5 for 20 seconds. As control, PBS alone or a mixture of 100 nM ECDHER2 and 1 μM hDM-αH-C6.5 MH3B1 incubated at 25°C for 30 minutes was injected on the surface. Binding of ECDHER2 to immobilized hDM-αH-C6.5 BKM120 MH3B1 was monitored in real time by following the association and dissociation phases
on the experimental surface with control surface subtracted. Binding parameters were determined using the 1:1 binding model by BIAevalution 3.0 software. Flow cytometry analysis of hDM-αH-C6.5 MH3B1 binding to HER2/neu expressing cells CT26, CT26HER2/neu or MCF-7HER2 cells (5 × 105 cells/sample) were incubated with either biotinylated or Alexa-fluor labeled hDM-αH-C6.5 MH3B1 for 30 minutes on ice and then washed twice with FACS buffer [PBS pH 6.8 with 1% calf-serum]. If biotinylated hDM-αH-C6.5 MH3B1 was used, cells were then stained for thirty minutes with PE-labeled streptavidin at final concentration of 0.3 μg/ml (BD Bioscience; Franklin Lakes, NJ), LEE011 research buy and washed twice. Fluorescence was measured on a cytofluorometer (FACSCalibur; BD Bioscience) and the mean fluorescence was analyzed using the Flowjo software (Treestar, Ashland, OR). Biotin (catalog
number: 21336; DNA Damage inhibitor PIERCE; Rockford, IL) or Alexa-fluor (catalog number: A10235; Invitrogen) conjugation of hDM-αH-C6.5 MH3B1 was carried out according to the manufacturer’s recommendation. Results Construction and purification of hDM-αH-C6.5 MH3B1 We have previously shown that hPNP with the two mutations Glu201Gln:Asn243Asp, unlike wild-type hPNP, converts a relatively non-toxic prodrug, F-dAdo to the cytotoxic drug F-Ade [5]. With the goal of being able to target hDM to
the tumor site, we fused it at its C-terminus to a human anti-HER2/neu single chain Fv (C6.5 MH3-B1) [7] through a rigid α-helical linker [10, 11] (Fig. 1A). C6.5 MH3B1 has been reported to bind to HER2/neu with high affinity and specificity [7]. The Progesterone available crystal structure of hPNP [12–14] suggested that fusing C6.5 MH3B1 to the C-terminus of the enzyme would have minimal affect on its enzymatic activity, since the C-terminus is distal from the enzyme active site. The rigid α-helical linker [11, 12], instead of a flexible GlySer linker was used to restrict the flexibility of the fusion protein. The plasmid encoding the hDM-αH-C6 MH3B1 was transiently expressed in 293T cells, the supernatant harvested and the protein purified by passage through an affinity column composed of ECDHER2 conjugated to Sepharose beads. The eluted protein was 99% pure as judged by Coomasie blue staining with 300 μg of protein obtained from 150 ml of culture supernatant (Fig. 1B). Analysis of the protein by size exclusion chromatography indicated that the fusion protein mainly existed as a 180 kDa homotrimer (Fig. 1C) of 60 kDa subunits. Figure 1 Schematic presentation and purity ofhDM-αH-C6 MH3B1. (A), Schematic diagram of hDM-αH-C6 MH3B1. Each monomer of hDM is shown as filled oval.