UNIGE document Scientific Article
previous document  unige:4075  next document
add to browser collection
Title

Covalent modifications of antitetanus F(ab')2 fragments with natural and synthetic polyamines and their effects on the antibody endocytosis in cultured HL60 cells

Authors
Hervé, Françoise
Ghinea, Nicolae
D'Athis, Philippe
Scherrmann, Jean-Michel
Published in Bioconjugate Chemistry. 2008, vol. 19, no. 8, p. 1543-55
Abstract For antibody therapeutics to succeed when intracellular target molecules are involved, a strategy must be applied to increase the delivery of antibodies into cells to reach their targets. Antibody cationization by chemical conjugation of a polyamine could be one such strategy. Both natural polyamines with increasing net charge valencies (putrescine, PUT; spermidine, SPD; and spermine, SPM) and a synthetic polyamine (hexamethylenediamine, HMD) can be used to cationize antibodies, but no comparison of the respective effects of these polyamines on intracellular delivery of antibodies has been performed yet. This study describes the covalent modification of antitetanus F(ab') 2 with these four polyamines using different reaction conditions, and compares the effects of these modifications on antibody interaction with cultured HL60 cells. The cationized antibodies retained > or =80% of the binding activity of the unmodified F(ab') 2 with regard to tetanus toxin, as measured by an antigen-binding capture enzyme immunoassay. This same method was used to quantify the amount of cell-associated F(ab') 2 following incubation with HL60 cells. Cationization was shown to enhance cell interaction of the F(ab') 2 : the higher the number of coupled polyamine molecules, the greater the amount of antibody associated with the cells. Moreover, coupling the F(ab') 2 to the SPD and SPM polyamines had greater effect on cell interaction than coupling the F(ab') 2 to the PUT and HMD diamines. Internalization of the cationized antibodies by the HL60 cells was demonstrated by confocal microscopy. This technique also showed that SPD and SPM were more effective than PUT and HMD in terms of intracellular delivery of the F(ab') 2. It follows from all these results that electrostatic interaction involving charge density plays a predominant role in the endocytic transport mechanism of the F(ab') 2 modified with these polyamines. However, coupling the F(ab') 2 to SPM and SPD yielded the same maximum effects in terms of cell interaction, although coupling SPM was expected to increase the antibody net charge valency more than coupling SPD. This finding suggests that the effective global charge for the cell interaction and uptake of polyamine-modified antibodies does not simply correspond to the addition of the ionizable amine functions on the coupled polyamines, and that other factors may come into play.
Keywords Antigens/immunologyBiological Products/metabolismCells/immunology/metabolismElectrophoresis, Polyacrylamide GelEndocytosisEnzyme-Linked Immunosorbent AssayHL-60 CellsHumansImmunoglobulin Fab Fragments/immunology/metabolismIsoelectric FocusingMicroscopy, ConfocalPolyamines/chemical synthesis/metabolismTetanus Antitoxin/immunology/metabolism
Identifiers
PMID: 18630939
Full text
Article (Published version) (1 MB) - document accessible for UNIGE members only Limited access to UNIGE
Structures
Citation
(ISO format)
HERVÉ, Françoise et al. Covalent modifications of antitetanus F(ab')2 fragments with natural and synthetic polyamines and their effects on the antibody endocytosis in cultured HL60 cells. In: Bioconjugate Chemistry, 2008, vol. 19, n° 8, p. 1543-55. https://archive-ouverte.unige.ch/unige:4075

183 hits

0 download

Update

Deposited on : 2009-11-20

Export document
Format :
Citation style :