An Autoantibody is Modified for Use as a Delivery System to Target the Cell Nucleus: Therapeutic Implications

• Published in: Journal of autoimmunity Vol. 11; no. 5; pp. 539 - 546
• Main Authors: Weisbart, Richard H., Stempniak, Mariusz, Harris, Scott, Zack, Debra Jeske, Ferreri, Kevin
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.10.1998
• Subjects: Animals; antibodies; Antibodies, Antinuclear - administration & dosage; Antibodies, Antinuclear - genetics; Antibodies, Antinuclear - therapeutic use; Antibodies, Monoclonal - administration & dosage; Antibodies, Monoclonal - genetics; Antibodies, Monoclonal - therapeutic use; Base Sequence; Cell Nucleus - immunology; Cell Nucleus - metabolism; cellular and nuclear transport; CHO Cells; COS Cells; Cricetinae; DNA Primers - genetics; Drug Delivery Systems; Green Fluorescent Proteins; Immunoglobulin Fab Fragments - administration & dosage; Immunoglobulin Fab Fragments - genetics; Immunoglobulin Fab Fragments - metabolism; Immunoglobulin Heavy Chains - administration & dosage; Immunoglobulin Heavy Chains - genetics; Immunoglobulin Heavy Chains - metabolism; Luminescent Proteins - immunology; Mice; molecular biology; Point Mutation; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - immunology; Recombinant Fusion Proteins - therapeutic use; molecular biology; cellular and nuclear transport; antibodies
• ISSN: 0896-8411; 1095-9157
• DOI: 10.1006/jaut.1998.0212

A murine monoclonal anti-dsDNA antibody was found to penetrate living cells and localize in the nucleus without pathologic effects. A single mutation in VHmarkedly enhanced cellular penetration. The mutant antibody was produced as recombinant Fab and single chain antibody fragments to investigate its use as a delivery system to target the cell nucleus. Complexes were made containing Fab fragments and alkaline phosphatase conjugated goat antibodies to mouse |gk chains. Fab fragments transported 305kDa goat antibody–enzyme complexes into the nucleus in COS-7 and CHO cells. A single chain antibody cDNA was constructed by splice overlap extension PCR and expressed in COS-7 cells. Binding of the single chain antibody to dsDNA was shown by ELISA, and cellular penetration and nuclear localization were demonstrated in COS-7 and CHO cells. The single chain antibody cDNA was ligated into the expression vector, pEGFP, to produce a fusion protein with green fluorescent protein. The fusion protein penetrated COS-7 cells and localized in the cell nucleus. The single chain antibody produced during sustained expression in CHO cells re-entered antibody-producing cells and localized in the nucleus without affecting cell viability. Our results demonstrate the potential use of a modified autoantibody as a delivery system to target the cell nucleus.