PURPOSE: Our objective was to study the cellular and nuclear uptake of (123)I-mouse IgG ((123)I-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 NaIO(4), then reacted with a 40-fold excess of peptides. Displacement of binding of anti-mouse IgG (Fab specific; alpha-mFab) to (123)I-mIgG by tat-mIgG or mIgG was compared. Internalization and nuclear translocation of (123)I-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 (123)I-alpha-mFab to cell lysate and by displacement of binding of (123)I-mIgG to alpha-mFab by cell lysate. Biodistribution and nuclear uptake of (123)I-tat-mIgG, (123)I-mIgG and (123)I-tat were compared in mice bearing s.c. MDA-MB-468 tumours. RESULTS: There was a 15-fold decrease in affinity of alpha-mFab for tat-mIgG compared with mIgG. Internalized radioactivity imported into the nucleus for (123)I-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 (123)I-alpha-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 (123)I-mIgG to alpha-mFab. Conjugation of mIgG to tat peptides did not change tissue distribution. Nuclear localization for (123)I-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: (123)I-tat-mIgG radioimunoconjugates suggest a route to the design of radiopharmaceuticals exploiting intracellular and nuclear epitopes.
PURPOSE: Our objective was to study the cellular and nuclear uptake of (123)I-mouse IgG ((123)I-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 NaIO(4), then reacted with a 40-fold excess of peptides. Displacement of binding of anti-mouse IgG (Fab specific; alpha-mFab) to (123)I-mIgG by tat-mIgG or mIgG was compared. Internalization and nuclear translocation of (123)I-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 (123)I-alpha-mFab to cell lysate and by displacement of binding of (123)I-mIgG to alpha-mFab by cell lysate. Biodistribution and nuclear uptake of (123)I-tat-mIgG, (123)I-mIgG and (123)I-tat were compared in mice bearing s.c. MDA-MB-468 tumours. RESULTS: There was a 15-fold decrease in affinity of alpha-mFab for tat-mIgG compared with mIgG. Internalized radioactivity imported into the nucleus for (123)I-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 (123)I-alpha-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 (123)I-mIgG to alpha-mFab. Conjugation of mIgG to tat peptides did not change tissue distribution. Nuclear localization for (123)I-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: (123)I-tat-mIgG radioimunoconjugates suggest a route to the design of radiopharmaceuticals exploiting intracellular and nuclear epitopes.
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