Phage displayed peptide libraries were used to identify amino acid sequences that bind specifically to irradiated cancers. The peptide ligand HVGGSSV bound within irradiated lung cancers. Co-precipitation of the HVGGSSV peptide revealed a putative receptor, taxinteracting protein-1 (TIP-1) that binds HVGGSSV. Membrane protein western blots showed a significant increase in the expression of TIP-1 protein at 4 and 24 hrs following irradiation with 3 Gy as compared to 0 Gy untreated control tumors (). Immunohistochemical staining showed significant levels of the TIP-1 membrane protein present in irradiated tumors, but not in untreated controls (). Near-infrared imaging studies showed significant targeting and binding of rabbit anti-TIP-1 IgG polyclonal antibody to irradiated tumors compared to untreated tumors and rabbit IgG controls at 72 hrs (). Further studies performed using guinea pig and mouse anti-TIP-1 IgG antibodies validated binding within irradiated tumors (). Results from near-infrared imaging studies showed that the radiance from anti-TIP-1 antibodies was 1.93 ± 2.04 photons/s/cm2/sr greater radiance compared to control IgG antibodies in irradiated tumors (p ≤ 0.08) ().
TIP-1 receptor targeting studies
The purpose of this study was to determine whether targeting nab-paclitaxel to radiation-inducible receptors improved tumor-specific drug delivery. To evaluate the tumor targeting ability of radiation-guided nab-paclitaxel, we studied in vivo near-infrared fluorescence imaging of the biodistribution in mice with heterotopic lung cancer tumors. We conjugated the HVGGSSV peptide to nab-paclitaxel via a bifunctional polyethylene glycol linker. The HVGGSSV peptide was then labeled with a fluorescent probe (Alexa fluor 750) for near-infrared fluorescence imaging studies. A scrambled sequence peptide (SGVSGHV) was used as a negative control. Nab-paclitaxel particles with no peptide conjugation were also fluorescently labeled and used as controls. Nude mice bearing subcutaneous Lewis lung carcinoma (LLC) tumors were treated with 3 Gy or 0 Gy radiation and administered HVGGSSV-nab-paclitaxel, SGVSGHV-nab-paclitaxel, or nab-paclitaxel. Images were acquired at 24, 48 and 72 hrs post-injection. Near-infrared images taken at 72 hrs after nab administration showed binding in irradiated tumors treated with HVGGSSV-nab-paclitaxel, whereas tumors treated with SGVSGHV-nab-paclitaxel or nab-paclitaxel alone showed minimal radiance (). Untreated (0 Gy) control tumors showed similar levels of radiance across all treatment groups (). Irradiated tumors treated with HVGGSSV-nab-paclitaxel showed 1.48 ± 1.66 photons/s/cm2/sr greater radiance compared to SGVSGHVnab-paclitaxel, and 1.49 ± 1.36 photons/s/cm2/sr greater than nab-paclitaxel alone (p<0.05) (). We found no significance difference in radiance from tumors treated with 2 Gy compared to 3 Gy during imaging of HVGGSSV-nab-paclitaxel (data not shown). To determine whether HVGGSSV binds to TIP-1 in tumor microvasculature, antibody blocking studies were performed. Near-infrared imaging showed that pre-blocking the TIP-1 receptor by administration of rabbit anti-TIP-1 IgG polyclonal antibody, followed by injection of HVGGSSV-nab-paclitaxel produced a marked decrease in radiance in irradiated tumors compared to control IgG antibody (). Blocking of TIP-1 resulted in a 47.6 fold drop in binding as compared to control IgG ().
HVGGSSV-nab-paclitaxel targeted to irradiated tumors
Targeting nab-paclitaxel to irradiated cancer improves biodistribution
To determine whether HVGGSSV-nab-paclitaxel binds within tumor microvasculature, we labeled HVGGSSV-nab-paclitaxel, SGVSGHV-nab-paclitaxel and nab-paclitaxel with fluorescent probe Alexa fluor 594 prior to injection. Approximately 3 hrs after treatment, mice were sacrificed, and tumors were excised and cryopreserved for fluorescence microscopy. Tissue slices were fluorescently stained for von Willebrand factor (vWF), an endothelial cell marker. shows strong colocalization of targeted HVGGSSV-nab-paclitaxel with vascular endothelium in irradiated tumors (), but not in unirradiated tumors () or TIP-1 blocked tumors. Scrambled SGVSGHV-nab-paclitaxel did not show significant colocalization in either irradiated or unirradiated tumors. Some colocalization was observed with nab-paclitaxel treatment in both irradiated and unirradiated groups. This suggests that HVGGSSV peptide enables binding of nab-paclitaxel within irradiated tumor microvasculature as early as 3 hrs after irradiation.
