PET Imaging of Tumor PD-L1 Expression with a Highly Specific Nonblocking Single-Domain Antibody.

Although immunotherapy through programmed death 1/programmed death ligand 1 (PD-1/PD-L1) checkpoint blockade has shown impressive clinical outcomes, not all patients respond to it. Recent studies have demonstrated that the expression level of PD-L1 in tumors is one of the factors that correlate with PD-1/PD-L1 checkpoint blockade therapy. Herein, a Ga-labeled single-domain antibody tracer, Ga-NOTA-Nb109, was designed and developed for specific and noninvasive imaging of PD-L1 expression in a melanoma-bearing mouse model. The single-domain antibody Nb109 was labeled with the radionuclide Ga through a NOTA chelator. An in vitro binding assay was performed to assess the affinity and binding epitope of Nb109 to PD-L1. The clinical application value of Ga-NOTA-Nb109 was evaluated by a stability assay; by biodistribution and pharmacokinetics studies; and by PET imaging, autoradiography, and immunohistochemical staining studies on tumor-bearing models with differences in PD-L1 expression. Ga-NOTA-Nb109 was obtained with a radiochemical yield of more than 95% and radiochemical purity of more than 98% in 10 min. It showed a highly specific affinity for PD-L1, with an equilibrium dissociation constant of 2.9 × 10 M. A competitive binding assay indicated Nb109 to have a binding epitope different from that of PD-1 and PD-L1 antibody. All biodistribution, PET imaging, autoradiography, and immunohistochemical staining studies revealed that Ga-NOTA-Nb109 specifically accumulated in A375-hPD-L1 tumor, with a maximum uptake of 5.0% ± 0.35% injected dose/g at 1 h. Ga-NOTA-Nb109 holds great potential for noninvasive PET imaging of the PD-L1 status in tumors and for timely evaluation of the effect of immune checkpoint targeting treatment.