An autoantibody is modified for use as a delivery system to target the cell nucleus: therapeutic implications.
A murine monoclonal anti-dsDNA antibody was found to penetrate living cells and localize in the nucleus without pathologic effects. A single mutation in VH markedly 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 305 kDa 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.
Weisbart
R H
RH
Veterans Affairs Medical Center, Sepulveda, CA, 91343, USA.
Stempniak
M
M
Harris
S
S
Zack
D J
DJ
Ferreri
K
K
D000818:Animals; D000974:Antibodies, Antinuclear / Q000008:administration & dosage* / Q000235:genetics / Q000627:therapeutic use*; D000911:Antibodies, Monoclonal / Q000008:administration & dosage* / Q000235:genetics / Q000627:therapeutic use*; D001483:Base Sequence; D016466:CHO Cells; D019556:COS Cells; D002467:Cell Nucleus / Q000276:immunology / Q000378:metabolism; D006224:Cricetinae; D017931:DNA Primers / Q000235:genetics; D016503:Drug Delivery Systems; D049452:Green Fluorescent Proteins; D007140:Immunoglobulin Fab Fragments / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000276:immunology; D051379:Mice; D017354:Point Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000627:therapeutic use
D000974:Antibodies, Antinuclear; D000911:Antibodies, Monoclonal; D017931:DNA Primers; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins