Brain tumor eradication and prolonged survival from intratumoral conversion of 5-fluorocytosine to 5-fluorouracil using a nonlytic retroviral replicating vector

Abstract

Patients with the most common and aggressive form of high-grade glioma, glioblastoma multiforme, have poor prognosis and few treatment options. In 2 immunocompetent mouse brain tumor models (CT26-BALB/c and Tu-2449-B6C3F1), we showed that a nonlytic retroviral replicating vector (Toca 511) stably delivers an optimized cytosine deaminase prodrug activating gene to the tumor lesion and leads to long-term survival after treatment with 5-fluorocytosine (5-FC). Survival benefit is dose dependent for both vector and 5-FC, and as few as 4 cycles of 5-FC dosing after Toca 511 therapy provides significant survival advantage. In the virally permissive CT26-BALB/c model, spread of Toca 511 to other tissues, particularly lymphoid tissues, is detectable by polymerase chain reaction (PCR) over a wide range of levels. In the Tu-2449-B6C3F1 model, Toca 511 PCR signal in nontumor tissues is much lower, spread is not always observed, and when observed, is mainly detected in lymphoid tissues at low levels. The difference in vector genome spread correlates with a more effective antiviral restriction element, APOBEC3, present in the B6C3F1 mice. Despite these differences, neither strain showed signs of treatment-related toxicity. These data support the concept that, in immunocompetent animals, a replicating retroviral vector carrying a prodrug activating gene (Toca 511) can spread through a tumor mass, leading to selective elimination of the tumor after prodrug administration, without local or systemic pathology. This concept is under investigation in an ongoing phase I/II clinical trial of Toca 511 in combination with 5-FC in patients with recurrent high-grade glioma (www.clinicaltrials.gov NCT01156584).

Figures

Fig. 1.

Comparison of CT26 and Tu-2449 in vitro susceptibility to Toca 511 (511) or MLV expressing GFP (MLV-GFP) vectors. Sensitivity to 5-FC was determined by measuring cell viability (MTS assay) over time in the absence or presence of titrating amounts of 5-FC (A). IC50 values were calculated for CT26 and Tu-2449 cell lines at 4.2 μM (0.5 μg/mL) and 1.5 μM (0.19 μg/mL), respectively. Flow cytometry analysis was done to measure GFP positive cells transduced by MLV-GFP at an MOI of 1.0 or 0.1 (B).

Fig. 2.

CT26 in BALB/c brain metastasis tumor model survival analysis. Groups of 10 female BALB/c mice (9 weeks of age) were implanted IC with 1E4 CT26 tumor cells then dosed IC with vehicle (Control) or IC with Toca 511 at 3 dose levels expressed as transducing units per gram of brain (TU/g) ranging from a dose level of E4 to a dose level of E6 TU/g. Following 9 days, to allow spread of Toca 511, mice were treated IP BID for 7 consecutive days with either PBS or 5-FC (500 mg/kg) as indicated. The cycle of 7 days on drug followed by 10 days off drug was repeated until the conclusion of the study. Survival analysis up to day 95 was performed. DNA and RNA PCR analysis to detect integrated vector in various tissues and viral particles in sera were performed on the mice terminated at day 95.

Fig. 3.

Tu-2449 in B6C3F1 glioma model survival analysis. Groups of 10 female B6C3F1 mice (8 weeks of age) were implanted IC with 1E4 Tu-2449 tumor cells then dosed IC with vehicle (Control) or IC with Toca 511. Two separate experiments were performed with doses ranging from E3 to a dose level of E6 TU/g (A and B). Following 7 days, to allow spread of Toca 511, mice were treated IP BID for 4 consecutive days with either PBS or 5-FC 500 mg/kg or 50 mg/kg (low) as indicated. This cycle of 4 days on drug followed by 10 days off drug was repeated until the conclusion of the study. Survival analysis up to day 180 (A) or day 100 (B) was performed on each group.

Fig. 4.

Tissue profiles of qPCR positivity from tumor-bearing murine studies after IC administration of Toca 511. Copies/μg of MLV DNA detected per tissue (triplicate measures per mouse sample, averaged for cohorts of n = 10) are presented. Breaks in the X-axis represent the distribution of low (<15 000 copies/μg), medium (15 000–600 000 copies/μg) and high (>600 000 copies/μg). (A) Day 90 tissue positivity profile of tumor bearing BALB/c mice. (B) Day 180 tissue positivity profile of tumor bearing B6C3F1.

Fig. 5.

5-FC to 5-FU conversion analysis (A), CD expression (B), and vector copy number (C). Six groups of female B6C3F1 mice (16 mice, 8 weeks of age) were implanted IC with Tu-2449 glioma tumor cells then injected IC with vehicle (Group 2) or IC with Toca 511 (E6 TU, Groups 1 and 3–6). On day 20, when mice started losing weight, they were treated IP or by oral gavage (OG) BID for 2 consecutive days with either PBS (Group 1) or 5-FC (500 mg/kg, Groups 3 and 6; or 250 mg/kg, Groups 2, 4, and 5). After 2 days of 5-FC dosing the mice were given 1 final 5-FC dose 1 h before sacrifice. Tu-2449 tumors were surgically isolated from the brains for HPLC processing (A) Select tumors (B) were further trimmed for Western blot analysis (tumors greater than 0.05 g). RIPA lysis supernatants were processed for HPLC analysis and, when available, RIPA lysis pellets were analyzed by PCR after DNA extraction. (C) Extracted DNA was obtained from 1, 3, 2, and 2 mice in groups 1, 2, 4, and 5, respectively.

Fig. 6.

Volume and histological analysis of Tu-2449 tumors before 5-FC cycles. (A) Average tumor size (+/− sem, Dorsal-Ventral (DV) or Lateral (L) in μM) from before 1st 5-FC dose, before 2nd 5-FC dose, and before 4th 5-FC dose, N ≥ 4. (B) H&E stains of tumor sections from brains of mice from the three groups analyzed.

Fig. 7.

Tu-2449 in B6C3F1 glioma model survival analysis after only 4 cycles of 5-FC. Groups of 10 female B6C3F1 mice (8 weeks of age) were implanted IC with 1E4 Tu-2449 tumor cells then dosed IC with vehicle (Control) or IC with Toca 511. Doses of E3, E4, or E5 TU/g of Toca 511 were given as indicated. Following 7 days, to allow spread of Toca 511, mice were treated IP once a day (SID) for 4 consecutive days with either PBS or 5-FC (500 mg/kg) as indicated. This cycle of 4 days on drug followed by 10 days off drug was repeated for only 4 cycles with treatment stopping at day 57. After the last 5-FC dosing regimen, survival analysis was carried out further to day 180.