A systemically deliverable Vaccinia virus with increased capacity for intertumoral and intratumoral spread effectively treats pancreatic cancer

Background Pancreatic cancer remains one of the most lethal cancers and is refractory to immunotherapeutic interventions. Oncolytic viruses are a promising new treatment option, but current platforms demonstrate limited efficacy, especially for inaccessible and metastatic cancers that require systemically deliverable therapies. We recently described an oncolytic vaccinia virus (VV), VVLΔTKΔN1L, which has potent antitumor activity, and a regime to enhance intravenous delivery of VV by pharmacological inhibition of pharmacological inhibition of PI3 Kinase δ (PI3Kδ) to prevent virus uptake by macrophages. While these platforms improve the clinical prospects of VV, antitumor efficacy must be improved. Methods VVLΔTKΔN1L was modified to improve viral spread within and between tumors via viral B5R protein modification, which enhanced production of the extracellular enveloped virus form of VV. Antitumor immunity evoked by viral treatment was improved by arming the virus with interleukin-21, creating VVL-21. Efficacy, functional activity and synergy with α-programmed cell death protein 1 (α-PD1) were assessed after systemic delivery to murine and Syrian hamster models of pancreatic cancer. Results VVL-21 could reach tumors after systemic delivery and demonstrated antitumor efficacy in subcutaneous, orthotopic and disseminated models of pancreatic cancer. The incorporation of modified B5R improved intratumoural accumulation of VV. VVL-21 treatment increased the numbers of effector CD8+ T cells within the tumor, increased circulating natural killer cells and was able to polarize macrophages to an M1 phenotype in vivo and in vitro. Importantly, treatment with VVL-21 sensitized tumors to the immune checkpoint inhibitor α-PD1. Conclusions Intravenously administered VVL-21 successfully remodeled the suppressive tumor-microenvironment to promote antitumor immune responses and improve long-term survival in animal models of pancreatic cancer. Importantly, treatment with VVL-21 sensitized tumors to the immune checkpoint inhibitor α-PD1. Combination of PI3Kδ inhibition, VVL-21 and α-PD1 creates an effective platform for treatment of pancreatic cancer.

(metastatic colon adenocarcinoma) and LLC (Lewis lung carcinoma) were obtained from the Cancer Research UK central cell bank (CRUK, Clare Hall, Herts, UK) and maintained in DMEM supplemented with 10% FBS Cell lines were routinely tested for mycoplasma and were maintained at 37°C with 5% CO2.

Reagents
The selective PI3Kδ inhibitor CAL101 was purchased from Selleckchem, re-suspended at 30mg/ml with 30% PEG 400, 0.5% Tween 80, 5% Propylene glycol and administered via oral gavage at 10mg/kg. The VV antibody used for the immuno-histochemical (IHC) experiments was a rabbit anti-VV polyclonal antibody supplied by AbD Serotec, batch number 250906, and was diluted 1:400. α-PD1 antibody (RMP1-14) was purchased from Bioxcell and  Viral burst titers were converted to PFU per cell based on the number of cells present at viral infection. One-way ANOVA followed by Bonferroni post-test was used to assess significance.

Cell cytotoxicity assay
The cytotoxicity of the viruses in each cell line was assessed in triplicate 6 days after infection with virus using an MTS non-radioactive cell proliferation assay kit (Promega) according to the manufacturers' instructions. Cell viability was determined by measuring absorbance at 490nm using a 96-well plate absorbance reader (Dynex) and a dose response curve created by non-linear regression allowing determination of an EC50 value (dose required to kill 50% of cells) as previously described 3 . Quantification of viral genome copy number was achieved using the TaqMan® PCR system provided by Applied Biosystems. For VV quantification, the primers and probe were designed for the Vaccinia virus late transcription factor 1 (VLTF-1) gene: Forward; 5'-AACCATAGAAGCCAACGAATCC, Reverse; 5'-TGAGACATACAAGGGTGGTGAAGT, Probe;

Detection of IL-21 expression by ELISA
ATTTTAGAACAGAAATACCC. The primers were supplied by Sigma-Aldrich. The standard was WT VV DNA, and 40ng of DNA was used per sample as the template. Viral genome copy number was normalized by total DNA loaded. Conditions were analysed in triplicate and two biological replicates were carried out.
Total RNA was extracted from cells using the RNeasy Mini Kit from Qiagen. Total RNA was resuspended in nuclease-free water and quantified using the spectrophotometer NANODROP ND-1000. 1μg of RNA was reverse-transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) following the manufacturer's instructions. cDNA was analyzed through quantitative Real-Time PCR using the Fast SYBR Green system. Triplicate samples were analysed. Primers used were:
Red blood cells were lysed using RBC lysis buffer (Sigma-Aldrich) and re-suspended in complete T-cell medium.

Tumor cell preparation
Tumor cell suspensions were prepared by incubation with 2mg/ml collagenase plus 0.1mg/ml hyaluronidase for 30 minutes at 37°C. Cells were separated using a 70-mm cell strainer and re-suspended in complete T-cell medium.

Immunophenotyping of splenocytes and tumors
Single cell suspensions of tumor, spleen or blood were prepared in FACS buffer (FB) as described above. All fluorophore-conjugated antibodies used at 1:200 dilutions. Cells were blocked with anti-CD16/32 prior to incubation with fluorophore-conjugated antibody for 30 minutes. Cells were fixed in 2% formalin and analyzed using an LSRFortessa™ multichannel flow cytometer (Beckton Dickinson (BD) Biosciences). Raw data were analyzed using FloJo v10 (FloJo, LLC). Three samples/group were analysed and three biological repeats carried out.

Imaging
Magnetic Resonance Imaging (MRI) was performed once a week starting 10 days after tumor injection. Mice were anesthetized as described above and T2 image acquired using ParaVision software. Images were analyzed using VivoQuant software by an independent researcher.