Affibody-DyLight Conjugates for <i>In Vivo</i> Assessment of HER2 Expression by Near-Infrared Optical Imaging

<div><p>Purpose</p><p>Amplification of the HER2/neu gene and/or overexpression of the corresponding protein have been identified in approximately 20% of invasive breast carcinomas. Assessment of HER2 expression <i>in vivo</i> would advance development of new HER2-targeted therapeutic agents and, potentially, facilitate choice of the proper treatment strategy offered to the individual patient. We present novel HER2-specific probes for <i>in vivo</i> evaluation of the receptor status by near-infrared (NIR) optical imaging.</p><p>Experimental Design</p><p>Affibody molecules were expressed, purified, and labeled with NIR-fluorescent dyes. The binding affinity and specificity of the obtained probe were tested <i>in vitro</i>. For <i>in vivo</i> validation, the relationship of the measured NIR signal and HER2 expression was characterized in four breast cancer xenograft models, expressing different levels of HER2. Accumulation of Affibody molecules in tumor tissue was further confirmed by <i>ex vivo</i> analysis.</p><p>Results</p><p>Affibody-DyLight conjugates showed high affinity to HER2 (K_D = 3.66±0.26). No acute toxicity resulted from injection of the probes (up to 0.5 mg/kg) into mice. Pharmacokinetic studies revealed a relatively short (37.53±2.8 min) half-life of the tracer in blood. Fluorescence accumulation in HER2-positive BT-474 xenografts was evident as soon as a few minutes post injection and reached its maximum at 90 minutes. On the other hand, no signal retention was observed in HER2-negative MDA-MB-468 xenografts. Immunostaining of extracted tumor tissue confirmed penetration of the tracer into tumor tissue.</p><p>Conclusions</p><p>The results of our studies suggest that Affibody-DyLight-750 conjugate is a powerful tool to monitor HER2 status in a preclinical setting. Following clinical validation, it might provide complementary means for assessment of HER2 expression in breast cancer patients (assuming availability of proper NIR scanners) and/or be used to facilitate detection of HER2-positive metastatic lesions during NIR-assisted surgery.</p></div

Confocal microscopy of HER2-positive (upper panel) and HER2-negative (lower panel) cells exposed to ZHER2-DyLight-488.

<p>SK-BR-3 and MDA-MB-468 cells were plated at 2×10⁴ cells/chamber and exposed to Z_HER2-DyLight-488 at 0.5 µg/ml. After a 30 min loading step, cells were rinsed and incubation was continued for indicated period of time at 37°C. A half hour before imaging, the nuclei were counterstained with Hoechst 33342 at 0.2 µg/ml. Images were acquired using Zeiss LSM 510 microscope.</p

IHC analysis of tumor tissue extracted from four different breast tumor models 24 hours post Z_HER2-DyLight-750 injection.

<p>Tumor tissues were extracted from animals 24 hours post probe injection, fixed in 10%NBF and analyzed by IHC. Adjacent sections were stained for detection of HER2 (upper panel) and Affibody as described in Material and Methods section. Arrows point positively stained cell membranes in the tumor tissue.</p

Relationship between normalized rates of accumulation of the probe and HER2 level. A.

<p>Relationship between normalized rates of accumulation of the probe and HER2 level (hours) in three types of HER2-positive tumors (BT-474, MDA-MB361, MCF7), estimated from <i>in vivo</i> fluorescence imaging and Affibody-DL-488 retention for corresponding cell lines to the same probe, found <i>in vitro</i>, using FACS <b>B.</b> Relationship between normalized rates of accumulation of the probe in individual HER2-positive tumors (MDA-MB-361, MCF7), estimated from <i>in vivo</i> fluorescence imaging and ELISA readings for the same tumor (ng/mg), dashed line is linear regression of MDA-MB-361 data, passing through the origin <b>C.</b> Relationship between normalized rates of accumulation of the probe in individual HER2 tumor BT474 (3+), estimated from <i>in vivo</i> fluorescence imaging and ELISA readings for the same tumor, dashed line is the linear regression, passing through the origin.</p

Z_HER2 binding affinity.

