VCN-01 disrupts pancreatic cancer stroma and exerts antitumor effects

Abstract

Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by dense desmoplastic stroma that limits the delivery of anticancer agents. VCN-01 is an oncolytic adenovirus designed to replicate in cancer cells with a dysfunctional RB1 pathway and express hyaluronidase. Here, we evaluated the mechanism of action of VCN-01 in preclinical models and in patients with pancreatic cancer.

Methods: VCN-01 replication and antitumor efficacy were evaluated alone and in combination with standard chemotherapy in immunodeficient and immunocompetent preclinical models using intravenous or intratumoral administration. Hyaluronidase activity was evaluated by histochemical staining and by measuring drug delivery into tumors. In a proof-of-concept clinical trial, VCN-01 was administered intratumorally to patients with PDAC at doses up to 1×10¹¹ viral particles in combination with chemotherapy. Hyaluronidase expression was measured in serum by an ELISA and its activity within tumors by endoscopic ultrasound elastography.

Results: VCN-01 replicated in PDAC models and exerted antitumor effects which were improved when combined with chemotherapy. Hyaluronidase expression by VCN-01 degraded tumor stroma and facilitated delivery of a variety of therapeutic agents such as chemotherapy and therapeutic antibodies. Clinically, treatment was generally well-tolerated and resulted in disease stabilization of injected lesions. VCN-01 was detected in blood as secondary peaks and in post-treatment tumor biopsies, indicating virus replication. Patients had increasing levels of hyaluronidase in sera over time and decreased tumor stiffness, suggesting stromal disruption.

Conclusions: VCN-01 is an oncolytic adenovirus with direct antitumor effects and stromal disruption capabilities, representing a new therapeutic agent for cancers with dense stroma.

Trial registration number: EudraCT number: 2012-005556-42 and NCT02045589.

Keywords: gastrointestinal neoplasms; immunity; investigational; oncolytic viruses; therapies; tumor microenvironment.

Figure 1

VCN-01 sensitizes tumors to chemotherapy leading to enhanced antitumor activity. (A) Antitumor activity against subcutaneous HP-1 Syrian hamster pancreatic tumors, implanted in immunocompetent hamsters, was evaluated after three intratumoral administrations of 2.5×10¹⁰ vp of VCN-01 per tumor alone (days 0, 9 and 18) or in combination with intraperitoneal (IP) G for seven doses (50 mg/kg, days 0, 3, 6, 9, 12, 15 and 18). n=8–12 tumors/group. (B) Antitumor activity against subcutaneous human NP-9 pancreatic tumors, implanted in immunodeficient mice, was evaluated after single intravenous administration of 4×10¹⁰ viral particles (VP) of VCN-01 alone (day 0) or in combination with IP gemcitabine (G) (50 mg/kg, days 0, 3, 6 and 9). n=7–13 tumors/group. (C) Viral E1A expression and hyaluronic acid (HA) degradation indicative of viral replication as determined by immunohistochemistry on serial tumor sections of VCN-01 treated tumors of (B) on day 79. (D) Antitumor activity against subcutaneous human pancreatic NP-18 tumors, implanted in immunodeficient mice, was evaluated after single intravenous administration of VCN-01 (4×10¹⁰vp/animal, day 0) with and without IP G with nab-paclitaxel plus gemcitabine (GA) (G 50 mg/kg and a 41.6 mg/kg, days 0, 7 and 14). n=10–12 tumors/group. (E) Tumor volumes for each tumor treated with VCN-01 +GA. For A, B, D, a parametric t-student test was used to determine statistical significance of the different treatments at each time point. (F) Antitumor activity against the orthotopic human TP-11 pancreatic model, implanted in immunodeficient mice, after single intravenous administration of VCN-01 (4×10¹⁰ vp/animal, day 0) with and without IP GA (a 41.6 mg/kg & g 50 mg/kg, days 0, 7 and 14) and measured tumor volume at the experiment end (day 28). n=6–13 tumors/group. Mean±SEM is plotted (black lines). One-way ANOVA (Tukey’s multiple comparisons test) was used to determine statistical significance of the different treatments. ANOVA, analysis of variance; GA, gemcitabine with albumin.

