Conditional regulation of protein stability in vivo. (a) SCID mice bearing HCT116 L106P-tsLuc xenografts (50-100 mm³) were either untreated (top) or treated ip with Shield-1 (10 mg/kg, bottom) and bioluminescent signals were imaged over time. (b) Quantification of tumor signals from panel A. (c) Mice as in a were treated ip with Shield-1 at 3 mg/kg (diamonds), 6 mg/kg (inverted triangles), or 10 mg/kg (triangles) and imaged over time. (d) Mice bearing HCT116-tsLuc xenografts were either untreated (squares) or treated with Shield-1 (10 mg/kg, triangles) every 48 hr and imaged over time. Data for panels b-d are presented as the average bioluminescence detected within regions of interest drawn around the tumors ± SEM (n=4 to 10).

Systemic, targeted-delivery of a conditionally stabilized protein. (a) HCT116 cells were infected with vvDD L106P-tsLuc and then mock treated (squares) or treated with Shield-1 at 1 μM (triangles), 100 nM (inverted triangles), or 10 nM (diamonds). Data are represented as the average luminescence ± SEM (n=3). (b) SCID mice bearing subcutaneous HCT116 xenografts (50-100 mm³) received a single tail vein injection of vvDD L106P-tsLuc (1×10⁸ PFU/mouse). After 72 hr, mice were either untreated (C, control) or treated with Shield-1 (10 mg/kg, #1-4). Bioluminescent signals were imaged over time. (c) Quantification of bioluminescent signal produced from regions of interest drawn around tumors as in panel b. Data presented are the average bioluminescence ± SEM (n=4).

Conditional regulation of a secreted immunomodulatory protein leads to a reduction of tumor burden in vivo. (a) HCT116 L-L106P-IL-2 cells were treated with various concentrations of Shield-1 and culture media was assayed for the presence of IL-2. Data are represented as the average IL-2 concentration ± SEM (n=3). (b) CD1 nu-/nu- mice bearing subcutaneous HCT116 L-L106P-IL-2 tumors were either untreated (diamonds) or treated ip with Shield-1 at 5 mg/kg (triangles) or 10 mg/kg (inverted triangles) every 48 hr beginning 5 days post-transplantation (arrow). Alternatively, mice received HCT116 L-L106P-IL-2 cells that had been pre-treated with 1 μM Shield-1 for 24 hr, and were then treated with Shield-1 (10 mg/kg) every 48 hr beginning on day 0 (squares). Tumor volume was determined by caliper measurement and monitored over time. Data are represented as the average tumor volume ± SEM (n=5). At Day 16, all Shield-1 treated groups displayed significantly reduced tumor burden relative to controls (p=0.0019 for 10 mg/kg; p=0.0002 for 5 mg/kg and p=0.0046 for pre-treat group). (c) Tumors from mice treated with Shield-1 post-transplantation were collected 48 hr after the start of Shield-1 treatment. Tumors were weighed ex vivo and then homogenized, and the concentration of IL-2 per gram tumor tissue was determined by ELISA (n=4). Tumors from mice treated with Shield-1 at 10 mg/kg contained significantly higher levels of IL-2 than tumors from mice treated at 5 mg/kg (p=0.032), which in turn produced more IL-2 than tumors from untreated mice (p=0.0028). (d) Mice treated with Shield-1 post-transplantation were bled 48 hr after the start of Shield-1 treatment, and the concentration of IL-2 in the serum was determined by ELISA. Shield-1 treatment at 10 mg/kg (n=7) produced significantly higher levels of serum IL-2 relative to control mice (n=6, p=0.0004), whereas treatment at 5 mg/kg (n=6) did not produce any increase in serum IL-2 levels.

Antitumor benefit of conditional regulation of a targeted gene therapy vector. CD1 nu-/nu- mice bearing subcutaneous HCT116 tumors (150-250 mm³) were treated via a single tail vein injection with either PBS (circles) or the vaccinia strain vvDD expressing both luciferase as well as the L-L106P-TNF-α fusion protein (1 × 10⁸ PFU/mouse). Treated mice also received Shield-1 (10 mg/kg) every 48 hr by three different protocols, starting (i) 24 hr prior to vvDD therapy (diamonds); (ii) 72 hr post-vvDD therapy (squares) or (iii) not at all (triangles) (n=8 mice/group). Tumor-bearing mice that did not receive vvDD therapy were also treated with Shield-1 (10 mg/kg) as a negative control (inverted triangles, n=5). (a) Viral load within the tumor was assayed by measuring constitutive viral gene expression (bioluminescence) for each group at the indicated time points after vvDD treatment. Shield-1 treatment starting 72 hr after vvDD (vvDD + Shield-1) resulted in significantly greater levels of viral gene expression in the tumor than when Shield-1 treatment was started prior to vvDD therapy (vvDD Pre-Shield-1; i.e., constitutive TNF-α expression) (p=0.035 at 2 days; p=0.035 at 4 days and p=0.002 at 7 days). (b) Tumor volume was determined by caliper measurement and mice were sacrificed once tumor volume reached 1.44 cm³. Kaplan-Meier survival graphs are shown, and all surviving mice at 50 days had no detectable tumor. Mice treated with Shield-1 prior to vvDD therapy (i.e., constitutive TNF-α expression) produced significantly enhanced survival relative to mice treated with vvDD alone (p=0.017). Addition of Shield-1 72 hr post-vvDD (vvDD + Shield-1) produced a further significant increase in survival relative to mice treated with Shield-1 prior to vvDD (vvDD pre-Shield-1) (p=0.031).