In vivo monitoring of intranuclear p27(kip1) protein expression in breast cancer cells during trastuzumab (Herceptin) therapy.
BACKGROUND
Trastuzumab, a humanized antibody directed against the Her2 receptor, induces the expression of p27(kip1), an intranuclear cyclin-dependent kinase inhibitor in some breast cancer cells. The aim of this study was to develop a radioimmunoconjugate (RIC) to monitor trastuzumab-induced p27(kip1) protein up-regulation in vivo.


METHODS
Anti-p27(kip1) IgG was purified, and conjugated to diethylenetriaminopentaacetate, to allow radiolabeling with (111)In for in vivo detection. Then tat peptide (GRKKRRQRRRPPQGYG), containing a nuclear localization sequence (underlined), was conjugated to the Fc-domain of IgG, using NaIO(4) oxidation of carbohydrates and the resulting Schiff base stabilized with NaCNBH(3). The conjugate was radiolabeled with (111)In, yielding [(111)In]-anti-p27(kip1)-tat. (111)In labeling efficiency, purity and p27(kip1) binding were measured. Trastuzumab-induced p27(kip1) up-regulation was assessed in a panel of breast cancer cell lines by Western blot analysis. Uptake and retention of [(111)In]-anti-p27(kip1)-tat were measured in MDA-MB-361 and SKBr3 cells after exposure to trastuzumab. Uptake of [(111)In]-anti-p27(kip1)-tat was determined at 72 h postintravenous injection in MDA-MB-361 xenografts in athymic mice treated with trastuzumab or saline.


RESULTS
[(111)In]-anti-p27(kip1)-tat was synthesized to 97% purity. The RIC was able to bind to p27(kip1) protein and internalized in the cells and was transported to the nuclei of MDA-MB-361 cells. The level of p27(kip1) protein in MDA-MB-361 cells was increased after exposure to clinically relevant doses of trastuzumab for 3 days. Trastuzumab-mediated induction of p27(kip1) was not associated with increased cellular uptake or nuclear localization of [(111)In]-anti-p27(kip1)-tat (6.53+/-0.61% vs. 6.98+/-1.36% internalized into trastuzumab-treated vs. control cells, respectively). However, retention of [(111)In]-anti-p27(kip1)-tat at 72 h was increased approximately twofold (13.5+/-1.3% vs. 6.6+/-0.6% of internalized [(111)In]-anti-p27(kip1)-tat was retained in trastuzumab-treated vs. control cells, respectively; P=.016). Immunohistochemistry showed up-regulation of p27(kip1) in trastuzumab-treated xenografts. Tumour uptake of [(111)In]-anti-p27(kip1)-tat was significantly higher in trastuzumab-treated compared to control animals (6.5+/-0.9 vs. 4.8+/-0.1 %ID/g at 72 h postinjection, respectively; P=.0065).


CONCLUSIONS
[(111)In]-Anti-p27(kip1)-tat may be useful for monitoring changes in the expression of the intranuclear protein, p27(kip1). Up-regulation of p27(kip1) resulted in increased retention of [(111)In]-anti-p27(kip1)-tat in cells treated with trastuzumab. Modest, but statistically significantly higher, retention was also observed in tumours in mice treated with trastuzumab. Since responsiveness to trastuzumab correlated to up-regulation of p27(kip1), it may be possible to use [(111)In]-anti-p27(kip1)-tat to guide treatment with Herceptin and other drugs which alter p27(kip1) expression.
Cornelissen
Bart
B
Division of Nuclear Medicine, University Health Network, Toronto, ON, Canada M5S 3E2. bart.cornelissen@rob.ox.ac.uk
Kersemans
Veerle
V
McLarty
Kristin
K
Tran
Lara
L
Vallis
Katherine A
KA
Reilly
Raymond M
RM
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology* / Q000627:therapeutic use; D061067:Antibodies, Monoclonal, Humanized; D001943:Breast Neoplasms / Q000235:genetics / Q000473:pathology* / Q000628:therapy; D045744:Cell Line, Tumor; D002471:Cell Transformation, Neoplastic; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000276:immunology / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D018796:Immunoconjugates / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism / Q000493:pharmacokinetics; D042581:Intranuclear Space / Q000187:drug effects* / Q000378:metabolism*; D007553:Isotope Labeling; D051379:Mice; D008969:Molecular Sequence Data; D004369:Pentetic Acid / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D013997:Time Factors; D014018:Tissue Distribution; D000068878:Trastuzumab; D016896:Treatment Outcome; D015854:Up-Regulation / Q000187:drug effects
D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D018796:Immunoconjugates; D010455:Peptides; D050760:Cyclin-Dependent Kinase Inhibitor p27; D004369:Pentetic Acid; D000068878:Trastuzumab