Site-specific conjugation of HIV-1 tat peptides to IgG: a potential route to construct radioimmunoconjugates for targeting intracellular and nuclear epitopes in cancer.
OBJECTIVE
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.


CONCLUSIONS
(123)I-tat-mIgG radioimunoconjugates suggest a route to the design of radiopharmaceuticals exploiting intracellular and nuclear epitopes.
Hu
Meiduo
M
Division of Nuclear Medicine, University Health Network, Toronto, Ontario, Canada.
Chen
Paul
P
Wang
Judy
J
Chan
Conrad
C
Scollard
Deborah A
DA
Reilly
Raymond M
RM
D001943:Breast Neoplasms / Q000276:immunology / Q000378:metabolism*; D016503:Drug Delivery Systems / Q000379:methods*; D000939:Epitopes / Q000276:immunology; D005240:Feasibility Studies; D015696:Gene Products, tat / Q000276:immunology / Q000493:pharmacokinetics*; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D018796:Immunoconjugates / Q000276:immunology / Q000493:pharmacokinetics*; D007074:Immunoglobulin G / Q000276:immunology / Q000378:metabolism*; D007457:Iodine Radioisotopes / Q000276:immunology / Q000493:pharmacokinetics; D054322:tat Gene Products, Human Immunodeficiency Virus
D000939:Epitopes; D015696:Gene Products, tat; D018796:Immunoconjugates; D007074:Immunoglobulin G; D007457:Iodine Radioisotopes; D054322:tat Gene Products, Human Immunodeficiency Virus