Introduction 1 2 3 4 5 6 7 8 9 11 11 15 16 17 The approach presented in this paper differs to the above-discussed strategies in several aspects. First, we now equipped TRAIL with targeting ligands directed to angiogenic endothelium rather than to tumor cells, allowing an enhanced binding to the tumor blood vessels. The vascular wall is in direct contact with the systemic circulation, which makes it an easily attainable target for therapeutic proteins. Furthermore, intratumoral pressure, which hampers the penetration of macromolecules into tumor tissue will not affect the homing to of RGD-equipped complexes to the angiogenic endothelium. v 3 18 19 1 v 3 Fig. 1 v 3  Materials and methods Preparation and characterization of RGDPEG-avidin Avidin was modified with heterobifunctional PEG groups that can react with primary amino groups in avidin, after which RGD-peptide groups can be conjugated to their distal ends. The hydrophilic PEG groups of 3.4 kDa will furthermore reduce non-specific protein interactions. N ε-S 20 21 Expression and purification of recombinant human His-tagged TRAIL Nde BamH Escherichia coli 22 g 4 Preparation and characterization of bio-NHS-TRAIL and bio-ULS-TRAIL ™ His-TRAIL in storage buffer was extensively dialyzed against PBS containing 10% glycerol at 4°C using Slide-A-Lyzer dialysis cassettes (10,000 MWCO, Pierce) to remove DTT and other components of the storage buffer that can interfere in the reactions. Typically, the purified His-TRAIL (500 ug, 7.6 nmol) at 0.7 mg/ml was mixed either with Sulfo-NHS biotin (Pierce) (10 mg/ml in DMF) or with biotin-ULS (10 mg/ml in 20 mM NaCl), after which the mixtures were incubated for 4 h at 37°C. Unreacted biotinylation reagent was removed by dialysis against PBS at 4°C. The products were sterilized by filtration via disposable 0.2 μm filters and stored at −20°C. Biotin-TRAIL conjugates were characterized for protein content using BCA protein assay (Pierce). The relative number of coupled biotin molecules was determined by anti-biotin ELISA. Wells were coated with serial dilutions of the biotinylated proteins for 1 h at room temperature, washed with PBS containing 0.05% Tween20 and incubated for 1 h at room temperature with streptavidin-peroxidase complex (Dako, 1:2500 in PBS), followed by a standard incubation with OPD. The protein concentration at which 50% of the maximum absorbance was measured (EC50) was calculated by nonlinear regression (Graphpad Prism), and used to calculate relative biotin:protein ratios in each conjugate. The grade of biotinylation was furthermore assayed by MALDI-TOF analysis as described above for RGDPEG-avidin. Cells l l 23 2 l Binding of RGDPEG-avidin to HUVEC v 3 125 v 3  24 125 2 2 2 Complexation of biotinylated TRAIL with RGDPEG-avidin We studied the complexation of biotinylated TRAIL with RGDPEG-avidin in an ELISA-like experimental setup. Wells were coated with 50 ng of avidin or RGDPEG-avidin overnight at room temperature, blocked with BSA (1% in PBS) and incubated with 10 ng of biotinylated TRAIL for 2 h at room temperature. After the wells were washed with PBS/0.05% Tween20, the amount of TRAIL associated with the avidin was detected by anti-TRAIL immunodetection (anti-TRAIL antibody 2E5, Alexis, Breda, The Netherlands) for 1 h, followed by standard detection with GARPO/OPD. Apoptotic activity of biotinylated TRAIL The apoptosis inducing activity of the biotinylated products was evaluated on Jurkat T cells and on endothelial cells, using a cell viability assay and a caspase activity assay. Experiments were conducted with the single cell types, Jurkat tumor cells or HUVEC, or with a combination of tumor and endothelial cells. Assessment of TRAIL activity by MTS cell viability assay 4 4 Assessment of TRAIL activity by caspase 3/7 assay We investigated the induction of apoptosis in Jurkat T cells using a caspase 3/7 activity assay (Promega) employing luminescence detection. Different experimental settings were evaluated either reflecting direct incubation of the products with tumor cells, or settings in which biotinylated TRAIL was complexed to RGDPEG-avidin and/or anchored onto endothelial cells. 