Toward the Clinical Development and Validation of a Thy1-Targeted Ultrasound Contrast Agent for the Early Detection of Pancreatic Ductal Adenocarcinoma
Authors: Bam Rakesh , Daryaei Iman , Abou-Elkacem Lotfi , Vilches-Moure Jose G. , Meuillet Emmanuelle J. , Lutz Amelie , Marinelli Edmund R. , Unger Evan C. , Gambhir Sanjiv S. , Paulmurugan Ramasamy 
Published: June 2020

Objectives
Early detection of pancreatic ductal adenocarcinoma (PDAC) represents the most significant step toward the treatment of this aggressive lethal disease. Previously, we engineered a preclinical Thy1-targeted microbubble (MBThy1) contrast agent that specifically recognizes Thy1 antigen overexpressed in the vasculature of murine PDAC tissues by ultrasound (US) imaging. In this study, we adopted a single-chain variable fragment (scFv) site-specific bioconjugation approach to construct clinically translatable MBThy1-scFv and test for its efficacy in vivo in murine PDAC imaging, and functionally evaluated the binding specificity of scFv ligand to human Thy1 in patient PDAC tissues ex vivo.

Materials and Methods
We recombinantly expressed the Thy1-scFv with a carboxy-terminus cysteine residue to facilitate its thioether conjugation to the PEGylated MBs presenting with maleimide functional groups. After the scFv-MB conjugations, we tested binding activity of the MBThy1-scFv to MS1 cells overexpressing human Thy1 (MS1Thy1) under liquid shear stress conditions in vitro using a flow chamber setup at 0.6 mL/min flow rate, corresponding to a wall shear stress rate of 100 seconds−1, similar to that in tumor capillaries. For in vivo Thy1 US molecular imaging, MBThy1-scFv was tested in the transgenic mouse model (C57BL/6J − Pdx1-Cretg/+; KRasLSL-G12D/+; Ink4a/Arf−/−) of PDAC and in control mice (C57BL/6J) with l-arginine–induced pancreatitis or normal pancreas. To facilitate its clinical feasibility, we further produced Thy1-scFv without the bacterial fusion tags and confirmed its recognition of human Thy1 in cell lines by flow cytometry and in patient PDAC frozen tissue sections of different clinical grades by immunofluorescence staining.

Results
Under shear stress flow conditions in vitro, MBThy1-scFv bound to MS1Thy1 cells at significantly higher numbers (3.0 ± 0.8 MB/cell; P < 0.01) compared with MBNontargeted (0.5 ± 0.5 MB/cell). In vivo, MBThy1-scFv (5.3 ± 1.9 arbitrary units [a.u.]) but not the MBNontargeted (1.2 ± 1.0 a.u.) produced high US molecular imaging signal (4.4-fold vs MBNontargeted; n = 8; P < 0.01) in the transgenic mice with spontaneous PDAC tumors (2–6 mm). Imaging signal from mice with l-arginine–induced pancreatitis (n = 8) or normal pancreas (n = 3) were not significantly different between the two MB constructs and were significantly lower than PDAC Thy1 molecular signal. Clinical-grade scFv conjugated to Alexa Fluor 647 dye recognized MS1Thy1 cells but not the parental wild-type cells as evaluated by flow cytometry. More importantly, scFv showed highly specific binding to VEGFR2-positive vasculature and fibroblast-like stromal components surrounding the ducts of human PDAC tissues as evaluated by confocal microscopy.

Conclusions
Our findings summarize the development and validation of a clinically relevant Thy1-targeted US contrast agent for the early detection of human PDAC by US molecular imaging.