Abstract 576: Discovery of FL801, a B7-H3-targeting nanobody-based radioligand vector, using Clear-XTM linker technology
Authors: Xiaozhu Wang, Ming-Jin Jheng, Pengyun Li, Dian Li, Yihu Xie, Biao Guo, Ruyi Hu, Fa Liu
Journal: Cancer Research (2025)

Abstract B7-H3, also known as CD276, is a type I transmembrane protein and a member of the B7 family, which includes immune checkpoints such as PD-L1. B7-H3 is over-expressed in multiple solid tumors including lung, esophageal, prostate and breast cancers. B7-H3 antibody-drug conjugates (ADCs) have demonstrated promising early clinical antitumor activity. In contrast to ADCs, radionuclide drug conjugates (RDCs) offer not only a different therapeutic approach but also allow tumor imaging. VHH (variable heavy domain of heavy chain) is a highly attractive binder modality, however, one persistent challenge of VHH-based RDCs is the low tumor uptake together with a high kidney uptake prohibiting its application in RDC therapy. Our Clear-X™ linker technology was developed to specifically address such therapeutically sub-optional biodistribution profiles that are nearly without exception shared by all VHHs and other similar-sized protein-based vectors such as affibodies and DARPins. Using Clear-X™, we developed our lead B7-H3 radioligand vector FL801. The binding affinity of FL801 was assessed in vitro. In the cell-based binding assays, FL801 exhibited specific B7-H3 binding with sub-nanomolar affinity. The radiolabeling of FL801 with 177Lu resulted in radiochemical purities (RCP) of &amp;gt;98% and molar activities of 18.5 MBq/nmol. [177Lu]Lu-FL801 was found to be stable in the formulation buffers at 4°C for 5 days. The in vivo biodistribution profile of [177Lu]Lu-FL801 was characterized by SPECT/CT imaging and cut-and-count biodistribution studies in B7-H3-positive tumor-bearing mice. The SPECT/CT imaging of 11.1 MBq [177Lu]Lu-FL801 revealed an intense tumor uptake that was significantly higher than in all normal organs at 24 and 72 hour. In the cut-and-count biodistribution studies, [177Lu]Lu-FL801 demonstrated a therapeutically favorable profile including a tumor uptake of &amp;gt;30% ID/g and a tumor-to-kidney (T/K) ratio of &amp;gt;2.0 at 24 hour. This was a significant improvement over our starting-point VHH lead without Clear-X™ modification which exhibited a tumor uptake of 2.3% ID/g and a T/K ratio of &amp;lt;0.01 at 24 hour. In addition, the anti-tumor activity of [177Lu]Lu-FL801was evaluated in the same model using a single dose of 11.1 MBq [177Lu]Lu-FL801 per mice. It was found that the treatment was well tolerated, and all treated animals achieved a complete remission of the tumor. In summary, the preclinical data package collectively supports to proceed towards clinical studies of [177Lu]Lu-FL801 as a potential therapeutic option for B7-H3 positive tumors. In addition, this work validated the effectiveness of Clear-X™ linker technology in optimizing VHH-RDCs, and we expect Clear-X™ is also applicable to other protein-based RDCs with similar molecular sizes. Citation Format: Xiaozhu Wang, Ming-Jin Jheng, Pengyun Li, Dian Li, Yihu Xie, Biao Guo, Ruyi Hu, Fa Liu. Discovery of FL801, a B7-H3-targeting nanobody-based radioligand vector, using Clear-XTM linker technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 576.