3 v 3 3 Statistical analysis t P Results Preparation and characterization of RGDPEG-avidin 2 2 2 Fig. 2 a b  v 3 v 3 v 3 125 125 3 3 50 50 Fig. 3 v 3 P  Preparation and characterization of biotinylated TRAIL 4 4 1 Fig. 4 Characterization of biotinylated TRAILs. Panel A: Anti-biotin ELISA with streptavidin-HRP. Signals were corrected for background and expressed relative to the maximum detected intensity. Panel B: MALDI-TOF analysis of TRAIL and biotin-NHS-TRAIL  Table 1 Characteristics of biotinylated TRAIL variants  a 50 b c TRAIL – 40 100 Bio-NHS-TRAIL 1.6 123 35 Bio-ULS-TRAIL 0.8 60 126 a b c 5 Fig. 5 Association of biotinylated TRAIL with RGDPEG-avidin. 96-well plates were coated overnight with 50 ng RGDPEG-avidin and incubated with indicated compounds for 2 h. TRAIL binding was assessed by anti-TRAIL immunodetection and expressed relative to the highest signal obtained with biotin-NHS-TRAIL. Experiments were performed in triplicate  Cytotoxic and proapoptotic activity of TRAIL complexes 6 50  1 6 6 Fig. 6 TRAIL activity assays. Panel A: Cell viability of Jurkat T cells after 48 h of incubation with indicated compounds. Cell viability was assessed by MTS assay. Experiments were performed in triplicate. Panel B: Caspase 3/7 activity in Jurkat T cells after 4 h of incubation with 100 ng/ml of the indicated TRAIL derivatives. RLU: relative light units. Panel C: Cell viability (MTS assay) of HUVEC after 48 h of incubation with 100 ng/ml of indicated compounds  v 3 7 6 Fig. 7 P P  A striking result was observed when the excess of RGDPEG-avidin was removed before addition of TRAIL and Jurkat cells, leaving only endothelial-associated RGDPEG-avidin available for complexation with biotinylated TRAIL. This procedure significantly potentiated the proapoptotic effect for both Bio-NHS-TRAIL and Bio-ULS-TRAIL, the latter even beyond the activity of unmodified TRAIL. Discussion In the present study we describe the development and functional evaluation of RGD-avidin:TRAIL complexes. These targeted complexes of TRAIL showed enhanced binding to endothelial cells and maintained their pro-apoptotic activity. As a result of the binding to angiogenic endothelial cells of these complexes, an improved tumor accumulation and enhanced residence within tumor tissue can be expected. Such an improvement in the pharmacokinetic profile of TRAIL may further enhance its therapeutic efficacy and enable a reduction in dosing frequency. v 3 v 3 d v 25 18 26 v 3 27 28 7 29 10 30 31 7 32 18 v 3 v 3 18 v 3 To summarize, we have prepared a novel type of RGD-targeted TRAIL with binding specificity for angiogenic endothelium and maintenance of strong cytotoxic activity. Whether in vivo application of this product  will lead to an enhanced tumor accumulation still needs to be investigated. We now succeeded in coupling the well known homing device RGD to the TRAIL molecule, without losing the apoptosis-inducing activity of TRAIL or the receptor-binding properties of RDG peptides. An enhanced activity could even be demonstrated in vitro. This may, in combination with an improved pharmacokinetic profile induced by the PEG and RGD moieties, lead to improved effects of TRAIL in vivo. Lastly, the followed synthetic approach offers opportunities for the complexation with alternative targeting ligands that bind directly to tumor cells, such as tumor-specific antibodies or peptide ligands.