<p>A. BT-474 cells were incubated for one hour with increasing concentration of Z_HER2-GSC-DyLight-488 or Z_Taq-GSC-DyLight-488. B. BT-474 cells were pre-incubated with increasing concentration of non-modified Z_HER2 molecules with or without flexible spacer Z(HER2-GS-Cys and Z_HER2-Cys respectively), followed by addition of 0.5 µg/ml of Z_HER2-DyLight-48. Fluorescence intensity was measured in triplicates using LSR II flow cytometer. FlowJo (Tree Star Inc, Ashland, OR) was used for FACS data analysis. K_D and IC50 values were calculated using GraphPad Prism (GraphPad Software, Inc., San Diego, CA).</p

Histological (IHC) images of tumor tissues for tumor with no HER2 expression (A–B) MDA-MB-468, and highly expressed HER2 tumor (C–D) BT-474 and (E–F) NCI-N87.

<p>Tumor tissue was extracted from animals 24 hours post HER2-Affibody-DyLight750 injection, fixed in 10%NBF and analyzed by IHC for detection of HER2 status (<b>A,C and E</b>) and Affibody presence (<b>B,D and F</b>).</p

<i>In vivo</i> fluorescence imaging of xenograft mouse with high HER2-expressing human tumor model (NCI-N87) after injection of the HER2 specific Affibody® (His6-Z_HER2:GS-Cys) conjugated to Dylight750.

<p>(<b>A</b>) Fluorescence intensity map at the tumor region. (<b>B</b>)Fluorescence intensity map at the contralateral site. (<b>C</b>) The difference of fluorescence intensity at the tumor region and the contralateral site, mapped on the tumor region. (<b>D</b>) Pharmacokinetics of the fluorescence intensity at the tumor region and contralateral site after the injection over time. The data in Figs. (<b>D</b>) and (<b>H</b>) are the average data of three mice. Markers show the average and bars show the standard deviation. (<b>E</b>)Fluorescence lifetime map at the tumor region. (<b>F</b>) Fluorescence lifetime map at the contralateral site. (<b>G</b>) The difference of fluorescent lifetime at the tumor and the contralateral site mapped on the tumor region. (<b>E</b>) Pharmacokinetics of the fluorescent lifetime at the tumor region and contralateral site after injection over time.</p

Statistical significance of the measurements between 30 min to 5 hours at the tumor and contralateral site after injection, using Wilcox Two-Sample U-test.

<p>Statistical significance of the measurements between 30 min to 5 hours at the tumor and contralateral site after injection, using Wilcox Two-Sample U-test.</p

<i>In vivo</i> fluorescence imaging of xenograft mouse with no HER2 expressing human tumor model (MDA-MB-468) after injection of the HER2-specific Affibody® (His6-Z_HER2:GS-Cys) conjugated to Dylight750.

<p>(<b>A</b>) Fluorescence intensity map at the tumor region. (<b>B</b>) Fluorescence intensity map at the contralateral site. (<b>C</b>) The difference of fluorescence intensity at the tumor region and the contralateral site, mapped on the tumor region. (<b>D</b>) Pharmacokinetics of the fluorescence intensity at the tumor region and contralateral site after the injection over time. The data in Figs. (<b>D</b>) and (<b>H</b>) are the average data of three mice. Markers show the average and bars show the standard deviation. (<b>E</b>)Fluorescence lifetime map at the tumor region. (<b>F</b>) Fluorescence lifetime map at the contralateral site. (<b>G</b>) The difference of fluorescent lifetime at the tumor and the contralateral site mapped on the tumor region. (<b>E</b>) Pharmacokinetics of the fluorescent lifetime at the tumor region and contralateral site after injection over time.</p