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 ¹¹¹In for in vivo detection. Then tat peptide (GRKKRRQRRRPPQGYG), containing a nuclear localization sequence (underlined), was conjugated to the Fc-domain of IgG, using NaIO₄ oxidation of carbohydrates and the resulting Schiff base stabilized with NaCNBH₃. The conjugate was radiolabeled with ¹¹¹In, yielding [¹¹¹In]-anti-p27^kip1-tat. ¹¹¹In 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 [¹¹¹In]-anti-p27^kip1-tat were measured in MDA-MB-361 and SKBr3 cells after exposure to trastuzumab. Uptake of [¹¹¹In]-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

[¹¹¹In]-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 [¹¹¹In]-anti-p27^kip1-tat (6.53±0.61% vs. 6.98±1.36% internalized into trastuzumab-treated vs. control cells, respectively). However, retention of [¹¹¹In]-anti-p27^kip1-tat at 72 h was increased approximately twofold (13.5±1.3% vs. 6.6±0.6% of internalized [¹¹¹In]-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 [¹¹¹In]-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

[¹¹¹In]-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 [¹¹¹In]-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 [¹¹¹In]-anti-p27^kip1-tat to guide treatment with Herceptin and other drugs which alter p27^kip1 expression.

Keywords: Herceptin, Radioimmunoconjugates, Tat, Nuclear, p27^kip1