobilization (Walsh and Swaisgood, 1994;Clare et al, 2001). Although the Escherichia coli expression system is inexpensive and easy to work with, many (strept)avidin fusion proteins have been expressed in insect cells (Karp et al, 1996;Airenne et al, 1999a,b) or mammalian cells (Shin et al, 1997;Ng et al, 2002;Boado et al, 2008). One of the reason is that in E. coli (strept)avidin and their fusion proteins are expressed as insoluble inclusion bodies in most cases (Sano and Cantor, 1991;Sano et al, 1992;Kipriyanov et al, 1996;Li et al, 1999), and renaturation and tedious downstream processing are required for the preparation of active proteins.…”

Preparation of unnatural N-to-N and C-to-C protein fusions

“…Besides being useful biochemical tools, chimeric proteins are also promising as treatment options for cancer, autoimmune diseases, lysosomal storage diseases, and brain disorders (7)(8)(9)(10). Toxins have been conjugated to antibodies, growth factors, and cytokines as a means of delivering these payloads to malignant cells that express the counterstructures recognized by such fusion proteins, to kill tumor cells while minimizing collateral damage (10)(11)(12).…”