Site-specific conjugation of HIV-1 tat peptides to IgG: a potential route to construct radioimmunoconjugates for targeti
- PDF / 287,078 Bytes
- 10 Pages / 595.276 x 785.197 pts Page_size
- 77 Downloads / 133 Views
Division of Nuclear Medicine, University Health Network, Toronto, Ontario, Canada Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario, Canada 3 Department of Medical Imaging, University of Toronto, Toronto, Ontario, Canada 4 Leslie Dan Faculty of Pharmacy, University of Toronto, 19 Russell Street, Toronto, Ontario, M5S 2S2, Canada 2
Received: 20 December 2004 / Accepted: 4 July 2005 / Published online: 29 October 2005 © Springer-Verlag 2005
Abstract. Purpose: Our objective was to study the cellular and nuclear uptake of 123I-mouse IgG (123I-mIgG) linked to peptides [GRKKRRQRRRPPQGYGC] harbouring the membrane-translocating and nuclear import sequences of HIV-1 tat protein. Methods: Carbohydrates on mIgG were oxidized by NaIO4, then reacted with a 40-fold excess of peptides. Displacement of binding of anti-mouse IgG (Fab specific; α-mFab) to 123I-mIgG by tat-mIgG or mIgG was compared. Internalization and nuclear translocation of 123I-tatmIgG in MDA-MB-468, MDA-MB-231 or MCF-7 breast cancer cells were measured. The immunoreactivity of imported tat-mIgG was evaluated by measuring binding of 123 I-α-mFab to cell lysate and by displacement of binding of 123I-mIgG to α-mFab by cell lysate. Biodistribution and nuclear uptake of 123I-tat-mIgG, 123I-mIgG and 123I-tat were compared in mice bearing s.c. MDA-MB-468 tumours. Results: There was a 15-fold decrease in affinity of α-mFab for tat-mIgG compared with mIgG. Internalized radioactivity imported into the nucleus for 123I-tat-mIgG in MDA-MB468, MDA-MB-231 and MCF-7 cells was 61.5±0.6%, 60.3± 3.6% and 64.7±1.0%, respectively. The binding of 123I-αmFab to lysate from MDA-MB-468 cells importing tatmIgG was 17-fold higher than that for cells not exposed to tat-mIgG. Imported tat-mIgG competed with tat-mIgG for displacement of binding of 123I-mIgG to α-mFab. Conjugation of mIgG to tat peptides did not change tissue distribution. Nuclear localization for 123I-tat-mIgG in MDA-MB-468 tumours was 28.1±5.6%, and for liver, spleen and kidneys it was 41.7±2.7%, 13.8±0.8% and 36.9± 3.3%, respectively.
.
Raymond M. Reilly (*) Leslie Dan Faculty of Pharmacy, University of Toronto, 19 Russell Street, Toronto, Ontario, M5S 2S2, Canada e-mail: [email protected] Tel.: +1-416-9465522, Fax: +1-416-9788511
Conclusion: 123I-tat-mIgG radioimunoconjugates suggest a route to the design of radiopharmaceuticals exploiting intracellular and nuclear epitopes. Keywords: Immunoglobulins – Nuclear translocation – HIV-1 tat peptides – Iodine-123 – Breast cancer Eur J Nucl Med Mol Imaging (2006) 33:301–310 DOI 10.1007/s00259-005-1908-7
Introduction Monoclonal antibodies (mAbs) targeting cell surface proteins have been extensively studied for radioimmunodetection (RID) [1] or radioimmunotherapy (RIT) of cancer [2]. Intracellular protein targets have not yet been exploited because most immunoglobulins are unable to penetrate the cell membrane and it was presumed that mAbs internalized into cells through endocytosis would be degraded in lysosomes, destroying their abili
Data Loading...
