⁸⁹Zr-labeled anti-PD-L1 antibody fragment for evaluating in vivo PD-L1 level in melanoma mouse model

Objectives: Malignant melanoma is one of the most common malignancies and its metastases are highly aggressive. The survival time for patients with metastatic melanoma averages 3-15 months. Recently, the rise of immunotherapy has been one of the most startling and promising developments in cancer research. Especially, the drugs called “immune checkpoint inhibitors” were used to lift the natural brakes that restrain immune effector cells, allowing them to go to town on tumors. With such immunotherapy, cancer patients who were given months to live are still here years later. Currently, immune checkpoint inhibitors have been approved by the FDA to treat advanced or metastatic melanoma. Unfortunately, not all the melanoma patients respond to immune checkpoint blockade. Despite impressive treatment outcomes in a subset of patients (10-40% with monotherapy), many patients with melanoma fail to respond to immune checkpoint inhibitor therapy. Many challenges exist including appropriate patient selection and therapeutic efficacy monitoring. The objective of this study is to develop radiolabeled anti-PD-L1 antibody fragments for evaluating the in vivo PD-L1 levels in melanoma mouse model.

Methods: Anti-PD-L1 antibody can specifically bind to cell surface PD-L1. This specific recognition between the antibody and antigen provides a unique opportunity for in vivo PET imaging of PD-L1 levels. Furthermore, the F(ab’)₂ fragment of antibody had been showed to maintain the antigen specificity, and at the same time, with better in vivo pharmacokinetics for imaging. Hence in this study, we will first synthesize the Df (deferoxamine) modified anti-mouse PD-L1 antibody F(ab’)₂ fragment. Then we will prepare the radiolabeled fragment and test its PD-L1 targeting ability in vitro and in vivo. PD-L1 expression level on murine melanoma cells will be verified with flow cytometry analysis in vitro, and the murine melanoma mouse model (B16F10/C57BL6) will be used for in vivo PET/CT imaging.

Results: We have successfully prepared the F(ab’)₂ fragment of the anti-PD-L1 antibody by enzymatic digestion and characterized it with fPLC, gel electrophoresis, and mass spectrum. We then successfully conjugated the bifunctional chelator Df onto the fragment and radiolabeled it with ⁸⁹Zr. The final product, ⁸⁹Zr-Df-F(ab’)₂, was purified and characterized by radioactive fPLC. The radiochemical yield was higher than 95% and the ⁸⁹Zr-Df-F(ab’)₂ possessed radioactive specific activity as high as 10 µCi/µg. The fPLC result showed that ⁸⁹Zr-Df-F(ab’)₂ was stable for up to at least a week in vitro. ⁸⁹Zr-Df-F(ab’)₂ was taken up by PD-L1 positive B16F10 cells, and this uptake was specifically blocked by unmodified anti-PD-L1 antibody (10^-6 M). The high specific binding affinity of ⁸⁹Zr-Df-F(ab’)₂ on PD-L1 was as high as 4 nM. These in vitro cell binding data indicated that the fragment ⁸⁹Zr-Df-F(ab’)₂ maintained the high and specific binding of the full anti-PD-L1 antibody. The PET/CT imaging of ⁸⁹Zr-Df-F(ab’)₂ in B16F10 melanoma-bearing mice has been successfully conducted, and compared with the corresponding ⁸⁹Zr-labeled full antibody. The PET images clearly showed that ⁸⁹Zr-Df-F(ab’)₂processes superior pharmacokinetics and imaging contrast over its radiolabeled full antibody counterpart, with earlier and much higher tumor uptake (5.5 times more at 2 hrs post injection) and much lower liver background (51% reduction at 2 hrs post injection). Flow cytometry analysis of the ex vivo tumor samples indicated that the accumulation of ⁸⁹Zr-Df-F(ab’)₂ in tumor foci was contributed by both B16F10 cells and PD-L1-positive immune cells. Conclusion: We have successfully synthesized the radiolabeled antibody fragment ⁸⁹Zr-Df-F(ab’)₂ and the promising in vitro and in vivo data suggest its potential for in vivo PET imaging of PD-L1 levels in melanoma mouse model.