PET imaging of Very Late Antigen-4 (VLA-4) in mouse models of multiple myeloma

Objectives The bone marrow microenvironment (BMM) plays a key role in the pathogenesis of multiple myeloma (MM). The integrin VLA-4 (CD49d/CD29) is present on MM cells and mediates adhesion to the BM stroma through the CS-1 region of fibronectin and VCAM-1 ligands. We investigate the MM cell-BMM interaction by targeting the VLA-4/VCAM-1 axis using a VLA-4 targeted, high affinity PET imaging probe, ⁶⁴Cu-CB-TE1A1P-LLP2A.

Methods The peptidomimetic LLP2A, with pM affinity for VLA-4, was conjugated with a methylphosphonate-based cross-bridged chelator (CB-TE1A1P) for labeling with ⁶⁴Cu at room temperature. Cellular uptake data were obtained using nM amounts of ⁶⁴Cu-CB-TE1A1P-LLP2A in RPMI 8226-luc-GFP, U266-luc-GFP, B16 (+ control) and MDA-MB-231 (- control) cells. Mouse models of human MM cells, RPMI 8226-luc-GFP and U266-luc-GFP (n=5 each), were used to evaluate the targeting specificity of ⁶⁴Cu-CB-TE1A1P-LLP2A. Tumor cells were implanted intratibially (IT) in bg-nu-Xid mice, and the tumor cells metastasized to spleen and bone. Additionally, the effect of sub-lethal irradiation on the tumor spread was evaluated in two separate cohorts of mice (n=5 each). All mice were imaged by small animal PET/CT and ⁶⁴Cu-CB-TE1A1P-LLP2A (35µCi; 23ng/mouse, SA: 1.5mCi/µg) during 3 weeks post-tumor cell injection. Biodistribution data were collected post PET/CT imaging.

Results PET/CT imaging showed high tracer uptake in the legs and spleen, the areas of maximum tumor burden. Cumulative SUV data from these tissues demonstrated an increased uptake of ⁶⁴Cu-CB-TE1A1P-LLP2A compared to control mice (p<.001). Irradiated mice showed higher uptake than non-irradiated. Low uptake in the clearance organs provided high tumor:background contrast which included uptake in malignant lymph nodes. MM cell uptake data validated specificity and receptor mediated endocytosis (p<0.001).

Conclusions ⁶⁴Cu-CB-TE1A1P-LLP2A can effectively image VLA-4 on MM cells and may be highly effective in monitoring therapy