Newcastle disease virus (NDV) is a single-stranded, negative-sense RNA virus in the Paramyxoviridae family. Although primarily an avian pathogen, NDV is a potent oncolytic virus that has been shown to be safe and effective in a variety of preclinical cancer models and human clinical trials. To produce virus for oncolytic trials, NDV is commonly amplified in embryonated chicken eggs and purified from the allantoic fluid. Conventional methods for purifying virus from allantoic fluid often result in relatively low-titer preparations containing high levels of impurities, including immunogenic chicken host cell proteins from allantoic fluid. However, large quantities of virus need to be delivered intravenously to administer oncolytic NDV systemically to mice. This route of administration requires virus preparations that are both highly concentrated (to enable delivery of small volumes) and highly pure (to limit toxic effects from contaminants). Given the accumulation of promising preclinical and clinical data demonstrating the efficacy of NDV as an oncolytic agent, strategies for increasing the titer and purity of NDV preparations are sorely needed to allow for effective intravenous administration in mice. Here, we describe an optimized protocol for the rescue, production, and purification of high-titer in vivo-grade NDV for preclinical studies in mouse models.

The avian paramyxovirus, Newcastle disease virus (NDV), is a promising oncolytic agent that has been shown to be safe and effective in a variety of pre-clinical cancer models and human clinical trials. NDV preferentially replicates in tumor cells due to signaling defects in apoptotic and antiviral pathways acquired during the transformation process and is a potent immunostimulatory agent. However, when used as a monotherapy NDV lacks the ability to consistently generate durable remissions.Here we investigate the use of viral sensitizer-mediated combination therapy to enhance the anti-neoplastic efficacy of NDV. Intratumoral injection of vanadyl sulfate, a pan-inhibitor of protein tyrosine phosphatases, in combination with NDV significantly increased the number and activation status of natural killer (NK) cells in the tumor microenvironment, concomitant with increased expression of interferon-b, granulocyte-macrophage colony-stimulating factor, and monocyte chemoattractant protein-1, leading to rapid tumor regression and long-term cures in mice bearing syngeneic B16-F10 melanomas. The anti-tumor efficacy of this combination therapy was abrogated when NK cells were depleted and when interferon-b expression was transiently suppressed. Tumor-specific CD8 + T cell responses were not detected, nor were mice whose tumors regressed protected from re-challenge. This suggested efficacy of the combination therapy predominantly relied on the innate immune system. Importantly, efficacy was not limited to melanoma; it was also demonstrated in a murine prostate cancer model. Taken together, these results suggest that combining NDV with vanadyl sulfate potentiates an innate immune response that can potentiate rapid clearance of tumors, with type I interferon signaling and NK cells being important mechanisms of action.

Immunotherapies are at the forefront of the fight against cancers, and researchers continue to develop and test novel immunotherapeutic modalities. Ideal cancer immunotherapies induce a patient’s immune system to kill their own cancer and develop long-lasting immunity. Research has demonstrated a critical requirement for CD8+ and CD4+ T cells in achieving durable responses. In the path to the clinic, researchers require robust tools to effectively evaluate the capacity for immunotherapies to generate adaptive anti-tumor responses. To study functional tumor-specific T cells, researchers have relied on targeting tumor-associated antigens (TAAs) or the inclusion of surrogate transgenes in pre-clinical models, which facilitate detection of T cells by using the targeted antigen(s) in peptide re-stimulation or tetramer-staining assays. Unfortunately, many pre-clinical models lack a defined TAA, and epitope mapping of TAAs is costly. Surrogate transgenes can alter tumor engraftment and influence the immunogenicity of tumors, making them less relevant to clinical tumors. Further, some researchers prefer to develop therapies that do not rely on pre-defined TAAs. Here, we describe a method to exploit major histocompatibility complex expression on murine cancer cell lines in a co-culture assay to detect T cells responding to bulk, undefined, tumor antigens. This is a tool to support the preclinical evaluation of novel, antigen-agnostic immunotherapies.

BackgroundNovel therapies are needed to improve outcomes for women diagnosed with ovarian cancer. Oncolytic viruses are multifunctional immunotherapeutic biologics that preferentially infect cancer cells and stimulate inflammation with the potential to generate antitumor immunity. Herein we describe Parapoxvirus ovis (Orf virus (OrfV)), an oncolytic poxvirus, as a viral immunotherapy for ovarian cancer.MethodsThe immunotherapeutic potential of OrfV was tested in the ID8 orthotopic mouse model of end-stage epithelial ovarian carcinoma. Immune cell profiling, impact on secondary lesion development and survival were evaluated in OrfV-treated mice as well as in Batf3 knockout, mice depleted of specific immune cell subsets and in mice where the primary tumor was removed. Finally, we interrogated gene expression datasets from primary human ovarian tumors from the International Cancer Genome Consortium database to determine whether the interplay we observed between natural killer (NK) cells, classical type 1 dendritic cells (cDC1s) and T cells exists and influences outcomes in human ovarian cancer.ResultsOrfV was an effective monotherapy in a murine model of advanced-stage epithelial ovarian cancer. OrfV intervention relied on NK cells, which when depleted abrogated antitumor CD8+ T-cell responses. OrfV therapy was shown to require cDC1s in experiments with BATF3 knockout mice, which do not have mature cDC1s. Furthermore, cDC1s governed antitumor NK and T-cell responses to mediate antitumor efficacy following OrfV. Primary tumor removal, a common treatment option in human patients, was effectively combined with OrfV for optimal therapeutic outcome. Analysis of human RNA sequencing datasets revealed that cDC1s correlate with NK cells in human ovarian cancer and that intratumoral NK cells correlate positively with survival.ConclusionsThe data herein support the translational potential of OrfV as an NK stimulating immunotherapeutic for the treatment of advanced-stage ovarian cancer.

Background: Oncolytic viruses are playing an increasingly important role in cancer immunotherapy applications. Given the preclinical and clinical efficacy of these virus-based therapeutics, there is a need for fast, simple, and inexpensive downstream processing methodologies to purify biologically active viral agents that meet the increasingly higher safety standards stipulated by regulatory authorities like the Food and Drug Administration and the European Agency for the Evaluation of Medicinal Products. However, the production of virus materials for clinical dosing of oncolytic virotherapies is currently limited-in quantity, quality, and timeliness-by current purification technologies. Adsorption of virus particles to solid phases provides a convenient and practical choice for large-scale fractionation and recovery of viruses from cell and media contaminants. Indeed, chromatography has been deemed the most promising technology for large-scale purification of viruses for biomedical applications. The implementation of new chromatography media has improved process performance, but low yields and long processing times required to reach the desired purity are still limiting. Results: Here we report the development of an interference chromatography-based process for purifying high titer, clinical grade oncolytic Newcastle disease virus using NatriFlo® HD-Q membrane technology. This novel approach to optimizing chromatographic performance utilizes differences in molecular bonding interactions to achieve high purity in a single ion exchange step. Conclusions: When used in conjunction with membrane chromatography, this high yield method based on interference chromatography has the potential to deliver efficient, scalable processes to enable viable production of oncolytic virotherapies.