E1A-expressing adenoviral E3B mutants act synergistically with chemotherapeutics in immunocompetent tumor models

Abstract

The majority of clinical trials evaluating replication-selective oncolytic adenoviruses utilized mutants with immunomodulatory E3B genes deleted, likely contributing to the attenuated efficacy. We investigated whether an intact immune response could contribute to the observed improved efficacy in response to combinations with chemotherapeutics. Seven carcinoma cell lines were evaluated by combining viral mutants; dl309 (DeltaE3B), dl704 (DeltaE3gp19K), dl312 (DeltaE1A) or wild-type Ad5 with the commonly used clinical drugs cisplatin and paclitaxel. Synergistic effects on cell death were determined by generation of combination indexes in cultured cells. In vivo tumor growth inhibition was achieved by virotherapy alone and was most efficacious with wild-type virus and least with the DeltaE3B mutant. Significantly higher efficacy was observed when the viruses were combined with drugs. The greatest enhancement of tumor inhibition was in combination with the DeltaE3B mutant restoring potency to that of Ad5 wild-type levels, observed only in animals with intact immune response. Increases in infectivity, viral gene expression and replication were identified as potential mechanisms contributing to the synergistic effects. Our results suggest that the attenuation of DeltaE3B mutants can be overcome by low doses of chemotherapeutics only in the presence of an intact immune response indicating a role for T-cell-mediated functions.

Figure 1

Representative isobolograms from combination treatment of wild-type virus and paclitaxel or cisplatin in murine and human cell lines. (a–c) Cells were infected with virus either 24 h prior to (V–T), (V–C) or 24 h after (T–V), (C–V) addition of paclitaxel (T) or cisplatin (C) at predetermined concentration ratios as in Materials and methods, (a) CMT-93, (b) CMT-64, (c) LNCaP. (d) Paclitaxel and cisplatin were added simultaneously with virus to the CMT-93 and CMT-64 cells. EC₅₀ values for each combination ratio were determined from dose–response curves and represented in isobolograms. The straight line connects the EC₅₀ values for each agent alone and illustrates the theoretical values resulting in additive effects. Data points below the line represent synergistic (CI≤0.8) and above the line antagonistic (CI≥1.2) interactions. Data are from one representative experiment in triplicate.

Figure 2

Effects of paclitaxel and cisplatin on viral replication, infectivity and gene expression. (a) Cells were treated with paclitaxel at 10 nm and cisplatin at 10 μm 24 h prior to Ad5 infection at 100 ppc (H460 and LNCaP) and 1000 ppc (CMT-64 and JC) and harvested 24 h (white bars), 48 h (striped bars) and 72 h (black bars) postinfection. Replication rate was determined by limiting dilution assays. Results are expressed as percent increase in replication in each cell line compared to cells treated with virus alone at the respective time points. Averages are from two experiments in triplicate ± s.e.m. (b) Cells were pretreated with 10 nm paclitaxel (black bars) and 10 μm cisplatin (white bars) 24 h prior to infection with nonreplicating AdGFP at 100 ppc (H460) and 1000 ppc (CMT-64, CMT-93 and JC). Changes in infectivity are expressed as fold increase compared to cells treated with virus alone, presented as averages of two experiments ± s.e.m. (a, b) P-values were determined by the t-test for treated versus untreated cells, P<0.05 (*). (c) CMT-64 and CMT-93 cells were infected at 1000 ppc 24 h after treatment with 10 nm paclitaxel or 10 μm cisplatin and harvested 24 and 48 h postinfection. Immunoblotting was performed for detection of penton and E1A using β-actin as a loading control (U = uninfected cells, ctrl = HEK293 cells infected with Ad5wt). (d) CMT-64 cells and tumors, in athymic (nu/nu) and immunocompetent (C57Bl/6) animals were infected with Ad5 and dl309 and harvested as described in Materials and methods. Viral burst was determined by the TCID₅₀ assay and replication expressed as ratios of dl309 to Ad5. Data presented as averages from six tumors in each group ± s.d. Significance was tested with t-test, P<0.005 (*) when compared to initial time points (24 h and 2 days, respectively).

Figure 3

Growth inhibition in response to single and combination treated CMT-64 and CMT-93 tumors in animals with normal and defective immune responses. Animals with intact (a, b) and defective immune response (c) bearing CMT-64 (a, c) and CMT-93 (b) tumors treated with viral mutants and paclitaxel (T) or cisplatin (C). Tumor ratios were calculated comparing the averages for each treatment group to untreated tumors (PBS) on day 15 after inoculation. Average tumor volumes ± s.d. were calculated for each group (n = 10 animals per group), data were analyzed by t-test and are representative of three separate studies. P-values <0.05 (*) when single agent treatment was compared to PBS or dl312 and combination treatments compared to both single agent treatments.

Figure 4

Tumor progression analysis for combination therapies with paclitaxel. Kaplan–Meier curves for treatment groups in C57Bl/6 with intact immune system. (a) Animals with CMT-64 tumors were treated with Ad5 (1 × 10¹⁰ vp) (filled diamonds, (a) left), dl309 (1 × 10¹⁰ vp) (filled diamonds, (a) right), paclitaxel (T; open triangles), combination treatments (filled squares) and PBS (filled circles). Combinations of Ad5 or dl309 with paclitaxel were significantly different from single agent treatments with P<0.05. (b) Animals with CMT-93 tumors were treated with Ad5 or dl309 (filled diamonds), paclitaxel (T; open triangles), virus with paclitaxel (filled squares). Combinations of dl309 with paclitaxel were significantly different from single agent treatments with P<0.05. The percentage of mice free from tumor progression (tumor volume <500 μl) at each time point was estimated using the Kaplan–Meier method, 10 animals per group.

Figure 5

Replication of Ad5 and the ΔE3B mutant in CMT-64 tumor xenografts. Replication ratios in tumors grown in intact mice treated with virus and combinations of virus and paclitaxel as described in Materials and methods. Tumors were harvested five days post-inoculation with virus. Data are expressed as change in replication compared to treatment with wild-type virus (Ad5) alone. Tumors were from six animals analyzed in triplicate. P-values were determined by t-test, P<0.005 when combination treatments were compared to each virus alone (*) and with dl309 alone compared to Ad5 combined with paclitaxel.