Oncolytic Viruses: The Power of Directed Evolution

Attempts at developing oncolytic viruses have been primarily based on rational design. However, this approach has been met with limited success. An alternative approach employs directed evolution as a means of producing highly selective and potent anticancer viruses. In this method, diverse viruses are grown under conditions that maximize diversity and then passaged under conditions meant to mimic those encountered in the human cancer microenvironment. Viruses which evolve to thrive under this selective pressure are isolated and tested to identify those with increased potency (i.e., ability to replicate and spread) and/or an increased therapeutic window (i.e., differentiated replication and spread on tumor versus normal cells), both of which have potential value but the latter of which defines an oncolytic virus. Using ColoAd1, an oncolytic virus derived by this approach as a prototype, we highlight the benefits of directed evolution, discuss methods to “arm” these novel viruses, and introduce techniques for their genetic modulation and control

Effect of different UCOE-promoter combinations in creation of engineered cell lines for the production of Factor VIII

<p>Abstract</p> <p>Background</p> <p>The most common approach used in generating cell lines for the production of therapetic proteins relies on gene amplification induced by a drug resistance gene e. g., DHFR and glutamine synthetase. Practically, this results in screening large number of clones for the one that expresses high levels of the biologic in a stable manner. The inefficiency of mammalian vector systems to express proteins in a stable manner typically involves silencing of the exogenous gene resulting from modifications such as methylation of CpG DNA sequences, histone deacetylation and chromatin condensation. The use of un-methylated CpG island fragments from housekeeping genes referred to as UCOE (ubiquitous chromatin opening elements) in plasmid vectors is now well established for increased stability of transgene expression. However, few UCOE-promoter combinations have been studied to date and in this report we have tested 14 different combinations.</p> <p>Findings</p> <p>In this report we describe studies with two different UCOEs (the 1.5 Kb human RNP fragment and the 3.2 Kb mouse RPS3 fragment) in combination with various promoters to express a large protein (B domain deleted factor VIII; BDD-FVIII) in a production cell line, BHK21. We show here that there are differences in expression of BDD-FVIII by the different UCOE-promoter combinations in both attached and serum free suspension adapted cells. In all cases, the 1.5 Kb human RNP UCOE performed better in expressing BDD-FVIII than their corresponding 3.2 Kb mouse RPS3 UCOE. Surprisingly, in certain scenarios described here, expression from a number of promoters was equivalent or higher than the commonly used and industry standard human CMV promoter.</p> <p>Conclusion</p> <p>This study indicates that certain UCOE-promoter combinations are better than others in expressing the BDD-FVIII protein in a stable manner in BHK21 cells. An empirical study such as this is required to determine the best combination of UCOE-promoter in a vector for a particular production cell line.</p

Directed Evolution Generates a Novel Oncolytic Virus for the Treatment of Colon Cancer

Background Viral-mediated oncolysis is a novel cancer therapeutic approach with the potential to be more effective and less toxic than current therapies due to the agents selective growth and amplification in tumor cells. To date, these agents have been highly safe in patients but have generally fallen short of their expected therapeutic value as monotherapies. Consequently, new approaches to generating highly potent oncolytic viruses are needed. To address this need, we developed a new method that we term “Directed Evolution” for creating highly potent oncolytic viruses. Methodology/Principal Findings Taking the “Directed Evolution” approach, viral diversity was increased by pooling an array of serotypes, then passaging the pools under conditions that invite recombination between serotypes. These highly diverse viral pools were then placed under stringent directed selection to generate and identify highly potent agents. ColoAd1, a complex Ad3/Ad11p chimeric virus, was the initial oncolytic virus derived by this novel methodology. ColoAd1, the first described non-Ad5-based oncolytic Ad, is 2–3 logs more potent and selective than the parent serotypes or the most clinically advanced oncolytic Ad, ONYX-015, in vitro. ColoAd1's efficacy was further tested in vivo in a colon cancer liver metastasis xenograft model following intravenous injection and its ex vivo selectivity was demonstrated on surgically-derived human colorectal tumor tissues. Lastly, we demonstrated the ability to arm ColoAd1 with an exogenous gene establishing the potential to impact the treatment of cancer on multiple levels from a single agent. Conclusions/Significance Using the “Directed Evolution” methodology, we have generated ColoAd1, a novel chimeric oncolytic virus. In vitro, this virus demonstrated a &gt;2 log increase in both potency and selectivity when compared to ONYX-015 on colon cancer cells. These results were further supported by in vivo and ex vivo studies. Furthermore, these results have validated this methodology as a new general approach for deriving clinically-relevant, highly potent anti-cancer virotherapies

