Dual-labeled pertuzumab for multimodality image-guided ovarian tumor resection

Abstract

Pertuzumab is clinically employed in the treatment of cancers over-expressing human epidermal growth factor receptor 2 (HER2). Herein, we developed dual-labeled pertuzumab with a radionuclide (⁸⁹Zr) and a near-infrared fluorophore (IRDye 800CW) to investigate the feasibility of utilizing dual-labeled monoclonal antibodies (mAbs) with numerous imaging modalities for preoperative imaging and image-guided surgery in ovarian cancer models. MAbs were dually-labeled with ⁸⁹Zr and IRDye 800CW to generate ⁸⁹Zr-Df-pertuzumab-800CW or ⁸⁹Zr-Df-IgG-800CW. Serial positron emission tomography (PET) and near-infrared fluorescence (NIRF) images were acquired up to 72 hours after injection of dual-labeled mAbs to map the tracers' biodistributions. After the last time point, image-guided tumor resection was executed using different modalities (NIRF, Cerenkov luminescence [CL], and β particle imaging) and ex vivo studies including biodistribution assays and histology analysis were performed to confirm the in vivo imaging data. SKOV3 ovarian cancer cells showed high expression of HER2 and pertuzumab conjugated with Df and IRDye 800CW maintained its binding affinity for these cells. For PET imaging in subcutaneous xenograft ovarian cancer models, ⁸⁹Zr-Df-pertuzumab-800CW showed a significantly higher tumor-to-muscle ratio compared to the nonspecific ⁸⁹Zr-Df-IgG-800CW from 24 hours after injection through the last time point (72 h: 30.7 ± 7.4 vs. 7.5 ± 1.8, P < 0.01, n = 3-4). During image-guided surgery, three imaging modalities including NIRF, CL, and β particle imaging could detect ovarian cancer in both subcutaneous and orthotopic models and each exhibited its own imaging characteristics. In addition, ex vivo imaging and biodistribution studies as well as histology analysis corroborated the in vivo imaging results. Therefore, we concluded that this single radiolabeled tracer can provide all-in-one contrast for multiple imaging modalities. The dual-labeled mAbs may hold promise to be employed for image-guided tumor surgery as well as diagnosis and staging through balancing out the strengths and weaknesses of various modalities such as PET/CT, NIRF, CL, and β particle imaging.

Keywords: 89Zr; Human epidermal growth factor receptor 2 (HER2); image-guided surgery; near-infrared fluorescence (NIRF) imaging; ovarian cancer; pertuzumab; positron emission tomography (PET).

Figure 4

Image-guided tumor resection in the subcutaneous SKOV3 tumor model after the last time point (72 h) of serial PET/NIRF imaging. The same mouse shown in Figure 2A is here presented in Figure 4. (A) Schematic diagram of using multimodalities for verification of tumor development, preoperative and intraoperative/ex vivo imaging. The length of the scale does not reflect actual time. (B-D) Image-guided surgery with (B) Fluorescence-based imaging, (C) Cerenkov luminescence (CL) imaging, and (D) β particle imaging. Red boxes are the expected field of view during HARLI imaging. The actual field-of-view was 2.17 × 2.17 cm².

Figure 1

In vitro characterization and generation of monoclonal antibodies (mAbs) conjugated with ⁸⁹Zr and IRDye 800CW. (A) Flow cytometry results showing high HER2 expression on SKOV3 cells compared to Caov3 or OVCAR3. (B) Internalization profile of ⁸⁹Zr-Df-pertuzumab-800CW in SKOV3 cells. Cell-associated activity (%) was calculated as the radioactivity in the bound or internalized fractions over the total radioactivity associated to cells, n = 3. (C) ⁸⁹Zr-Df-mAb-800CW was prepared as reported previously [17]. Pertuzumab and a non-specific IgG were employed as the mAbs in this study.

