A Novel Engineered Small Protein for Positron Emission Tomography Imaging of Human Programmed Death Ligand-1 : Validation in Mouse Models and Human Cancer Tissues

Purpose: To design and evaluate a small engineered protein binder targeting human programmed death-1 ligand (hPD-L1) in vivo for PET imaging in four mouse tumor models, and in situ in human cancer specimens. Experimental Design: The hPD-L1 protein binder, FN3hPD-L1, was engineered using a 12 kDa human fibronectin type-3 domain (FN3) scaffold. The binder's affinity was assayed in CT26 mouse colon carcinoma cells stably expressing hPD-L1 (CT26/hPD-L1). 64Cu-FN3hPD-L1 was assayed for purity, specific activity, and immunoreactivity. Four groups of NSG mice (n=3-5/group) were imaged with 64Cu-FN3hPD-L1 PET imaging (1-24 hours post-injection of 3.7 MBq/7 μg of Do-FN3 in 200 µL PBS): Nod SCID Gamma (NSG) mice bearing (1) syngeneic CT26/hPD-L1tumors, (2) CT26/hPD-L1 tumors blocked (blk) by pre-injected non-radioactive FN3hPD-L1 binder, (3) hPD-L1-negative Raji xenografts, and (4) MDA-MB-231 xenografts. The FN3hPD-L1 binder staining was evaluated against validated hPD-L1 antibodies by immunostaining in human cancer specimens. Results: FN3hPD-L1 bound hPD-L1 with 1.4±0.3 nM affinity in CT26/hPD-L1 cells. 64Cu-FN3hPD-L1 radiotracer showed >70% yield and >95% purity. 64Cu-FN3hPD-L1 PET imaging of mice bearing CT26/hPD-L1 tumors showed tumor-to-muscle ratios of 5.6±0.9 and 13.1±2.3 at 1 and 4 hours post-injection, respectively. The FN3hPD-L1 binder detected hPD-L1 expression in human tissues with known hPD-L1 expression status based on two validated antibodies. Conclusions: The 64Cu-FN3hPD-L1 radiotracer represents a novel, small, and high-affinity binder for imaging hPD-L1 in tumors. Our data support further exploration and clinical translation of this binder for non-invasive identification of cancer patients who may respond to immune checkpoint blockade therapies.

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