68Ga-Labeled Peptide for Noninvasive Quantifying Tumor Exposure of PD-L1 Therapeutics.

Purpose: Targeting the programmed death protein 1/programmed death-ligand 1 (PD-1/PD-L1) immune checkpoint blockade therapy plays a critical role in cancer therapy. However, not all patients benefit from this approach, with PD-L1 expression levels being a significant contributing factor. Positron emission tomography (PET) imaging of PD-L1 offers a noninvasive, whole-body, and dynamic assessment of its expression. This study aims to develop a novel peptide-based PD-L1 tracer, [⁶⁸Ga]HF12, to quantitatively evaluate PD-L1 expression in tumors, thereby offering clinical guidance.

Methods: HF12 was successfully synthesized and radiolabeled with ⁶⁸Ga to yield [⁶⁸Ga]HF12. In vitro binding assays confirmed the specific binding affinity of HF12 for PD-L1 using CHO-hPD-L1 and CHO cell lines. Subsequent in vivo positron emission tomography (PET) imaging and biodistribution studies assessed [⁶⁸Ga]HF12 for monitoring PD-L1 expression levels in tumor-bearing mice, including those subjected to immunotherapy. Furthermore, PD-L1 expression in tumor tissues was evaluated by using autoradiography, Western blotting, and immunohistochemical (IHC) analysis.

Results: The synthesis of [⁶⁸Ga]HF12 was successfully achieved with a radiochemical purity and yield exceeding 95%. Cellular uptake studies indicated that [⁶⁸Ga]HF12 demonstrated both high specificity and significant uptake in PD-L1-positive CHO-hPD-L1 cells. Micro-PET imaging and biodistribution studies revealed that [⁶⁸Ga]HF12 was preferentially accumulated in CHO-hPD-L1 tumors compared to PD-L1-negative CHO tumors. Treatment with Atezolizumab resulted in a significant reduction in [⁶⁸Ga]HF12 uptake in CHO-hPD-L1 tumors relative to pretreatment levels, whereas no significant changes were observed in the phosphate-buffered saline (PBS) control group. Subsequent biodistribution studies, along with Western blotting and immunohistochemical analyses, confirmed that PD-L1 expression levels in tumors were reduced following immunotherapy, consistent with the results obtained from PET imaging.

Conclusions: [⁶⁸Ga]HF12 was successfully synthesized as a radiotracer for noninvasive quantitative PET imaging of PD-L1 expression levels. This radiotracer exhibited the potential to quantify PD-L1 expression across various tumors, thereby facilitating the prediction of patient response to anti-PD-1 and anti-PD-L1 immunotherapies and monitoring therapeutic efficacy.