Efficacy of Affibody-Based Ultrasound Molecular Imaging of Vascular B7-H3 for Breast Cancer Detection.

Abstract

Human B7-H3 (hB7-H3) is a promising molecular imaging target differentially expressed on the neovasculature of breast cancer and has been validated for preclinical ultrasound (US) imaging with anti-B7-H3-antibody-functionalized microbubbles (MB). However, smaller ligands such as affibodies (ABY) are more suitable for the design of clinical-grade targeted MB. Binding of ABY_B7-H3 was confirmed with soluble and cell-surface B7-H3 by flow cytometry. MB were functionalized with ABY_B7-H3 or anti-B7-H3-antibody (Ab_B7-H3). Control and targeted MB were tested for binding to hB7-H3-expressing cells (MS1_hB7-H3) under shear stress conditions. US imaging was performed with MB_ABY-B7-H3 in an orthotopic mouse model of human MDA-MB-231 coimplanted with MS1_hB7-H3 or control MS1_WT cells and a transgenic mouse model of breast cancer development. ABY_B7-H3 specifically binds to MS1_hB7-H3 and murine-B7-H3-expressing monocytes. MB_ABY-B7-H3 (8.5 ± 1.4 MB/cell) and MB_Ab-B7-H3 (9.8 ± 1.3 MB/cell) showed significantly higher (<i>P</i> < 0.0001) binding to the MS1_hB7-H3 cells compared with control MB_Non-targeted (0.5 ± 0.1 MB/cell) under shear stress conditions. <i>In vivo</i>, MB_ABY-B7-H3 produced significantly higher (<i>P</i> < 0.04) imaging signal in orthotopic tumors coengrafted with MS1_hB7-H3 (8.4 ± 3.3 a.u.) compared with tumors with MS1_WT cells (1.4 ± 1.0 a.u.). In the transgenic mouse tumors, MB_ABY-B7-H3 (9.6 ± 2.0 a.u.) produced higher (<i>P</i> < 0.0002) imaging signal compared with MB_Non-targeted (1.3 ± 0.3 a.u.), whereas MB_ABY-B7-H3 signal in normal mammary glands and tumors with B7-H3 blocking significantly reduced (<i>P</i> < 0.02) imaging signal. MB_ABY-B7-H3 enhances B7-H3 molecular signal in breast tumors, improving cancer detection, while offering the advantages of a small size ligand and easier production for clinical imaging.