Abstract 4923: Gallium-68- and Copper-64-labeled LLP2A conjugates for PET-CT imaging of integrin α4β1 in melanoma
Authors: Beaino Wissam , Anderson Carolyn J. 
Published: May 2015

Abstract
Introduction:
Melanoma is a malignant tumor derived from epidermal melanocytes, and it is known for its therapeutic resistance, aggressive clinical behavior, and predisposition for late metastasis. Integrin α4β1 is a transmembrane non-covalent heterodimer overexpressed in melanoma tumors that plays an important role in tumor growth, angiogenesis and metastasis by promoting adhesion and migration of cancer cells. There has been increasing interest in targeting this receptor for cancer imaging and therapy. In this study we evaluated three conjugates of a peptidomimetic ligand, LLP2A known to have high binding affinity for α4β1 for PET-CT imaging and potential radiotherapy.
Methods:
LLP2A was conjugated to three different chelators, CB-TE1A1P, NODAGA and DOTA, for 68Ga and 64Cu labeling. The conjugates were synthesized by solid phase peptide synthesis, purified by RP-HPLC and verified by LC-MS mass spectrometry. 68Ga and 64Cu labeling was done in acetate buffer pH 4 for 68Ga and pH 6.5 for 64Cu at 70°C for 20 min. High Specific activity (1 mCi/µg) and radiopurity (&gt;98%) were achieved. Saturation binding and competitive binding assays with B16F10 melanoma cells determined the binding affinity of the compounds. The biodistributions of the LLP2A conjugates were evaluated in B16F10 subcutaneous tumor bearing C57BL/6 mice. PET-CT imaging was performed at 2, 4 and 24 h post-injection for the 64Cu tracers and 1h post-injection for 68Ga tracer.
Results:
The competitive binding assay indicated that Ga-NODAGA-PEG4-LLP2A had higher affinity (IC50 = 0.68, Ki = 0.11 nM) compared to Ga-DOTA-PEG4-LLP2A (IC50 = 9.37, Ki = 1.56 nM); however, biodistribution showed similar tumor uptake for 68Ga-NODAGA-PEG4-LLP2A and 68Ga-DOTA-PEG4-LLP2A (8.7 ± 1.3 %ID/g and 9.1 ± 0.9 %ID/g respectively) but less renal and liver retention for the DOTA conjugate. Both 64Cu-labeled CB-TE1A1P-PEG4-LLP2A and NODAGA-PEG4-LLP2A showed high affinity to α4β1 integrin with a comparable Kd (0.28 nM vs 0.23 nM) and Bmax (296 fmol/mg vs 243 fmol/mg). The tumor uptake at 2 h post-injection was comparable for the two probes but 64Cu-CB-TE1A1P-PEG4-LLP2A had higher uptake compared to 64Cu-NODAGA-PEG4-LLP2A (16.9 ± 2.2 %ID/g vs13.4 ± 1.7 %ID/g). Tumor to muscle and tumor to blood ratios from biodistribution and PET-CT images were significantly higher for 64Cu-CB-TE1A1P-PEG4-LLP2A compared to 64Cu-NODAGA-PEG4-LLP2A. In addition, liver and kidney uptake was 2-fold lower for 64Cu-CB-TE1A1P-PEG4-LLP2A.
Conclusion:
These data demonstrate that 68Ga-labeled DOTA, NODAGA and 64Cu-labeled CB-TE1A1P, NODAGA LLP2A conjugates are excellent imaging agents for melanoma or other α4β1-positive tumors, with 64Cu-CB-TE1A1P-PEG4-LLP2A being the best. These data also suggest the potential for radiotherapy and radiotheranostics using radioisotopes like Lu-177, Y-90 and Cu-67.
Citation Format: Wissam Beaino, Carolyn J. Anderson. Gallium-68- and Copper-64-labeled LLP2A conjugates for PET-CT imaging of integrin α4β1 in melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4923. doi:10.1158/1538-7445.AM2014-4923