PET Imaging of Very Late Antigen-4 in Melanoma: Comparison of⁶⁸Ga- and⁶⁴Cu-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 68Ga and 64Cu 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 64Cu tracers and 1 h after injection for the 68Ga tracer. Results 64Cu-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 64Cu-CB-TE1A1P-PEG4-LLP2A trended toward higher uptake than 64Cu-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 64Cu-CB-TE1A1P-PEG4-LLP2A than 64Cu-NODAGA-PEG4-LLP2A (all P values < 0.05). PET/CT imaging of metastatic melanoma with 68Ga-NODAGA-PEG4-LLP2A and 64Cu-NODAGA-PEG4-LLP2A showed high uptake of the probes at the site of metastasis, correlating with the bioluminescence imaging of the tumor. Conclusion These data demonstrate that 64Cu-labeled CB-TE1A1P/NODAGA LLP2A conjugates and 68Ga-labeled NODAGA-LLP2A are excellent imaging agents for melanoma and potentially other VLA-4–positive tumors. 64Cu-CB-TE1A1P-PEG4-LLP2A had the most optimal tumor–to–nontarget tissue ratios for translation into humans as a PET imaging agent for melanoma.

Ex Vivo and In Vivo Evaluation of Overexpressed VLA-4 in Multiple Myeloma Using LLP2A Imaging Agents

“…CB-TE1A1P-PEG 4 -LLP2A was synthesized and radiolabeled as described previously (13). The supplemental data provide details (supplemental materials are available at http://jnm.snmjournals.org).…”

“…We previously demonstrated the proof-of-principle molecular imaging of VLA-4 in murine 5TGM1 myeloma subcutaneous tumors using the PET radiopharmaceutical 64 Cu-(CB-TE1A1P)-LLP2A (12). Recently Beaino et al demonstrated imaging of VLA-4–positive melanoma mouse models using 64 Cu-CB-TE1A1P-PEG 4 -LLP2A ( 64 Cu-LLP2A) (13). 64 Cu-LLP2A possesses ideal pharmacokinetic features such as optimal bioavailability (by virtue of added PEG chains), enhanced solubility, and high 64 Cu specific activity (~37 MBq/μg).…”

Withaferin A inhibits in vivo growth of breast cancer cells accelerated by Notch2 knockdown

“…Fasted mice were imaged on an Inveon Small Animal Multimodal PET/CT scanner 1 hour following intravenous (lateral tail vein) administration of 7.4 ± 0.74 MBq (200 ± 20 µCi; 100 µL) [ 18 F]-2-fluorodeoxyglucose (FDG; IBA Pharmaceuticals, Dulles, VA) and analyzed as described previously [24]. …”

New Bifunctional Chelator p-SCN-PhPr-NE3TA for Copper-64: Synthesis, Peptidomimetic Conjugation, Radiolabeling, and Evaluation for PET Imaging

“…11,12 Harsh labeling conditions such as high temperature and long labeling time were usually required for CB-TE2A chelators to get decent labeling yields, limiting their applications in labeling temperature-sensitive biomolecules such as antibodies, although the phosphonate-based cross-bridged chelators such as CB-TE1A1P can be labeled at lower temperatures and have demonstrated success in both antibody and peptide labeling. 13–16 There remains a need for a diverse array of BFCs, particularly those that can be readily synthesized, labeled with radiometals at low temperatures and show specficity for a particular radiometal. Derivatives of the polyaminocarboxylate NOTA were revisited, because of their convenient radiolabeling of 64 Cu and good in vivo stability.…”