64Cu-1,4,7-Triazacyclononane-1,4,7-triacetic acid-p-isothiocyanatobenzyl-ALT-836

64Cu-NOTA-ALT-836

Leung K.

Publication Details

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Table

In vitro Rodents

Background

[PubMed]

Tissue factor (TF) is a transmembrane receptor present in subendothelial tissue, platelets, and leukocytes (1). This factor binds and localizes the coagulation factor VII to the cell surface to initiate the coagulation cascade. TF is normally expressed only in adventitial fibroblasts and vascular smooth muscle cells surrounding blood vessels, providing a hemostatic barrier that activates coagulation when the tissue is injured (2). TF can be newly synthesized upon endotoxin and cytokine stimulation of endothelial cells and leukocytes. The expression of TF is upregulated in thrombosis, tumor growth, metastasis, and angiogenesis (3, 4). There is an aberrant expression of TF in pancreatic cancer, breast cancer, glioma, leukemia, and lung cancer, with strong association of several hemostatic complications (5). ALT-836 (formerly known as Sunol-cH36) is a human/mouse chimeric monoclonal antibody (mAb) that blocks the factor X/factor IX (FX/FIX) binding site of TF and inhibits the coagulation cascade (6). This mAb has been evaluated in clinical trials for treatments of acute lung injury/acute respiratory distress syndrome (7). Hong et al. (8) evaluated 64Cu-1,4,7-triazacyclononane-1,4,7-triacetic acid-p-isothiocyanatobenzyl-ALT-836 (64Cu-NOTA-p-Bn-SCN-ALT-836, or 64Cu-NOTA-ALT-836) for positron emission tomography (PET) imaging of TF expression in nude mice bearing human pancreatic tumors with specific accumulation.

Synthesis

[PubMed]

64Cu-NOTA-ALT-836 was prepared by conjugation of p-SCN-Bn-NOTA to ALT-836 in 25:1 molar ratio (8). 64Cu-NOTA-ALT-836 was purified with column chromatography. 64Cu-NOTA-ALT-836 (0.165 nmol) was radiolabeled using 37 MBq (1 mCi) 64CuCl2 in sodium acetate buffer (pH 5.0) for 30 min at 37°C. 64Cu-NOTA-ALT-836 was purified with PD-10 column chromatography. Total preparation time of the tracer was 70 ± 10 min (n = 6). The radiochemical yield was 79.6 ± 5.4%. The radiochemical purity was >95%. The specific activity of 64Cu-NOTA-ALT-836 was reported to be 180 MBq/nmol (4.86 mCi/nmol). The number of NOTA moieties per mAb conjugate was not reported.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

64Cu-NOTA-ALT-836 exhibited immunoreactivity similar to the unconjugated mAb as determined with binding to BXPC-3 (high TF expression) and ASPC-1 (moderate TF expression) pancreatic cancer cells with flow cytometry analysis (8). Little binding was observed in PANC-1 (TF-negative) pancreatic cells. The binding levels of both mAbs to the three cell lines correlated with TF expression levels in these cells (BXPC-3 > ASPC-1 > PNAC-1).

Animal Studies

Rodents

[PubMed]

PET scans were performed after intravenous injection of 5–10 MBq (135–270 μCi) 64Cu-NOTA-ALT-836 in nude mice (n = 4/group) bearing BXPC-3, ASPC-1, or PNAC-1 xenografts at 4, 24, and 48 h after injection (8). High radioactivity accumulation was evident in the TF-positive BXPC-3 and ASPC-1 tumors, with low accumulation in the TF-negative PNAC-1 tumors at 4, 24, and 48 h. The radioactivity levels in the BXPC-3 tumors increased from 5.7 ± 1.8% injected dose/gram (ID/g) at 4 h to 10.4 ± 0.8% ID/g at 24 h and 16.5 ± 2.6% ID/g at 48 h, whereas the radioactivity levels in the PNAC-1 tumors were 1.7 ± 0.8% ID/g, 2.8 ± 0.7% ID/g, and 2.2 ± 0.6% ID/g at 4, 24 and 48 h, respectively. Accumulation in the ASPC-1 tumors was lower than that in the BXPC-3 tumors but higher than that in the PNAC-1 tumors at all times (P < 0.05). The liver accumulation of radioactivity was 18.2 ± 1.8% ID/g, 16.7 ± 1.4% ID/g, and 10.2 ± 0.9% ID/g at 4, 24 and 48 h, respectively. The blood radioactivity was 10.6 ± 1.3% ID/g, 6.8 ± 1.5% ID/g, and 3.5 ± 0.7% ID/g at 4, 24 and 48 h, respectively. Overall, tracer accumulation in all major organs was similar among the three tumor groups. A blocking experiment was performed with mice bearing BXPC-3 tumors by administration of ALT-836 2 h before the tracer injection. Tumor accumulation was inhibited by ~80% at 4, 24 and 48 h (P < 0.05), whereas accumulation in all major organs and blood was similar between the blocking and non-blocking groups. Ex vivo biodistribution studies were performed at 48 h and showed that the radioactivity distribution patterns were similar with the PET imaging data. All major organs (such as the liver, spleen, kidney, lung, heart, and pancreas) exhibited lower radioactivity than that of the BXPC-3 tumors. Immunofluorescence staining of tumor sections confirmed the strong expression of TF in the BXPC-3 and ASPC-1 tumor cells, with weak expression in the PANC-1 tumor cells. TF staining was absent in the liver and spleen.

Other Non-Primate Mammals

[PubMed]

No publication is currently available.

Non-Human Primates

[PubMed]

No publication is currently available.

Human Studies

[PubMed]

No publication is currently available.

NIH Support

9U54TR000021

References

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Hong H., Zhang Y., Nayak T.R., Engle J.W., Wong H.C., Liu B., Barnhart T.E., Cai W. Immuno-PET of Tissue Factor in Pancreatic Cancer. J Nucl Med. 2012;53(11):1748–54. [PMC free article: PMC3489969] [PubMed: 22988057]