Figure 2

VCN-01 enhances extravasation of chemotherapy and monoclonal antibodies into tumors. (A) Single intravenous administration of VCN-01 alone or in combination with chemotherapy significantly increases tumor-uptake of gemcitabine (G) in an orthotopic pancreatic model, TP-11. Animals from the different experimental groups described in figure 1F were administered G IP at a dose of 50 mg/kg 1 hour before sacrifice. The intratumoral content of G in each tumor was measured by high-performance liquid chromatography (HPLC). Box and whiskers plots (mean is shown as +). n=6–13 tumors/group. One-way ANOVA (Tukey’s multiple comparisons test) was employed to determine statistical significance of the different treatments. (B) fluorescence in vivo imaging after intravenous administration of labeled anti-PD-L1 antibody (10 mg/kg). VCN-01 or the hyaluronidase negative virus ICOVIR15K was administered intravenous at a dose of 4×10¹⁰ vp/animal to NP-18 tumor-bearing immunodeficient mice on day 0. Labeled anti-PD-L1 (Vivotag 800) was administered intravenous at a dose of 10 mg/kg on day 14 and imaging performed after 48 hours. Upper panel: representative images from fluorescence in tumors show enhanced extravasation of labeled anti-PD-L1 in VCN-01 treated tumors compared with the ICOVIR15K group. Lower panel: quantification of average radiant efficiency at 800 nm in tumor areas is represented in the box and whiskers plot (mean is shown as +). n=6–8 tumors/group. One-way ANOVA (Tukey’s multiple comparisons test) was employed to determine statistical significance of the different treatments. (C) tumor growth curves of the TRAMPC-2 cancer model treated with 1×10¹⁰vp/tumor of VCN-01 or phosphate-buffered saline administered intratumorally alone or in combination with five intraperitoneal doses of 200 µg of anti-PD-L1 antibody (days 0, 3, 6, 8 and 10). n=8–9 tumors/group. A two-way ANOVA, followed by a Tukey’s multiple comparisons test, was employed to determine statistical significance of the different treatments for each day. ANOVA, analysis of variance; GA, gemcitabine with albumin.

Figure 3

VCN-01 pharmacokinetics in mice and intratumoral viral replication. (A) Pharmacokinetics and peaks of replication of VCN-01 in blood after single intratumoral administration of 5×10⁸ vp in athymic mice with the human pancreatic tumor NP-18 implanted subcutaneous (SC). Blood samples (20 µL) were obtained at the indicated time points postadministration and viral genomes were measured by qPCR. n=6–7 mice/time point. (B) Left panel: VCN-01 blood levels after administration of a single intravenous dose of 5×10¹⁰ vp/animal to immunodeficient mice bearing the SC human pancreatic NP-18 tumor. Blood samples (20 µL) were obtained at 2 and 15 min and at 1, 4, 24, and 48 hours. Post dose and genomes were measured by qPCR. Right panel: VCN-01 viral genomes in blood of immunodeficient mice with the SC NP-18 human pancreatic model after intravenous administration of 4×10¹⁰ vp/animal of VCN-01 on day 0. Blood samples (20 µL) were collected on days 2, 10 and 21. Results are expressed as viral genomes per mL of total blood. n=6–7 mice/time point. (C) Viral genomes in tumors and livers were analyzed at the indicated time points after one time intravenous administration of VCN-01 (4×10¹⁰ vp/animal) to mice with orthotopically implanted TP-11 human pancreatic tumor model. Animals were sacrificed on day 2 and 28 and VCN-01 genome measured by qPCR. n=6–8 tumors or livers/group/day. A Mann-Whitney U test was employed to analyze statistically significant differences (*p<0.05 vs D2).

Figure 4

Antitumor activity of Intratumor Injections of VCN-01 in patients. Percentage variation in the size of tumor lesions injected with VCN-01 compared with baseline in patients treated with VCN-01 in combination with either gemcitabine (G) or nab-paclitaxel plus gemcitabine (GA). Ech blue line represents the monitored tumor of each patient (eight patients).

Figure 5

VCN-01 is detected in patient blood over time showing viral replication peaks and in tumors 20–28 days after treatment. (A) Blood levels of VCN-01 at the indicated time points as determined by qPCR. Each black line represents a patient (six patients) and the mean of all patients treated with 1×10¹¹ vp + nab-paclitaxel plus gemcitabine (GA) per time point is shown in gray. (B) VCN-01 is detected in all tumors analyzed on days 20–28. Fine-needle aspiration biopsy of the tumor performed at the indicated times (pre-VCN-01 or post-treatment) on each patient and analyzed by qPCR to detect the number of viral genomes (six patients). Each tumor sample is represented by a symbol, performed either on day 0 (negative) or between day 20 and 28 post-VCN-01 it administration. G, gemcitabine.

Figure 6

Hyaluronidase PH20 expression and stroma disruption effects of VCN-01 in patients. (A) PH20 serum levels as determined by ELISA at the indicated time points in patients treated with VCN-01 in combination with nab-paclitaxel plus gemcitabine (GA). Each filled circle represents a serum sample per patient. Analyzed patients were injected at a dose of 1×10¹¹ vp of VCN-01 (six patients). One-way ANOVA (Tukey’s multiple comparisons test) was used to evaluate multiple comparisons between groups. PH20 expression in serum samples at different time points is expressed as pg/mL minus background levels detected for each sample at day 0 and represented in box and whiskers graphs. (B) Changes in strain ratio measured by endoscopic ultrasound (EUS) elastography in five patients prior to cycles 1, 2 and 3. Each line represents an individual patient. ANOVA, analysis of variance.