Practical Immuno-PET Radiotracer Design Considerations for Human Immune Checkpoint Imaging

Area of Science:

• Molecular Imaging
• Oncology
• Radiochemistry

Background:

• Immune checkpoint blockade is a cancer therapy, but many patients do not respond.
• Lack of imaging tools to assess dynamic immune checkpoint expression hinders biomarker validation.
• Programmed death-ligand 1 (PD-L1) is a key immune checkpoint target.

Purpose of the Study:

• To optimize noninvasive immuno-PET imaging of human PD-L1 expression.
• To develop and evaluate small, high-affinity engineered protein scaffolds (HAC-PD1) as radiotracers.
• To assess the impact of radiotracer design modifications on imaging performance.

Main Methods:

• Six HAC-PD1 radiotracer variants were synthesized with modifications in chelate, glycosylation, and radiometal.
• Preclinical imaging and biodistribution studies were performed in mice bearing human PD-L1 expressing tumors.
• Aglycosylated variants (HACA-PD1) were specifically evaluated.

Main Results:

• Aglycosylation was a critical design factor influencing tracer uptake, specificity, and clearance.
• Aglycosylated ⁶⁴Cu-NOTA-HACA-PD1 demonstrated accurate in vivo visualization of human PD-L1.
• ⁶⁸Ga-labeled HACA-PD1 variants showed promising tumor-to-background ratios at 1 hour post-injection.
• All HAC-PD1 variants enabled significantly earlier detection of PD-L1 expression compared to antibodies.

Conclusions:

• Small engineered protein scaffolds are effective for immuno-PET imaging of PD-L1.
• Optimized radiotracer design, particularly aglycosylation, enhances imaging capabilities.
• This work supports the translation of small engineered protein radiotracers for clinical immune checkpoint imaging.