Improved potency and selectivity of an oncolytic E1ACR2 and E1B19K deleted adenoviral mutant in prostate and pancreatic cancers

Abstract

Purpose: Replication-selective oncolytic adenoviruses are a promising class of tumor-targeting agents with proven safety in hundreds of patients. However, clinical responses have been limited and viral mutants with higher potency are needed. Here, we report on the generation of a novel set of mutants with improved efficacy in prostate and pancreatic carcinoma models. Currently, no curative treatments are available for late-stage metastatic prostate or rapidly progressing pancreatic cancers.

Experimental design: Adenovirus type 5 mutants were created with deletions in the E1ACR2 region for tumor selectivity and/or the E1B19K gene for attenuated replication in vivo; all constructs retain the E3 genes intact. Cell-killing efficacy, replication, and cytotoxicity in combination with chemotherapeutics were investigated in normal cells (PrEC and NHBE), seven carcinoma cell lines, and human (PC3 and DU145) and murine (TRAMPC, CMT-64, and CMT-93) tumor models in vivo.

Results: The double-deleted AdDeltaDelta (DeltaE1ACR2 and DeltaE1B19K) mutant had high cell-killing activity in prostate, pancreatic, and lung carcinomas. Replication was similar to wild-type in all tumor cells and was attenuated in normal cells to levels less than the single-deleted AdDeltaCR2 mutant. AdDeltaDelta combined with the chemotherapeutics docetaxel and mitoxantrone resulted in synergistically enhanced cell killing and greatly improved antitumor efficacy in prostate xenografts in vivo. In murine immunocompetent in vivo models efficacy was greater for mutants with the E3B genes intact even in the absence of viral replication, indicating attenuated macrophage-dependent clearance.

Conclusions: These data suggest that the novel oncolytic mutant AdDeltaDelta is a promising candidate for targeting of solid tumors specifically in combination with chemotherapeutics.

Figure 1. Deletions of the E1ACR2 region and the E1B19K gene improved cell killing potency of Ad5 in several tumor cell lines

A) Adenovirus wild type 5 (Ad5tg) and the respective mutants with the E1B19K (AdΔ19K), the E1ACR2 (AdΔCR2) or both gene-regions deleted (AdΔΔ) were generated. All mutants had intact E3-region. B) Cell-killing efficacy determined as EC₅₀ values in PC3, DU145, 22Rv1 and LNCaP prostate, PT45 and Suit2 pancreatic, and H460 lung carcinoma cell lines with Ad5tg, AdΔ19K, AdΔCR2 and AdΔΔ viruses. The decreases in EC₅₀ values are presented as percentages of the EC₅₀ for wild type Ad5tg, averages ±SEM, n ≥ 3, * p < 0.05.

Figure 2. Viral replication and gene expression of the novel mutants paralleled that of the wild type virus

A) Amplification of viral genomes was determined over time for all mutants with qPCR of the hexon gene in DU145, 22Rv1 and LNCaP cells infected at 5ppc and in PC3 cells at 100ppc, only cells were included in the DNA analysis. Data expressed as averages of three samples ± SEM, differences between viruses were not significant (p>0.5). B) Viral replication determined in burst assays by TCID₅₀ at 48 and 72h after infection with the respective mutants at 100ppc in all cell lines, both cells and media were analysed. C) Early E1A and late hexon gene expression in DU145 cells 24 and 48h after infection, representative immunoblot. D) Viral replication in the NHBE and PrEC normal cells determined by TCID₅₀ assay at 48 and 72h after infection with 100ppc. (B and D) Averages of three samples ± SEM, * p< 0.05.

Figure 3. The novel viral mutants synergistically enhanced docetaxel-induced cell killing in prostate cancer cell lines

A) DU145 and PC3 cells were treated with combinations of viral mutants and docetaxel at fixed ratios, EC₅₀-values were calculated for each condition and isobolograms generated to determine synergistic interactions on cell death. The straight line represents the theoretical line for additive effects and each data point the respective combination ratio. B) Caspase 3 activation in response to infection with viral mutants at 100ppc after 24 and 48h in 22Rv1 and DU145 cells. C) Mitochondrial depolarisation (Δψ; TMRE staining) to determine proportion of live 22Rv1 and DU145 cells 24-96h after infection at 10ppc, averages ±SD, n=3.

Figure 4. Combinations of low doses of the AdΔΔ mutant and docetaxel inhibited tumor progression in human prostate carcinoma xenografts in nude mice

A) Animals with DU145 subcutaneous tumor xenografts were treated with PBS (phosphate buffered saline; filled circle), the AdΔΔ mutant at 1×10⁹vp (intratumoral injections on day 1, 3, and 5) with and without docetaxel at 5mg/kg (intraperitoneal administration on day 2 and 8) (open and filled squares respectively) or docetaxel 5mg/kg alone (D5; triangle) and tumor growth was monitored. *p<0.001 for treatments compared to either single agent treatment and viral mutant compared to untreated animals. Significance determined by one-way Anova analysis. B) Animals with PC3 subcutaneous tumor xenografts were treated as above with the indicated suboptimal doses 1×10⁹vp (dl312; open circles, and AdΔΔ; filled diamond) and docetaxel at 10mg/kg (D10; open triangle) or combination of AdΔΔ and docetaxel (open square). Median time to tumor progression (tumor volume >500μl) was determined by Kaplan-Meier survival analysis, 6-10 animals per group. *p<0.002 for combination treated compared to single agent groups. C) Viral genome amplification in DU145 tumors after intratumoral administration of virus on day 0, treated as indicated for 3 and 10 days. Total DNA was extracted and quantified by qPCR using specific hexon primers. Data averaged from 3 tumors/group, analysed in triplicates, and expressed as hexon DNA copies/5ng total DNA ±SD. Right panel: Viral amplification in DU145 tumors for the AdΔΔ mutant with and without simultaneous docetaxel administration at 5mg/kg. D) Viral hexon expression in DU145 tumor sections after intratumoral injection of 1×10⁹vp of the respective mutant. One group treated with the AdΔΔ mutant and docetaxel at 10mg/kg. Three tumors were evaluated from each treatment group and analysed for expression of hexon 10 days after viral treatment, magnification 200x.

Figure 5. Retention of the E3B-genes in the AdΔΔ mutant improved efficacy in murine immunocompetent models

A) C57BL intact mice with subcutaneous TRAMPC murine prostate tumor xenografts were treated with dl309 or AdΔΔ at 1×10¹⁰ vp/injection on day 1, 3, and 5 with and without docetaxel at 15mg/kg administered intraperitoneally on day 2 and 8. Treatments were not significantly different (p>0.3). B) Representative micrographs of TRAMPC tumors stained for macrophages (CD68) and viral proteins (E1A) 15 days after intratumoral administration of one dose of the respective virus intratumorally at 1×10¹⁰vp. Magnification: 400x for CD68 and 200x for E1A. C) Survival curves for C57BL intact mice with murine colorectal CMT-93 (left panel) and lung CMT-64 (right panel) subcutaneous tumors, treated with 1×10¹⁰ vp/injection on day 1, 3, and 5 with dl922-947 (ΔE3B) (open diamond), AdΔCR2 (closed circle) and AdΔΔ (open circle) for CMT-93, and with dl922-947 and AdΔΔ for CMT-64; mock treated animals (closed square). Survival of AdΔΔ treated animals was significantly different from animals treated with dl922-947 (*p<0.02) only in the CMT-93 model, 8 animals/group.