The human cytomegalovirus ul11 protein interacts with the receptor tyrosine phosphatase cd45, resulting in functional paralysis of t cells

Human cytomegalovirus (CMV) exerts diverse and complex effects on the immune system, not all of which have been attributed to viral genes. Acute CMV infection results in transient restrictions in T cell proliferative ability, which can impair the control of the virus and increase the risk of secondary infections in patients with weakened or immature immune systems. In a search for new immunomodulatory proteins, we investigated the UL11 protein, a member of the CMV RL11 family. This protein family is defined by the RL11 domain, which has homology to immunoglobulin domains and adenoviral immunomodulatory proteins. We show that pUL11 is expressed on the cell surface and induces intercellular interactions with leukocytes. This was demonstrated to be due to the interaction of pUL11 with the receptor tyrosine phosphatase CD45, identified by mass spectrometry analysis of pUL11-associated proteins. CD45 expression is sufficient to mediate the interaction with pUL11 and is required for pUL11 binding to T cells, indicating that pUL11 is a specific CD45 ligand. CD45 has a pivotal function regulating T cell signaling thresholds; in its absence, the Src family kinase Lck is inactive and signaling through the T cell receptor (TCR) is therefore shut off. In the presence of pUL11, several CD45-mediated functions were inhibited. The induction of tyrosine phosphorylation of multiple signaling proteins upon TCR stimulation was reduced and T cell proliferation was impaired. We therefore conclude that pUL11 has immunosuppressive properties, and that disruption of T cell function via inhibition of CD45 is a previously unknown immunomodulatory strategy of CMV

Global substrate profiling of proteases in human neutrophil extracellular traps reveals consensus motif predominantly contributed by elastase.

Neutrophil extracellular traps (NETs) consist of antimicrobial molecules embedded in a web of extracellular DNA. Formation of NETs is considered to be a defense mechanism utilized by neutrophils to ensnare and kill invading pathogens, and has been recently termed NETosis. Neutrophils can be stimulated to undergo NETosis ex vivo, and are predicted to contain high levels of serine proteases, such as neutrophil elastase (NE), cathepsin G (CG) and proteinase 3 (PR3). Serine proteases are important effectors of neutrophil-mediated immunity, which function directly by degrading pathogenic virulent factors and indirectly via proteolytic activation or deactivation of cytokines, chemokines and receptors. In this study, we utilized a diverse and unbiased peptide library to detect and profile protease activity associated with NETs induced by phorbol-12-myristate-13-acetate (PMA). We obtained a "proteolytic signature" from NETs derived from healthy donor neutrophils and used proteomics to assist in the identification of the source of this proteolytic activity. In addition, we profiled each neutrophil serine protease and included the newly identified enzyme, neutrophil serine protease 4 (NSP4). Each enzyme had overlapping yet distinct endopeptidase activities and often cleaved at unique sites within the same peptide substrate. The dominant proteolytic activity in NETs was attributed to NE; however, cleavage sites corresponding to CG and PR3 activity were evident. When NE was immunodepleted, the remaining activity was attributed to CG and to a lesser extent PR3 and NSP4. Our results suggest that blocking NE activity would abrogate the major protease activity associated with NETs. In addition, the newly identified substrate specificity signatures will guide the design of more specific probes and inhibitors that target NET-associated proteases