Very late antigen-4 (VLA-4) is a transmembrane integrin protein that is highly expressed in aggressive forms of metastatic melanoma. A small-molecule peptidomimetic, LLP2A, was found to have a low pM affinity binding to VLA-4. Because LLP2A itself does not inhibit cancer cell proliferation and survival, it is an ideal candidate for the imaging and delivery of therapeutic payloads. An analog of [177Lu]Lu-labeled-LLP2A was previously investigated as a therapeutic agent in melanoma tumor-bearing mice, resulting in only a modest improvement in tumor growth inhibition, likely due to rapid clearance of the agent from the tumor. To improve the pharmacokinetic profile, DOTAGA-PEG4-LLP2A with a 4-(p-iodophenyl)butyric acid (pIBA) albumin binding moiety was synthesized. We demonstrate the feasibility of this albumin binding strategy by comparing in vitro cell binding assays and in vivo biodistribution performance of [177Lu]Lu-DOTAGA-PEG4-LLP2A ([177Lu]Lu-1) to the albumin binding [177Lu]Lu-DOTAGA-pIBA-PEG4-LLP2A ([177Lu]Lu-2). In vitro cell binding assay results for [177Lu]Lu-1 and [177Lu]Lu-2 showed Kd values of 0.40 ± 0.07 and 1.75 ± 0.40 nM, with similar Bmax values of 200 ± 6 and 315 ± 15 fmol/mg, respectively. In vivo biodistribution data for both tracers exhibited specific uptake in the tumor, spleen, thymus, and bone due to endogenous expression of VLA-4. Compound [177Lu]Lu-2 exhibited a much longer blood circulation time compared to [177Lu]Lu-1. The tumor uptake for [177Lu]Lu-1 was highest at 1 h (∼15%ID/g) and that for [177Lu]Lu-2 was highest at 4 h (∼23%ID/g). Significant clearance of [177Lu]Lu-1 from the tumor occurs at 24 h (<5%ID/g) while[177Lu]Lu-2 is retained for greater than 96 h (∼10%ID/g). An efficacy study showed that melanoma tumor-bearing mice receiving compound [177Lu]Lu-2 given in two fractions (2 × 14.8 MBq, 14 days apart) had a greater median survival time than mice administered a single 29.6 MBq dose of compound [177Lu]Lu-1, while a single 29.6 MBq dose of [177Lu]Lu-2 imparted hematopoietic toxicity. The in vitro and in vivo data show addition of pIBA to [177Lu]Lu-DOTAGA-PEG4-LLP2A slows blood clearance for a higher tumor uptake, and there is potential of [177Lu]Lu-2 as a theranostic in fractionated administered doses.
Keywords: VLA-4; albumin binding; theranostics.