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 (B_max=471 fmol/mg) in B16F10 melanoma tumor cells than ⁶⁴Cu-CB-TE2A-LLP2A (B_max=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 2h 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.