Evaluation of albumin-binding PD-L1-targeted VHH for PET imaging in colorectal cancer

Immunotherapy plays a pivotal role in the treatment of colorectal cancer (CRC). PD-L1 has been identified to be a significant biomarker of response to treatment. The assessment of PD-L1 presently depends on biopsy samples - a problem that may be resolved with the help of a molecular imaging approach. This study devised and evaluated an ¹⁸F-and ⁸⁹Zr-labelled albumin-binding PD-L1-targeted VHH for potential diagnostic imaging in CRC models.

PD-L1 expression was initially validated in HCT-116 cell-derived xenografts and CRC20-PDX tumors. An albumin-binding moiety was conjugated to a PD-L1-targeted VHH to construct SN-2D01. The ¹⁸F-labeled SN-2D01 was prepared using the AlF-RESCA chelation strategy and the probe was modified with DFO for a ⁸⁹Zr radiolabeling process. The evaluation of [¹⁸F]F-SN-2D01 and [⁸⁹Zr]Zr-SN-2D01 through PET imaging was performed on the two tumor models and a quantitative analysis of tumor uptake for each tracer. Ultimately, a biodistribution study of [⁸⁹Zr]Zr-SN-2D01 at 120 h post-injection (p.i.) was performed in both tumor models.

The HCT-116 cell-derived xenografts and CRC20-PDX tumors were both PD-L1-positive, with a higher expression observed in lesions from CRC20-PDX versus HCT-116 tumors. The 2 h p.i. of [¹⁸F]F-SN-2D01 PET showed clear visualization of HCT-116 and CRC20-PDX tumors, with tracer mostly cleared via hepatic and renal excretion. In PET imaging with [⁸⁹Zr]Zr-SN-2D01, the tumor uptake in both HCT-116 and CRC20-PDX slowly increased from 24 h to 120 h p.i, which peaked at 120 h. Biodistribution at 120 h showed tumor uptake of 11.49 ± 1.32 %ID/g for the CRC20-PDX model and 8.12 ± 1.54 %ID/g for the HCT-116 model.

In summary, we presented two PET probes to visualize PD-L1. Of these, [⁸⁹Zr]Zr-SN-2D01 was found to have significant capability to evaluate PD-L1 expression in CRC. The PD-L1-targeted SN-2D01 may provide a new approach in radionuclide therapy targeting PD-L1 in CRC.