Synthesis and preclinical evaluation of an Al18F radio-fluorinated bivalent PD-L1 nanobody

Immunotherapy targeting the programmed death 1/programmed death ligand 1 (PD-1/PD-L1) pathway has achieved remarkable clinical success, but there is a shortage of effective approaches for screening suitable patients. Recently developed PD-L1 nanobody probes have limitations, including limited availability of radionuclides, short tumor retention times, and accumulation in non-target organs. To enhance tumor retention and improve tumor-to-normal tissue contrast, we herein report the synthesis and preclinical evaluation of two Al 18 F-labeled bivalent PD-L1 nanobody probes ([ 18 F]TzTCO-BINb109 and [ 18 F]RESCA-BINb109). Preliminary results indicated that [ 18 F]TzTCO-BINb109 had a greater affinity for PD-L1 and better stability than [ 18 F]RESCA-BINb109. Micro-PET/CT revealed that [ 18 F]TzTCO-BINb109 uptake in A549-PDL1 tumors peaked at 240 min post-injection (3.19 ± 0.49 %ID/g) and demonstrated sustained retention without in vivo defluorination. In contrast, [ 18 F]RESCA-BINb109 exhibited shorter tumor retention (at 60 and 240 min, 2.08 ± 0.22 and 1.37 ± 0.26 %ID/g, respectively) and significant defluorination in vivo. Ex vivo biodistribution studies revealed that the tumor uptake of [ 18 F]TzTCO-BINb109 was consistent with the PET results, with the highest uptake by A549-PDL1 tumor cells (3.43 ± 0.94 %ID/g) compared with H1975 (0.93 ± 0.18 %ID/g) and A549 (0.68 ± 0.12 %ID/g) cells observed at 240 min post-injection. Compared with the previously reported monomeric PD-L1-targeting nanobody probe, [ 68 Ga]NOTA-Nb109, [ 18 F]TzTCO-BINb109 demonstrated enhanced tumor uptake, prolonged retention, and superior tumor-to-normal tissue contrast, contributing to higher imaging quality. These results confirmed that the bivalent PD-L1 nanobody radioligand, [ 18 F]TzTCO-BINb109, was a promising diagnostic probe for PD-L1 detection, efficacy evaluation, and prescription optimization of immune checkpoint inhibitor therapies. • A bivalent PD-L1 nanobody was successfully labeled via two distinct Al 18 F-based radiolabeling methods. • Compared with [ 18 F]RESCA-BINb109 obtained by direct chelation with RESCA, [ 18 F]TzTCO-BINb109 prepared indirectly by “click chemistry” has higher stability and binding affinity. • The bivalent nanobody probe targeting PD-L1, [ 18 F]TzTCO-BINb109, demonstrates selective accumulation in PD-L1-positive tumors in vivo. • Compared to the previously reported monomeric PD-L1-targeted nanobody probe [ 68 Ga]NOTA-Nb109, [ 18 F]TzTCO-BINb109 exhibits higher tumor uptake and prolonged tumor retention capabilities.