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

Clin Cancer Res. 2019 Mar 15;25(6):1774-1785. doi: 10.1158/1078-0432.CCR-18-1871. Epub 2018 Oct 29.


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 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 FN3hPD-L1 binder, (iii) hPD-L1-negative Raji xenografts, and (iv) 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 nmol/L 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 postinjection, 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 noninvasive identification of cancer patients who may respond to immune checkpoint blockade therapies.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Validation Study
  • Video-Audio Media

MeSH terms

  • Adult
  • Aged
  • Animals
  • B7-H1 Antigen / immunology
  • B7-H1 Antigen / metabolism*
  • Cell Line, Tumor / transplantation
  • Copper Radioisotopes / administration & dosage
  • Copper Radioisotopes / chemistry
  • Disease Models, Animal
  • Female
  • Fibronectin Type III Domain / genetics
  • Humans
  • Male
  • Mice
  • Molecular Imaging / methods*
  • Molecular Probes / administration & dosage*
  • Molecular Probes / chemistry
  • Molecular Probes / genetics
  • Neoplasms / diagnostic imaging*
  • Neoplasms / pathology
  • Positron Emission Tomography Computed Tomography / methods*
  • Protein Binding
  • Protein Engineering
  • Radiopharmaceuticals / administration & dosage
  • Radiopharmaceuticals / chemistry
  • Recombinant Proteins / administration & dosage
  • Recombinant Proteins / genetics
  • Tissue Distribution


  • B7-H1 Antigen
  • CD274 protein, human
  • Copper Radioisotopes
  • Copper-64
  • Molecular Probes
  • Radiopharmaceuticals
  • Recombinant Proteins