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

Introduction: Trastuzumab, a humanized antibody directed against the Her2 receptor, induces the expression of p27(kip1), ail 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 In-111, 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 In-111, yielding [In-111]-anti-p27(kip1)-tat. In-111 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 [In-111]-anti-p27(kip1)-tat were measured in MDA-MB-361 and SKBr3 cells after exposure to trastuzumab. Uptake of [In-111]-anti-p27(kip1)-tat was determined at 72 It postintravenous injection in MDA-MB-361 xenografts in athymic mice treated with trastuzumab or saline.

Results: [In-111]-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 [In-111]-anti-p27(kip1)-tat (6.53 +/- 0.61% vs. 6.98 +/- 1.36% internalized into trastuzumab-treated vs. control cells, respectively). However, retention of [In-111]-anti-p27(kip1)-tat at 72 h was increased approximately twofold (13.5 +/- 1.3% vs. 6.6 +/- 0.6% of internalized [In-111]-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 [In-111]-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: [In-111]-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 [In-111]-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 [In-111]-anti-p27(kip1)-tat to guide treatment with Herceptin and other drugs which alter p27(kip1) expression. (C) 2009 Elsevier Inc. All rights reserved.