Site-specific 64Cu-Labelling of Azido-modified Active Site Inhibited Factor Seven (ASIS-N3)

Objectives TF (Tissue Factor) is implicated in cancer progression, angiogenesis and metastasis via TF/FVIIa (activated Factor VII)-mediated activation of PARs (protease-activated receptors). TF is therefore a potential target for assessment of cancer aggressiveness.[1] FVIIa is a 50 kDa protein with an active site which can be inhibited using the small peptide D-Phe-L-Phe-L-Arginyl chloromethyl ketone (FFR-ck).[2] Active Site Inhibited Factor VIIa (ASIS) exhibits five times higher affinity towards TF than Factor VIIa and does not possess pro-coagulant activity.[3] We have recently randomly labelled the lysines of ASIS with [18F]SFB.[4,5] The aim of the current study was to enable site-specific labelling of ASIS in a single well-defined position. For this purpose an azide-modified inhibitor was synthesized and used to inhibit Factor VIIa to form ASIS-N3. This azide could be used as a selective handle for radiolabelling, for instance after reaction with ADIBO-PEG4-NOTA via Strain Promoted Azide-Alkyne Cycloaddition (SPAAC).

Methods 2-Azidoacetic acid (2) was produced as previously described.[6] The inhibitor N3-FFRck was produced by reacting azide (2) with FFRck. FVIIa was inhibited with N3-FFRck in gly-gly buffer at room temperature for 19 hours, producing ASIS-N3. Inhibition of FVIIa was quantified by a S-2288 colorimetric proteolytic assay. ASIS-N3 was reacted with ADIBO-PEG4-NOTA in an SPAAC reaction, yielding ASIS-PEG4-NOTA. 64Cu was chelated to ASIS-PEG4-NOTA, producing site specific labelled ASIS (1) (Figure 1). The corresponding product was evaluated by HPLC, SDS-PAGE and protein precipitation. Binding to soluble TF (sTF) and anti-FVIIa monoclonal antibody F1A2 was evaluated in a pull-down assay.

Results Inhibition of FVIIa to ASIS-N3 was 99.4% complete, assessed by the S-2288 assay. Up to 370 MBq 64Cu-ASIS (1) was produced in this way, with 67.6 ± 3.4% radiochemical yield. The purity was 98.6 ± 1.1% by HPLC and 97.2 ± 1.5% by protein precipitation. Binding to sTF and anti-FVIIa-antibody was 87.7% and 100% respectively.

Conclusions ASIS-N3 was produced to give a specific and site selective handle for further modifications. As an example a NOTA chelator could be introduced via SPAAC as a specific site for radiolabelling. After radiolabelling the binding to TF was intact. Thus ASIS-N3 can be used as a building block for specific radiolabelling of this 50 kDa protein with potential for in vivo TF imaging. Acknowledgements: Supported by the Innovation Fund Denmark

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