Comparison of a Series of ⁶⁸Ga-Labeled DOTA-LLP2A Conjugates for Positron Emission Tomography Imaging of Very Late Antigen-4 in Melanoma

Abstract

Melanoma, with its steadily rising global incidence, is characterized by high invasiveness, leading to poor prognosis in advanced stages. There remains an unmet clinical need for the development of radiolabeled PET imaging probes for the early diagnosis of melanoma. Integrin VLA-4, a key factor in melanoma metastasis, presents a promising protein target to address the specificity shortcomings of existing probes in melanoma imaging. This study evaluates 68 Ga-labeled DOTA-LLP2A PET probes for melanoma imaging by modifying different carboxyl sites and employing various polyethylene glycol (PEG) linkers based on the structure of the high-affinity ligand LLP2A for VLA-4. The ligand intermediates LLP2A-NH 2 and LLP2A(tBu)-OH, as well as their conjugates (probe precursors), were synthesized via solid-phase synthesis. The specificity and cytotoxicity of the probes were assessed in VLA-4-positive B16F10 cells and VLA-4-negative A375 cells. Targeting efficacy of the probes in B16F10 and A375 xenograft models was compared through PET imaging and biodistribution studies. VLA-4 expression in tissues was evaluated via immunofluorescence, while H&E staining was employed to assess the safety profile of the probes. The probe ([ 68 Ga]Ga-T-CH) modified at the Aminocyclohexane carboxylic acid (Ach) exhibited greater signal accumulation in B16F10 melanoma (3.90 ± 0.43% ID/g at 1 h) compared to the 2-aminoadipic acid (Aad) side-chain-modified probe ([ 68 Ga]Ga-T-AD) (1.43 ± 0.23%ID/g at 1 h). PET images of the three PEG conjugates derived from the Ach demonstrated bright tumor signals and low background noise, showing a progressive increase in tumor signal intensity from [ 68 Ga]Ga-T6 to [ 68 Ga]Ga-T4 and [ 68 Ga]Ga-T2. Tumor uptake, tumor-to-muscle ratio, and tumor-to-blood ratio from biodistribution were significantly higher for [ 68 Ga]Ga-T2 than for [ 68 Ga]Ga-T4 and [ 68 Ga]Ga-T6 (tumor: 3.58 ± 0.28 vs 2.90 ± 0.16 vs 1.87 ± 0.22%ID/g at 1 h; tumor/muscle: 13.38 ± 0.43 vs 10.62 ± 0.70 vs 7.19 ± 1.15 at 1 h; tumor/blood: 8.64 ± 1.12 vs 5.32 ± 0.91 vs 4.36 ± 0.59 at 1 h; P < 0.05). These data suggest that the series of PEG derivatives [ 68 Ga]Ga-T2, [ 68 Ga]Ga-T4, and [ 68 Ga]Ga-T6, linked at the Ach site, are excellent 68 Ga-labeled probes for melanoma and other potential VLA-4-positive tumors. Among them, [ 68 Ga]Ga-T2 shows the highest tumor-to-background contrast for melanoma, positioning it as the most promising candidate for clinical translation.