Abstract P1-02-01: HER2/neu antibody-fluorophore conjugate for intraoperative detection of HER2-positive breast cancer
Authors: Jo Simien, Julie Voss, A.M. Thompson, S.C. Ghosh, S. AghaAmiri, A. Azhdarinia, H.S. Tran-Cao
Journal: Cancer Research (2020)

Abstract Background: Although advances in neoadjuvant and adjuvant treatments have led to enhanced survival in patients with HER2 positive breast cancer, intraoperative detection of residual disease and tumor margins (including HER2 positive DCIS) remain challenging. Real-time fluorescence imaging is being used during surgery to improve tumor visualization and identify margins. However, the lack of an approved intraoperative imaging agent that is specific for HER2 expressing cancers limits the utility of fluorescence-guided surgery (FGS) in this patient population. We examined the potential of using clinically approved anti-HER2 monoclonal antibodies (mAbs), trastuzumab and pertuzumab, for the development of fluorescent immunoconjugates dual-labeled with radioactivity to enable quantitative assessment. Methods: Trastuzumab, and pertuzumab were conjugated to the near-infrared (NIR) fluorophore, IR800CW, and radiometal chelator, deferoxamine (DFO), to permit dual labeling with 89Zr (positron emitting radionuclide). To confirm retention of HER2 specificity and functionality, flow cytometry analysis of the dual-labeled immunoconjugates was performed in HER2+ (BT474, SKBR3), HER2 moderate (MCF7), and HER2− (MDA231) cell lines. Using the same cell lines, radioactive uptake studies were employed to quantitatively measure cellular uptake study using 89Zr with and without blocking doses of unlabeled mAb. In vivo optical imaging of each immunoconjugate and IgG controls was performed in BT474 and MCF7 xenografts in BALB/c (nu/nu) mice 48 hours post injection using the BrukerXtreme system. Results: In vitro results confirmed retention of receptor mediated binding on both flow cytometry and radioactive analyses. As shown in Table 1, uptake of each immunoconjugate correlated with the degree of HER2 expression. Importantly, the addition of unlabeled mAb led to a 79-93% reduction in binding to cells with the highest HER2 expression (BT474 and SKBR3). In MDA231 cells binding of the immunoconjugates was similar to the IgG control and indicates low non-specific binding. In vivo and ex vivo analysis of HER2+ and HER2 moderate xenografts demonstrated prominent tumor uptake with minimal signal in non-clearance organs. As a result, tumor-to-muscle, tumor-to-fat pad, and tumor-to-lung ratios were &amp;gt;5, indicating excellent contrast in key regions where surgical excisions are performed clinically. Table 1: Uptake of 89Zr-mAb-IR800 in breast cancer cell linesCell Lines% Uptake of 89Zr-PertuzumabPertuzumab Block (Unlabeled)% Uptake of 89Zr-TrastuzumabTrastuzumab Block (Unlabeled)% Uptake of 89Zr-IgGBT47449.2±14.83.7±0.036.9±5.84.6±2.61.3±0SKBR337.9±1.77.9±0.942.8±3.52.9±1.73.5±0.4MCF721.7±2.63.2±0.44.0±0.12.5±0.11.3±0.7MDA2311.9±0.40.8±0.31.0±0.31.9±0.51.3±0.7 Conclusion: This study demonstrates the potential utility of HER2-targeted FGS to improve tumor visualization, increase the likelihood of obtaining clean surgical margins, and ultimately reduce morbidity in patients with HER2 breast cancer. Citation Format: Jo Simien, Julie Voss, A.M. Thompson, S.C. Ghosh, S. AghaAmiri, A. Azhdarinia, H.S. Tran-Cao. HER2/neu antibody-fluorophore conjugate for intraoperative detection of HER2-positive breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-02-01.