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Random conjugation of therapeutic or diagnostic payloads to targeting proteins generates functionally heterogeneous products. Conjugation of payloads to an adapter that binds to a peptide tag engineered into a targeting protein provides an alternative strategy. To progress into clinical development, an adapter/docking tag system should include humanized components and be stable in circulation. We describe here an adapter/docking tag system based on mutated fragments of human RNase I that spontaneously bind to each other and form a conjugate with a disulfide bond between complimentary cysteine residues. This self-assembled "dock and lock" system utilizes the previously described fusion C-tag, a 1-15 aa fragment of human RNase I with the R4C amino acid substitution, and a newly engineered adapter protein (Ad-C), a 21-127-aa fragment of human RNase I with the V118C substitution. Two vastly different C-tagged recombinant proteins, human vascular endothelial growth factor (VEGF) and a 254-aa long N-terminal fragment of anthrax lethal factor (LFn), retain functional activities after spontaneous conjugation of Ad-C to N-terminal or C-terminal C-tag, respectively. Ad-C modified with pegylated phospolipid and inserted into the lipid membrane of drug-loaded liposomes (Doxil) retained the ability to conjugate C-tagged proteins, yielding targeted liposomes decorated with functionally active proteins. To further optimize the system, we engineered an adapter with an additional cysteine residue at position 88 for site-specific modification, conjugated it to C-tagged VEGF, and labeled with a near-infrared fluorescent dye Cy5.5, yielding a unique functionally active probe for in vivo molecular imaging. We expect that this self-assembled "dock and lock" system will provide new opportunities for using functionally active proteins for biomedical purposes.

We describe a new generation of protein-targeted contrast agents for multimodal imaging of the cell-surface receptors for vascular endothelial growth factor (VEGF). These receptors have a key role in angiogenesis and are important targets for drug development. Our probes are based on a single-chain recombinant VEGF expressed with a cysteine-containing tag that allows site-specific labeling with contrast agents for near-infrared fluorescence imaging, single-photon emission computed tomography or positron emission tomography. These probes retain VEGF activities in vitro and undergo selective and highly specific focal uptake into the vasculature of tumors and surrounding host tissue in vivo. The fluorescence contrast agent shows long-term persistence and co-localizes with endothelial cell markers, indicating that internalization is mediated by the receptors. We expect that multimodal imaging of VEGF receptors with these probes will be useful for clinical diagnosis and therapeutic monitoring, and will help to accelerate the development of new angiogenesis-directed drugs and treatments. [ABSTRACT FROM AUTHOR]

Purpose: We recently developed a cysteine-containing peptide tag (C-tag) that allows for site-specific modification of C-tag-containing fusion proteins with a bifunctional chelator, HYNIC (hydrazine nicotinamide)-maleimide. We then constructed and expressed C-tagged vascular endothelial growth factor (VEGF) and labeled it with HYNIC. We wished to test 99mTc-HYNIC-C-tagged VEGF (99mTc-HYNIC-VEGF) for the imaging of tumor vasculature before and after antiangiogenic (low continuous dosing, metronomic) and tumoricidal (high-dose) cyclophosphamide treatment. Methods: HYNIC-maleimide was reacted with the two thiol groups of C-tagged VEGF without any effect on biologic activity in vitro. 99mTc-HYNIC-VEGF was prepared using tin/tricine as an exchange reagent, and injected via the tail vein (200.300 µCi, 1.2 µg protein) followed by microSPECT imaging 1 h later. Results: Sequencing analysis of HYNIC-containing peptides obtained after digestion confirmed the site-specific labeling of the two accessible thiol groups of C-tagged VEGF. Tumor vascularity was easily visualized with 99mTc/VEGF in Balb/c mice with 4T1 murine mammary carcinoma 10 days after implantation into the left axillary fat pad in controls (12.3±5.0 tumor/bkg, n=27) along with its decrease following treatment with high (150 mg/kg q.o. d. ×4; 1.14±0.48 tumor/bkg, n=9) or low (25 mg/kg q.d. × 7; 1.03±0.18 tumor/bkg, n=9) dose cyclophosphamide. Binding specificity was confirmed by observing a 75% decrease in tumor uptake of 99mTc/biotin-inactivated VEGF, as compared with 99mTc-HYNIC-VEGF. Conclusion: 99mTc can be loaded onto C-tagged VEGF in a site-specific fashion without reducing its bioactivity. 99mTc-HYNIC-VEGF can be rapidly prepared for the imaging of tumor vasculature and its response to different types of chemotherapy. [ABSTRACT FROM AUTHOR]

Abstract: Developing tissue engineering scaffolds with immobilized growth factors requires facile and reliable methods for the covalent attachment of functionally active proteins. We describe here a new approach to immobilize recombinant proteins based on expression of the protein of interest with a 15-aa long fusion tag (Cys-tag), which avails a free sulfhydryl group for site-specific conjugation. To validate this approach, we conjugated a single-chain vascular endothelial growth factor expressed with an N-terminal Cys-tag (scVEGF) to fibronectin (FN) using a common thiol-directed bi-functional cross-linking agent. We found that the FN-scVEGF conjugate retains VEGF activity similar to that of free scVEGF when used as a soluble ligand. Cells expressing VEGF receptor VEGFR-2 grown on plates coated with FN-scVEGF displayed morphological phenotypes similar to those observed for cells grown on FN in the presence of equivalent amounts of free scVEGF. In addition, 293/KDR cell growth stimulation was observed in the same concentration range with either immobilized or free scVEGF. The effects of immobilized scVEGF, and soluble scVEGF were blocked by NVP-AAD777-NX, a VEGF receptor tyrosine kinase inhibitor. These data indicate that site-specific immobilization via Cys-tag provides a facile and reliable method for permanent deposition of functionally active growth factors on synthetic or protein scaffolds with applications for advanced tissue engineering. [Copyright &y& Elsevier]