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-<b>1</b>), [¹⁷⁷Lu]Lu-DOTAGA-pIBA-BCN-azide-PEG₄-LLP2A ([¹⁷⁷Lu]Lu-<b>2</b>), and [¹⁷⁷Lu]Lu-DOTAGA-pIBA-DBCO-azide-PEG₄-LLP2A ([¹⁷⁷Lu]Lu-<b>3</b>). 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-<b>1</b>, [¹⁷⁷Lu]Lu-<b>2</b>, and [¹⁷⁷Lu]Lu-<b>3</b> to VLA-4 in B16F10 cells, with <i>K</i>_d = 1.2 ± 0.2, 0.8 ± 0.4, and 1.6 ± 0.5 nM, respectively. Biodistribution of [¹⁷⁷Lu]Lu-<b>1</b> 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-<b>2</b> peaked at 48 h (13.5 ± 2.2%IA/g) and gradually decreased (9.93 ± 3.3%IA/g at 96 h). [¹⁷⁷Lu]Lu-<b>3</b> 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-<b>1</b> and [¹⁷⁷Lu]Lu-<b>3</b>, [¹⁷⁷Lu]Lu-<b>2</b> cleared more rapidly from normal tissues. [¹⁷⁷Lu]Lu-<b>2</b> showed higher tumor-to-kidney ratios compared to [¹⁷⁷Lu]Lu-<b>1</b> at all time points and higher tumor-to-liver ratios up to 96 h. [¹⁷⁷Lu]Lu-<b>2</b> also showed higher tumor-to-liver ratios compared to [¹⁷⁷Lu]Lu-<b>3</b> up to 48 h. The tumor can be clearly visualized with all compounds using SPECT/CT. The BCN click linkage ([¹⁷⁷Lu]Lu-<b>2</b>) will be applied in future compounds with other targeting ligands, radionuclides, albumin binders, and chelators.