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-tat-mIgG 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 123I-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-MB-468, 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-α-mFab to lysate from MDA-MB-468 cells importing tat-mIgG 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.
Conclusion
123I-tat-mIgG radioimunoconjugates suggest a route to the design of radiopharmaceuticals exploiting intracellular and nuclear epitopes.