title: PET imaging of very late antigen-4 in melanoma: comparison of 68Ga- and 64Cu-labeled NODAGA and CB-TE1A1P-LLP2A conjugates.
abstract: Melanoma is a malignant tumor derived from epidermal melanocytes, and it is known for its aggressiveness, therapeutic resistance, and predisposition for late metastasis. Very late antigen-4 (VLA-4; also called integrin α4β1) is a transmembrane noncovalent heterodimer overexpressed in melanoma tumors that plays an important role in tumor growth, angiogenesis, and metastasis by promoting adhesion and migration of cancer cells. In this study, we evaluated 2 conjugates of a high-affinity VLA-4 peptidomimetic ligand, LLP2A, for PET/CT imaging in a subcutaneous and metastatic melanoma tumor.
  
  
  METHODS
  LLP2A was conjugated to 1,4,8,11-tetraazacyclotetradecane-1-(methane phosphonic acid)-8-(methane carboxylic acid) (CB-TE1A1P) and 2-(4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl)pentanedioic acid (NODAGA) chelators for (68)Ga and (64)Cu labeling. The conjugates were synthesized by solid-phase peptide synthesis, purified by reversed-phase high-performance liquid chromatography, and verified by liquid chromatography mass spectrometry. Saturation and competitive binding assays with B16F10 melanoma cells determined the affinity of the compounds for VLA-4. The biodistributions of the LLP2A conjugates were evaluated in murine B16F10 subcutaneous tumor-bearing C57BL/6 mice. Melanoma metastasis was induced by intracardiac injection of B16F10 cells. PET/CT imaging was performed at 2, 4, and 24 h after injection for the (64)Cu tracers and 1 h after injection for the (68)Ga tracer.
  
  
  RESULTS
  (64)Cu-labeled CB-TE1A1P-PEG4-LLP2A and NODAGA-PEG4-LLP2A showed high affinity to VLA-4, with a comparable dissociation constant (0.28 vs. 0.23 nM) and receptor concentration (296 vs. 243 fmol/mg). The tumor uptake at 2 h after injection was comparable for the 2 probes, but (64)Cu-CB-TE1A1P-PEG4-LLP2A trended toward higher uptake than (64)Cu-NODAGA-PEG4-LLP2A (16.9 ± 2.2 vs. 13.4 ± 1.7 percentage injected dose per gram, P = 0.07). Tumor-to-muscle and tumor-to-blood ratios from biodistribution and PET/CT images were significantly higher for (64)Cu-CB-TE1A1P-PEG4-LLP2A than (64)Cu-NODAGA-PEG4-LLP2A (all P values < 0.05). PET/CT imaging of metastatic melanoma with (68)Ga-NODAGA-PEG4-LLP2A and (64)Cu-NODAGA-PEG4-LLP2A showed high uptake of the probes at the site of metastasis, correlating with the bioluminescence imaging of the tumor.
  
  
  CONCLUSIONS
  These data demonstrate that (64)Cu-labeled CB-TE1A1P/NODAGA LLP2A conjugates and (68)Ga-labeled NODAGA-LLP2A are excellent imaging agents for melanoma and potentially other VLA-4-positive tumors. (64)Cu-CB-TE1A1P-PEG4-LLP2A had the most optimal tumor-to-nontarget tissue ratios for translation into humans as a PET imaging agent for melanoma.
authors.affiliation: Department of Radiology, University of Pittsburgh, Pittsburgh, Pennsylvania.
authors.forename: Wissam
authors.initials: W
authors.lastname: Beaino
authors.affiliation: Department of Radiology, University of Pittsburgh, Pittsburgh, Pennsylvania Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, Pennsylvania; and Department of Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania andersoncj@upmc.edu.
authors.forename: Carolyn J
authors.initials: CJ
authors.lastname: Anderson
mesh_terms: D000085:Acetates / Q000737:chemistry; D000818:Animals; D001667:Binding, Competitive; D002264:Carboxylic Acids / Q000737:chemistry; D002614:Chelating Agents / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D003301:Copper Radioisotopes / Q000737:chemistry*; D004151:Dipeptides / Q000737:chemistry; D005710:Gallium Radioisotopes / Q000737:chemistry*; D006573:Heterocyclic Compounds, 1-Ring / Q000737:chemistry; D006574:Heterocyclic Compounds, 2-Ring / Q000737:chemistry; D039041:Integrin alpha4beta1 / Q000378:metabolism*; D013058:Mass Spectrometry; D008545:Melanoma / Q000000981:diagnostic imaging* / Q000378:metabolism*; D008546:Melanoma, Experimental; D051379:Mice; D008810:Mice, Inbred C57BL; D009368:Neoplasm Transplantation; D063065:Organophosphonates / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D010671:Phenylurea Compounds / Q000737:chemistry; D049268:Positron-Emission Tomography; D011485:Protein Binding
chemical_list: C000595578:1,4,8,11-tetraazacyclotetradecane-1-(methanephosphonic acid)-8-(methane carboxylic acid); C572723:1-(1,3-carboxypropyl)-4,7-carboxymethyl-1,4,7-triazacyclononane; D000085:Acetates; D002264:Carboxylic Acids; D002614:Chelating Agents; D003301:Copper Radioisotopes; D004151:Dipeptides; D005710:Gallium Radioisotopes; D006573:Heterocyclic Compounds, 1-Ring; D006574:Heterocyclic Compounds, 2-Ring; D039041:Integrin alpha4beta1; C512274:LLP2A compound; D063065:Organophosphonates; D010455:Peptides; D010671:Phenylurea Compounds
keywords: LLP2A; PET; VLA-4; melanoma; metastasis