Figure 1
Generation of a panel of targeted VEAs.
(A) Schematic representation of IL-2 and its deletion mutants which were fused to the C-terminal tail of the NHS76 heavy chain to create the VEA candidates being tested. The A, A′, B, C and D helices making up the secondary structure of IL-2 are represented by cylinders. The PEP region is colored red. Aspartic acid at residue 20 (Asp 20 or D20) is part of the “xDy motif” known to cause vascular leak7. Point mutations converting lysines to alanine (K8A, K9A) and cysteine to valine (C58V) improve product stability. (B) Purified PVLs were analyzed by SDS-PAGE on a 4–20% gradient gel. Samples were reduced with β-mercaptoethanol, heated to 95 °C for 5 minutes before being resolved into heavy and light chain bands and visualized with Coomassie stain. The heavy chain bands are shown here to highlight differences in migration patterns for each construct and their correlation to the estimated molecular weights in Table 1. The lanes marked Mol Wt contain proteins of known molecular weight and illustrate their migration pattern in this gradient gel. (C) Energy minimization analysis using SYBYL Dynamics software compares the expected PEP monomer structure in solution (green) with the homologous peptide fragment comprising the native IL-2 sequence (red) and finds the two structures nearly identical. (D) Overlay of the minimized structures of the PEP fragment, after simulated annealing from 700 K down to 0 K, illustrated with single green tubes representing each of the energy minimized PEP backbones after various annealing cycles. Note the overall similarity of the low energy conformations for PEP throughout the 50 simulated annealing cycles. (E) Interaction analysis of PEP fragment (light blue) with the IL-2 α receptor (blue) and IL-2 γ receptor (red) using SYBYL Biopolymer, Tripos Force Field and the Powell minimization algorithm. The separating surface (rendered by MOLCAD) between the PEP molecule and the individual receptor chains represent the interaction surface between molecules in contact with one another. The color gradient from red to pink represents the distance between interacting molecules with the closer proximity represented in deeper shades of red. Only a portion of the IL-2 α receptor is represented in the image.
