Comparative evaluation of radiolabeled bombesin analogs [177Lu]Lu-AMBA and [177Lu]Lu-RM2 for targeting GRPR in glioblastoma model

Introduction

Radiolabeled agonist ligands bind to and activate target receptors, inducing internalization and delivering radionuclides into the inner cancer cells. In contrast, receptor-bound antagonizing radiopeptides inhibit receptor signaling and remain outside of the cells, but often show more favorable pharmacokinetics. Our head-to-head preclinical comparison of the gastrin-releasing peptide receptor (GRPR) antagonist and the agonist radioligands was conducted in a human glioblastoma (GBM) model.

Methods

The cellular uptake of lutetium-177 labeled agonist [¹⁷⁷Lu]Lu-AMBA and antagonist [¹⁷⁷Lu]Lu-RM2 was evaluated through internalization assays in human GBM U-251 and breast cancer T47-D cells. Immunohistochemistry for γH2AX and the 2′,7′-dichlorofluorescein-diacetate (DCFH-DA) probe were used to assess the induction of DNA double strand breaks (DSB) and generation of reactive oxygen species (ROS), respectively. An in vivo biodistribution study of the radiopeptides was conducted using U-251 xenografted nude mice. Results: Both [¹⁷⁷Lu]Lu-AMBA and [¹⁷⁷Lu]Lu-RM2 showed GRPR-specific uptake, whereby [¹⁷⁷Lu]Lu-AMBA was internalized in contrast to [¹⁷⁷Lu]Lu-RM2, which remained bound to the cell membrane. In vitro studies showed no significant difference in the total cellular uptake of radiopeptides. [¹⁷⁷Lu]Lu-RM2 binding to the receptor was blocked by the unlabeled AMBA ligand. Quantification of [¹⁷⁷Lu]Lu-AMBA and [¹⁷⁷Lu]Lu-RM2-induced DSB and ROS levels revealed no significant difference in treated cells. In vivo, biodistribution showed increased tumor uptake of [¹⁷⁷Lu]Lu-RM2 compared to [¹⁷⁷Lu]Lu-AMBA, whereby washout from receptor positive organs like pancreas, intestine, stomach, and spleen were significantly faster in [¹⁷⁷Lu]Lu-RM2-treated mice.

Conclusion

This study established targeting GRPR with both agonist and antagonist radioligands in glioma U-251 in vitro and in vivo models and recommends further development of antagonizing radiolabeled bombesin analog RM2, which showed in vivo superiority in tumor uptake and tumor-to-normal organ ratios (TNRs) over agonist AMBA radioligand.