64Cu-labeled affibody molecules for imaging of HER2 expressing tumors

Abstract

Introduction: The development of molecular probes based on novel engineered protein constructs is under active investigation due to the great potential of this generalizable strategy for imaging a variety of tumor targets.

Discussion: In this report, human epidermal growth factor receptor type 2 (HER2)-binding Affibody molecules were radiolabeled with (64)Cu and their imaging ability was further evaluated in tumor mice models to understand the promise and limitations of such probes. The anti-HER2 Affibody molecules in monomeric (Z(HER2:477)) and dimeric [(Z(HER2:477))(2)] forms were site specifically modified with the maleimide-functionalized chelator, 1,4,7,10-tetraazacyclododecane-1,4,7-tris(acetic acid)-10-acetate mono (N-ethylmaleimide amide) (Mal-DOTA). The resulting DOTA-Affibody conjugates were radiolabeled with (64)Cu and evaluated in nude mice bearing subcutaneous SKOV3 tumors. Biodistribution experiments showed that tumor uptake values of (64)Cu-DOTA-Z(HER2:477) and (64)Cu-DOTA-(Z(HER2:477))(2) were 6.12 +/- 1.44% and 1.46 +/- 0.50% ID/g, respectively, in nude mice (n = 3 each) at 4 h postinjection. Moreover, (64)Cu-labeled monomer exhibited significantly higher tumor/blood ratio than that of radiolabeled dimeric counterpart at all time points examined in this study. MicroPET imaging of (64)Cu-DOTA-Z(HER2:477) in SKOV3 tumor mice clearly showed good and specific tumor localization. This study demonstrates that (64)Cu-labeled Z(HER2:477) is a promising targeted molecular probe for imaging HER2 receptor expression in living mice. Further work is needed to improve the excretion properties, hence dosimetry and imaging efficacy, of the radiometal-based probe.

Fig. 1

Synthetic schemes for bifunctional chelator Mal-DOTA preparation and conjugation of the chelator to the anti-HER2 Affibody molecules [Z_HER2:477 and (Z_HER2:477)₂].

Fig. 2

Biosensor binding studies of Affibody bioconjugates [DOTA-(Z_HER2:477)₂ (a) and DOTA-Z_HER2:477 (b)]. Sensorgrams were obtained after injection of the purified Affibody molecules over a sensor chip flow-cell surface containing amine-coupled Fc-HER2 chimeric protein.

Fig. 3

Western blot analysis of HER2 expression in SKOV3 ovarian cancer and MDA-MB-435 (abbreviated as MM435) melanoma in cultured tumor cells (labeled as Cell) and xenografted tumor samples (labeled as Tumor). Much higher level of HER2 is found in SKOV3 than that of in MDA-MB-435 for both cultured cells and tumor samples.

Fig. 4

Decay corrected coronal microPET images of nu/nu mice bearing SKOV3 (indicated by white arrows) and MDA-MB-435 tumor (indicated by red arrows) at 1, 4, and 20 h after tail vein injection of ⁶⁴Cu-DOTA-(Z_HER2:477)₂ (a) and ⁶⁴Cu-DOTA-Z_HER2:477 (b). Decay corrected coronal microPET images of SKOV3 bearing mice which were pretreated with PBS (c) or 300μg of Herceptin (d) 48 h before probe administration at 1, 3.5, and 24 h after tail vein injection of ⁶⁴Cu-DOTA-Z_HER2:477. Yellow arrows indicate location of kidneys.

Fig. 5

Tumor and muscle—time activity curves derived from the multitime point microPET imaging of mice bearing SKOV3 tumor. The regions of interests are shown as mean % ID/g ± SD (n=3).