Figure 2

Serial PET and NIRF images of subcutaneous SKOV3 tumor-bearing mice after intravenous injection of the radiolabeled probes. (A) ⁸⁹Zr-Df-pertuzumab-800CW clearly visualized tumors compared to (B) ⁸⁹Zr-Df-IgG-800CW as early as 24 h p.i. in both PET and NIRF imaging. For PET imaging, the images are presented as maximum intensity projections (MIPs). The yellow and blue arrows indicate the tumors detected by PET and NIRF imaging, respectively (n = 3-4).

Figure 3

Quantitative region-of-interest analysis of PET imaging over time and ex vivo biodistribution results at 72 h after injection of the tracers. Time-activity curves after administration of (A) ⁸⁹Zr-Df-pertuzumab-800CW and (B) ⁸⁹Zr-Df-IgG-800CW in subcutaneous SKOV3 tumor-bearing mice. (C) The tumor-to-muscle ratio was significantly higher in the ⁸⁹Zr-Df-pertuzumab-800CW group than in the ⁸⁹Zr-Df-IgG-800CW group after 24 h (*P < 0.05, **P < 0.01; n = 3-4). (D) Ex vivo biodistribution results also confirmed that tumor uptake of ⁸⁹ Zr-Df-pertuzumab-800CW was significantly higher than that of the control IgG group (***P < 0.001).

Figure 5

Preoperative/intraoperative imaging and quantitative analysis in orthotopic SKOV3 tumor-bearing mice. (A) PET/CT maximum intensity projection (MIP) and axial images of orthotopic SKOV3 tumor-bearing mice at 72 h after injection of ⁸⁹Zr-Df-pertuzumab-800CW. The yellow arrows indicate the location of the tumors. (B) Time-activity curve based on PET imaging of orthotopic SKOV3 tumor models after intravenous injection of ⁸⁹Zr-Df-pertuzumab-800CW (n = 5). The mean tumor uptake (%ID/g) peaked at 48 h after injection of the tracer. (C) Preoperative near-infrared fluorescence (NIRF) imaging at 72 h post-injection of the tracer and image-guided surgery based on white light and NIRF imaging. Both the blue arrow and the white dotted circle indicate the location of the tumor. (D, E) Image guided surgery with (D) Cerenkov luminescence (CL) and (E) β particle imaging. Red boxes are the expected field of view during β particle imaging. The actual field-of-view was 2.17 × 2.17 cm². (F) Examples of non-target signals detected by NIRF and CL imaging in the orthotopic model. The yellow-dotted circle indicates the SKOV3 orthotopic tumors, while white, pink, and orange arrows represent the stomach, bone, and liver of the SKOV3 tumor-bearing mice, respectively.

Figure 6

Ex vivo imaging and a biodistribution study of ⁸⁹Zr-Df-pertuzumab-800CW in orthotopic SKOV3 tumor-bearing mice were performed for confirmation of results of preoperative and intraoperative imaging. (A) Ex vivo PET images and near-infrared fluorescence images of the organs collected from the mouse in Figure 5A-E. (B) Biodistribution of ⁸⁹Zr-Df-pertuzumab-800CW in orthotopic SKOV3 tumor-bearing mice at 72 h post-injection of the tracer (n = 5). (C) Hematoxylin and eosin staining of normal ovary and excised ovarian tumor sections during surgery. The scale bars are 250 μm and 100 μm for low and high magnifications, respectively.

Figure 7

Immunofluorescent staining of orthotopic and subcutaneous SKOV3 tumors and a normal mouse ovary. Human HER2 signal is presented in green, and CD31 (red) and DAPI (blue) staining visualize the locations of vasculature and cell nuclei, respectively. Green signal was pronounced in both orthotopic and subcutaneous SKOV3, while it was undetectable in normal ovaries. Scale bar = 50 μm. (For images of other organs [liver, spleen, and kidney], the reader is referred to the supporting information).