Comparison of two cross-bridged macrocyclic chelators for the evaluation of 64Cu-labeled-LLP2A, a peptidomimetic ligand targeting VLA-4-positive tumors

Integrin α4β1 (also called very late antigen-4 or VLA-4) plays an important role in tumor growth, angiogenesis and metastasis, and there has been increasing interest in targeting this receptor for cancer imaging and therapy. In this study, we conjugated a peptidomimetic ligand known to have good binding affinity for α4β1 integrin to a cross-bridged macrocyclic chelator with a methane phosphonic acid pendant arm, CB-TE1A1P. CB-TE1A1P-LLP2A was labeled with 64Cu under mild conditions in high specific activity, in contrast to conjugates based on the “gold standard” di-acid cross-bridged chelator, CB-TE2A, which require high temperatures for efficient radiolabeling. Saturation binding assays demonstrated that 64Cu-CB-TE1A1P-LLP2A had comparable binding affinity (1.2 nM vs 1.6 nM) but more binding sites (Bmax = 471 fmol/mg) in B16F10 melanoma tumor cells than 64Cu-CB-TE2A-LLP2A (Bmax = 304 fmol/mg, p < 0.03). In biodistribution studies, 64Cu-CB-TE1A1P-LLP2A had less renal retention but higher uptake in tumor (11.4 ± 2.3 %ID/g versus 3.1 ± 0.6 %ID/g, p < 0.001) and other receptor-rich tissues compared to64Cu-CB-TE2A-LLP2A. At 2 h post-injection, 64Cu-CB-TE1A1P-LLP2A also had significantly higher tumor:blood and tumor:muscle ratios than 64Cu-CB-TE2A-LLP2A (CB-TE1A1P = 19.5 ± 3.0 and 13.0 ± 1.4, respectively, CB-TE2A = 4.2 ± 1.4 and 5.5 ± 0.9, respectively, p < 0.001). These data demonstrate that 64Cu-CB-TE1A1P-LLP2A is an excellent PET radiopharmaceutical for the imaging of α4β1 positive tumors and also has potential for imaging other α4β1 positive cells such as those of the pre-metastatic niche.