In vivo monitoring of intranuclear p27(kip1) protein expression in breast cancer cells during trastuzumab (Herceptin) therapy.

Abstract

INTRODUCTION:

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.

MATERIALS AND 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).

CONCLUSION:

[(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.

• Authors: Bart Cornelissen, Veerle Kersemans, Kristin McLarty, Lara Tran, Katherine A Vallis, Raymond M Reilly
• Journal: Nuclear medicine and biology (Nucl Med Biol) Vol. 36 Issue 7 Pg. 811-9 (Oct 2009) ISSN: 1872-9614 [Electronic] United States
• PMID: 19720293 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Antibodies, Monoclonal
• Antibodies, Monoclonal, Humanized
• Immunoconjugates
• Peptides
• Cyclin-Dependent Kinase Inhibitor p27
• Pentetic Acid
• Trastuzumab

Topics

• Amino Acid Sequence
• Animals
• Antibodies, Monoclonal (pharmacology, therapeutic use)
• Antibodies, Monoclonal, Humanized
• Breast Neoplasms (genetics, pathology, therapy)
• Cell Line, Tumor
• Cell Transformation, Neoplastic
• Cyclin-Dependent Kinase Inhibitor p27 (immunology, metabolism)
• Gene Expression Regulation, Neoplastic (drug effects)
• Humans
• Immunoconjugates (immunology, isolation & purification, metabolism, pharmacokinetics)
• Intranuclear Space (drug effects, metabolism)
• Isotope Labeling
• Mice
• Molecular Sequence Data
• Pentetic Acid (metabolism)
• Peptides (chemistry, metabolism)
• Protein Transport (drug effects)
• Time Factors
• Tissue Distribution
• Trastuzumab
• Treatment Outcome
• Up-Regulation (drug effects)