PURPOSE

To design and evaluate a small engineered protein binder targeting human programmed death-1 ligand (hPD-L1) for PET imaging in four mouse tumor models, and in human cancer specimens. The hPD-L1 protein binder, FN3, 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). Cu-FN3 was assayed for purity, specific activity, and immunoreactivity. Four groups of NSG mice ( = 3-5/group) were imaged with Cu-FN3 PET imaging (1-24 hours postinjection of 3.7 MBq/7 μg of Do-FN3 in 200 μL PBS): Nod SCID Gamma (NSG) mice bearing (i) syngeneic CT26/hPD-L1tumors, (ii) CT26/hPD-L1 tumors blocked (blk) by preinjected nonradioactive FN3 binder, (iii) hPD-L1-negative Raji xenografts, and (iv) MDA-MB-231 xenografts. The FN3 binder staining was evaluated against validated hPD-L1 antibodies by immunostaining in human cancer specimens.

RESULTS

FN3 bound hPD-L1 with 1.4 ± 0.3 nmol/L affinity in CT26/hPD-L1 cells. Cu-FN3 radiotracer showed >70% yield and >95% purity. Cu-FN3 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 postinjection, respectively. The FN3 binder detected hPD-L1 expression in human tissues with known hPD-L1 expression status based on two validated antibodies.

CONCLUSIONS

The Cu-FN3 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 noninvasive identification of cancer patients who may respond to immune checkpoint blockade therapies.