Colocalization HVGGSSV-nab-paclitaxel with tumor vascular endothelium
The ability of targeted HVGGSSV-nab-paclitaxel to localize and bind specifically to irradiated tumors was examined by biodistribution analysis in mice bearing LLC murine lung carcinoma. Paclitaxel levels were quantified in tumors, blood and organs 72 hrs following intravenous injection of either HVGGSSV-nab-paclitaxel, SGVSGHV-nab-paclitaxel or nab-paclitaxel. The results of this biodistribution analysis show that significantly higher levels of paclitaxel accumulated in irradiated tumors treated with HVGGSSV-nab-paclitaxel compared to SGVSGHV-nab-paclitaxel or nab-paclitaxel, and untreated control tumors (0 Gy) after 72 hrs. shows more than 5-fold increase in paclitaxel levels within irradiated tumors in HVGGSSV-nab-paclitaxel treated groups as compared to either nab-paclitaxel or SGVSGHV-nab-paclitaxel at 72 hrs. No significant difference in paclitaxel levels were observed in any unirradiated tumors. Biodistribution in organs showed similar paclitaxel levels distributed among heart, lungs, kidneys, liver and brain, with slightly less in the lungs and more in the brain. shows tumor to plasma ratios for HVGGSSV-nab-paclitaxel, SGVSGHV-nab-paclitaxel and nab-paclitaxel at 72 hours post-treatment. HVGGSSV nab-paclitaxel has a tumor/plasma ratio approximately 4-fold higher in irradiated tumors than unirradiated tumors, and 4-fold higher as compared to SGVSGHV-nab-paclitaxel. No significant difference was observed between irradiated and unirradiated SGVSGHV-nab-paclitaxel treated groups. A slight increase in tumor/plasma ratio was observed in nab-paclitaxel following tumor irradiation as compared to no irradiation. HVGGSSV-nab-paclitaxel had a tumor/plasma ratio approximately 3-fold higher than nab-paclitaxel in irradiated tumors.
Biodistribution of targeted HVGGSSV-nab-paclitaxel
shows immunohistochemical staining of paclitaxel in tumor tissues at 12 hrs post-treatment. Greater paclitaxel staining was observed in irradiated HVGGSSV-nab-paclitaxel treated tumors compared to unirradiated tumors, as well as SGVSGHV-nab-paclitaxel controls. Treatment with nab-paclitaxel resulted in similar paclitaxel staining between irradiated and unirradiated tumors. Irradiated tumors treated with HVGGSSV-nab-paclitaxel showed comparable levels of paclitaxel staining compared to nab-paclitaxel treated tumors. Targeting HVGGSSV-nab-paclitaxel to irradiated tumors increased the amount of paclitaxel delivered to tumors, and simultaneously decreased the amount of paclitaxel within other organs and tissues.
Targeted nab-paclitaxel enhances therapeutic efficacy
Therapeutic efficacy of HVGGSSV guided nab-paclitaxel was studied in two tumor models, murine Lewis lung carcinoma and NCI-H460 human large cell lung carcinoma grafted subcutaneously in C57 and athymic nude mice, respectively. On day 7 after tumor cell implantation, mice were injected with 10 mg/kg i.v. of either HVGGSSV-nab-paclitaxel, SGVSGHV-nab-paclitaxel, nab-paclitaxel, or saline. Tumor volumes for each treatment group were measured manually with calipers until they reached a 4-fold increase in volume. show radiation alone (9 Gy) achieved only a slight tumor growth delay (2 days) in LLC tumors, and 6 days in H460 tumors as compared to untreated controls. Negative SGVSGHV-nab-paclitaxel controls showed no significant growth delay in LLC tumors and only 2 days delay in H460. After subsequent irradiation, this was improved to 2 days (LLC) and 6 days (H460). Treatment with nab-paclitaxel alone produced tumor growth delay of 2 days (LLC) and 6 days (H460), and upon additional irradiation increased to 6 days (LLC) and 11 days (H460). Both LLC and H460 lung carcinoma showed significant tumor growth delay for HVGGSSV-nab-paclitaxel as compared to nab-paclitaxel, SGVSGHV-nab-paclitaxel and saline controls (). HVGGSSV-nab-paclitaxel treatment achieved a growth delay of 3.4 days (LLC) and 8 days (H460). Additional irradiation increased this to 10 days (LLC) and 15 days (H460) over untreated controls. Subsequent doses of radiation improved growth delay for both HVGGSSV-nab-paclitaxel and nab-paclitaxel control. HVGGSSV-nab-paclitaxel combined with irradiation resulted in significantly greater tumor growth delay compared to tumors treated with SGVSGHV-nab-paclitaxel and irradiation or nab-paclitaxel and irradiation (p<0.01, Kruskal-Wallis).
Therapeutic efficacy of targeted HVGGSSV-nab-paclitaxel in LLC and H460 xenografts
To evaluate the mechanism of cell death, tumor sections were immunostained for caspase-3 expression, an apoptosis marker. As shown in , HVGGSSV-nab-paclitaxel treatment induced significant apoptosis in the irradiated tumors but not in the unirradiated tumors. Treatment with nab-paclitaxel alone produced greater apoptosis in irradiated tumors compared to unirradiated tumors. Results for HVGGSSV-nab-paclitaxel and nab-paclitaxel were similar for both irradiated groups, as compared to SGVSGHV-nab-paclitaxel and negative controls. Tumor sections were stained for the endothelial cell marker, von Willebrand factor (vWF) to evaluate tumor vascularity. As shown in , HVGGSSV-nab-paclitaxel treatment induced a significantly greater loss in vasculature in irradiated tumors compared to unirradiated tumors, nab-paclitaxel, SGVSGHV-nab-paclitaxel, and untreated controls. Treatment with nab-paclitaxel decreased vascular density compared with negative controls, but did not show a significant decrease between irradiated and unirradiated tumors. There was no difference in the vessel density between the groups treated with SGVSGHV-nab-paclitaxel and untreated controls.