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Radiosynthesis, Quality Control, and Small Animal Positron Emission Tomography Imaging of 68Ga-Labelled Nano Molecules
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A Novel Engineered Small Protein for Positron Emission Tomography Imaging of Human Programmed Death Ligand-1:

Arutselvan Natarajan1, Chirag B Patel1,2, Sindhuja Ramakrishnan1

  • 1Molecular Imaging Program at Stanford (MIPS), Department of Radiology, Stanford University, Stanford, California.

Clinical Cancer Research : an Official Journal of the American Association for Cancer Research
|October 31, 2018
PubMed
Abstract

Insights

A novel small protein binder, 64Cu-FN3hPD-L1, effectively images human programmed death-1 ligand (hPD-L1) in tumors using PET. This tracer shows promise for identifying cancer patients eligible for immune checkpoint therapies.

Area of Science:

  • Biotechnology
  • Molecular Imaging
  • Oncology

Background:

  • Immune checkpoint blockade therapies, such as those targeting human programmed death-1 ligand (hPD-L1), have revolutionized cancer treatment.
  • Accurate identification of patients who will respond to these therapies is crucial for optimizing treatment outcomes.
  • Current imaging methods for hPD-L1 expression have limitations, necessitating the development of novel, high-affinity tracers.

Purpose of the Study:

  • To design and evaluate a small engineered protein binder, FN3hPD-L1, for targeting hPD-L1.
  • To assess the in vivo performance of the radiolabeled binder (64Cu-FN3hPD-L1) using Positron Emission Tomography (PET) imaging in mouse tumor models.
  • To evaluate the binder's utility for in situ imaging in human cancer specimens.

Main Methods:

  • Engineered a 12-kDa human fibronectin type-3 domain (FN3) scaffold to create the FN3hPD-L1 binder.
  • Assayed binder affinity using CT26 mouse colon carcinoma cells expressing hPD-L1 (CT26/hPD-L1).
  • Radiolabeled FN3hPD-L1 with Copper-64 (64Cu) and evaluated the resulting tracer (64Cu-FN3hPD-L1) for purity, specific activity, and immunoreactivity.
  • Conducted PET imaging in NSG mice bearing various tumors (hPD-L1-positive syngeneic, hPD-L1-negative xenografts, and hPD-L1-positive xenografts with blocking) and evaluated immunostaining in human cancer specimens.

Main Results:

  • FN3hPD-L1 demonstrated high affinity (1.4 ± 0.3 nmol/L) for hPD-L1.
  • The 64Cu-FN3hPD-L1 radiotracer exhibited high yield (>70%) and purity (>95%).
  • PET imaging revealed significant tumor uptake in mice with CT26/hPD-L1 tumors, with tumor-to-muscle ratios of 5.6 ± 0.9 at 1 hour and 13.1 ± 2.3 at 4 hours postinjection.
  • The binder successfully detected hPD-L1 expression in human tissues, correlating with validated antibody staining.

Conclusions:

  • The 64Cu-FN3hPD-L1 radiotracer is a novel, small, high-affinity binder suitable for imaging hPD-L1 in tumors.
  • The findings support the potential clinical translation of this binder for noninvasive identification of cancer patients who may benefit from immune checkpoint blockade therapies.

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