Effect of Click Chemistry Linkages on the Biological Behavior of Albumin-Binding ¹⁷⁷Lu-DOTAGA-pIBA-LLP2A Analogues Targeting Melanoma

The transmembrane integrin, very late antigen-4 (VLA-4), which is a critical integrin involved in promoting tumor progression, angiogenesis, and metastasis, is overexpressed in metastatic melanoma. The peptidomimetic LLP2A has a high binding affinity to VLA-4 and is used as a radiopharmaceutical targeting agent for imaging and therapy. Previous studies demonstrated that the albumin-binding compound, [177Lu]Lu-DOTAGA-pIBA-PEG4-LLP2A, significantly improved tumor retention and blood circulation time but resulted in lower tumor-to-nontumor tissue ratios compared to the nonalbumin-binding compound, [177Lu]Lu-DOTAGA-PEG4-LLP2A. To streamline the synthesis of VLA-4 targeting molecules as therapeutic agents and allow a modular approach, we investigated three click chemistry linkers for preparing DOTAGA-pIBA-PEG4-LLP2A analogues: [177Lu]Lu-DOTAGA-pIBA-TCO-tetrazine-PEG4-LLP2A ([177Lu]Lu-1), [177Lu]Lu-DOTAGA-pIBA-BCN-azide-PEG4-LLP2A ([177Lu]Lu-2), and [177Lu]Lu-DOTAGA-pIBA-DBCO-azide-PEG4-LLP2A ([177Lu]Lu-3). Determining the click linkage that provides optimal synthesis ease and pharmacokinetics will allow us to readily produce additional VLA-4 targeting radiopharmaceuticals. Saturation binding assays demonstrated high binding affinity of [177Lu]Lu-1, [177Lu]Lu-2, and [177Lu]Lu-3 to VLA-4 in B16F10 cells, with Kd = 1.2 ± 0.2, 0.8 ± 0.4, and 1.6 ± 0.5 nM, respectively. Biodistribution of [177Lu]Lu-1 showed peak tumor uptake at 24 h (12.2 ± 0.7%IA/g) and retention to 96 h (9.5 ± 1.7%IA/g), while [177Lu]Lu-2 peaked at 48 h (13.5 ± 2.2%IA/g) and gradually decreased (9.93 ± 3.3%IA/g at 96 h). [177Lu]Lu-3 peaked at 48 h (16.9 ± 3.9%IA/g) and was retained to 96 h (14.8 ± 3.8%IA/g). Compared with [177Lu]Lu-1 and [177Lu]Lu-3, [177Lu]Lu-2 cleared more rapidly from normal tissues. [177Lu]Lu-2 showed higher tumor-to-kidney ratios compared to [177Lu]Lu-1 at all time points and higher tumor-to-liver ratios up to 96 h. [177Lu]Lu-2 also showed higher tumor-to-liver ratios compared to [177Lu]Lu-3 up to 48 h. The tumor can be clearly visualized with all compounds using SPECT/CT. The BCN click linkage ([177Lu]Lu-2) will be applied in future compounds with other targeting ligands, radionuclides, albumin binders, and chelators.