Very late antigen-4 (α(4)ß(1) Integrin) targeted PET imaging of multiple myeloma.

Biomedical imaging techniques such as skeletal survey and (18)F-fluorodeoxyglucose (FDG)/Positron Emission Tomography (PET) are frequently used to diagnose and stage multiple myeloma (MM) patients. However, skeletal survey has limited sensitivity as it can detect osteolytic lesions only after 30-50% cortical bone destruction, and FDG is a marker of cell metabolism that has limited sensitivity for intramedullary lesions in MM. Targeted, and non-invasive novel probes are needed to sensitively and selectively image the unique molecular signatures and cellular processes associated with MM. Very late antigen-4 (VLA-4; also called α(4)ß(1) integrin) is over-expressed on MM cells, and is one of the key mediators of myeloma cell adhesion to the bone marrow (BM) that promotes MM cell trafficking and drug resistance. Here we describe a proof-of-principle, novel molecular imaging strategy for MM tumors using a VLA-4 targeted PET radiopharmaceutical, (64)Cu-CB-TE1A1P-LLP2A. Cell uptake studies in a VLA-4-positive murine MM cell line, 5TGM1, demonstrated receptor specific uptake (P<0.0001, block vs. non-block). Tissue biodistribution at 2 h of (64)Cu-CB-TE1A1P-LLP2A in 5TGM1 tumor bearing syngeneic KaLwRij mice demonstrated high radiotracer uptake in the tumor (12±4.5%ID/g), and in the VLA-4 rich organs, spleen (8.8±1.0%ID/g) and marrow (11.6±2.0%ID/g). Small animal PET/CT imaging with (64)Cu-CB-TE1A1P-LLP2A demonstrated high uptake in the 5TGM1 tumors (SUV 6.6±1.1). There was a 3-fold reduction in the in vivo tumor uptake in the presence of blocking agent (2.3±0.4). Additionally, (64)Cu-CB-TE1A1P-LLP2A demonstrated high binding to the human MM cell line RPMI-8226 that was significantly reduced in the presence of the cold targeting agent. These results provide pre-clinical evidence that VLA-4-targeted imaging using (64)Cu-CB-TE1A1P-LLP2A is a novel approach to imaging MM tumors.

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

Integrin α(4)ß(1) (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 α(4)ß(1) integrin to a cross-bridged macrocyclic chelator with a methane phosphonic acid pendant arm, CB-TE1A1P. CB-TE1A1P-LLP2A was labeled with (64)Cu 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 (64)Cu-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 (64)Cu-CB-TE2A-LLP2A (B(max)=304 fmol/mg, p<0.03). In biodistribution studies, (64)Cu-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 to(64)Cu-CB-TE2A-LLP2A. At 2h post-injection, (64)Cu-CB-TE1A1P-LLP2A also had significantly higher tumor:blood and tumor:muscle ratios than (64)Cu-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 (64)Cu-CB-TE1A1P-LLP2A is an excellent PET radiopharmaceutical for the imaging of α(4)ß(1) positive tumors and also has potential for imaging other α(4)ß(1) positive cells such as those of the pre-metastatic niche.

Molecular imaging of very late antigen-4 (α4ß1 integrin) in the premetastatic niche.

UNLABELLED: Despite advances in cancer treatment over the past few decades, metastatic disease remains the primary cause of morbidity and mortality. Recent reports suggest the formation of a "premetastatic niche" before the metastatic cascade, where niche is defined as the microenvironment for tumor cells to be able to engraft and proliferate at secondary sites. Bone marrow-derived (BMD) cells that express vascular endothelial growth factor receptor-1 and very late antigen-4 (VLA-4) have been shown to arrive at sites of metastasis to form a receptive environment for tumor cells. Here we describe experiments toward imaging of VLA-4-positive BMD cells using a high-affinity PET probe, (64)Cu-labeled 11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2] hexadecane (CB-TE2A)-LLP2A. METHODS: VLA-4-negative MDA-MB-231/firefly luciferase (fluc) human breast tumor cells were injected intraarterially in the left ventricle in nude mice. Tumor metastasis in mice was monitored for 30 d by bioluminescence imaging and small-animal PET/CT. Small-animal PET images were collected 2 h after mice were injected in the tail vein with (64)Cu-CB-TE2A-LLP2A (5.6-11.1 MBq [150-300 µCi; specific activity, 400 µCi/µg]). Cellular uptake of (64)Cu-CB-TE2A-LLP2A was determined in VLA-4-positive B16F10 mouse melanoma cells and VLA-4-negative MDA-MB-231/fluc human breast cancer tumor cells. Biodistribution experiments in nude mice bearing VLA-4-positive B16F10 subcutaneous tumors in the flank were conducted to validate targeting of VLA-4-positive cells in vivo. RESULTS: Uptake of (64)Cu-CB-TE2A-LLP2A was higher in VLA-4-positive human melanoma B16F10 cells than in VLA-4-negative MDA-MB-231 cells (P < 0.05). In B16F10 tumor-bearing mice, (64)Cu-CB-TE2A-LLP2A had high uptake in the VLA-4-rich organs marrow, spleen, and tumor (11.26% ± 2.59%, 8.36% ± 2.15%, and 3.09% ± 0.58% injected dose/g, respectively). Cumulative standardized uptake value data from 2 independent studies (n = 7 and 8 mice) on nude mice implanted with VLA-4-negative MDA-MB-231/fluc human breast tumor cells suggested an influx of VLA-4-positive BMD cells that corresponded to metastasis (P < 0.05). Immunohistochemical analysis and flow cytometry also showed upregulation of VLA-4-positive cell clusters and BMD cells at the metastatic sites, providing evidence for noninvasive imaging of BMD cells in the premetastatic niche. CONCLUSION: The results of the study demonstrated the potential of PET with VLA-4-targeted (64)Cu-CB-TE2A-LLP2A to visualize BMD cell reorganization and expansion noninvasively in vivo.