Divergent modulation of activated protein C pleiotropic functions by antibodies that differ by a single amino acid.

Activated protein C (APC) is a pleiotropic plasma protease with diverse functions derived from its anticoagulant, anti-inflammatory, and cytoprotective activities. The selective uncoupling and/or modulation of these APC activities by antibodies may have therapeutic benefit in diseases such as traumatic bleeding, hemophilia, sepsis, and ischemia. TPP-26870 is an antibody that targets a nonactive site of APC for the selective modulation of APC activities.

CT-001, a novel fast-clearing factor VIIa, enhanced the hemostatic activity in postpartum samples.

The hemostatic system is upregulated to protect pregnant mothers from hemorrhage during childbirth. Studies of the details just before and after delivery, however, are lacking. Recombinant factor VIIa (rFVIIa) has recently been granted approval by the European Medicines Agency for the treatment of postpartum hemorrhage (PPH).

Rapid clearing CT-001 restored hemostasis in mice with coagulopathy induced by activated protein C.

Background: Activated protein C (APC) is one of the mechanisms contributing to coagulopathy, which is associated with high mortality. The counteraction of the APC pathway could help ameliorate bleeding. However, patients also transform frequently from a hemorrhagic state to a prothrombotic state at a later time.

Selective modulation of activated protein C activities by a nonactive site-targeting nanobody library.

Activated protein C (APC) is a pleiotropic coagulation protease with anticoagulant, anti-inflammatory, and cytoprotective activities. Selective modulation of these APC activities contributes to our understanding of the regulation of these physiological mechanisms and permits the development of therapeutics for the pathologies associated with these pathways. An antibody library targeting the nonactive site of APC was generated using llama antibodies (nanobodies).

CT-001 is a rapid clearing factor VIIa with enhanced clearance and hemostatic activity for the treatment of acute bleeding in non-hemophilia settings.

Introduction: Acute bleeding leads to significant morbidity and mortality. Recombinant wildtype Factor VIIa (WT FVIIa) had been reported to have some therapeutic effects in some clinical trials, however, its use was associated with thromboembolic events. We sought to develop a novel FVIIa molecule (CT-001) with enhanced activity and lowered thrombogenicity risk.

In vitro characterization of CT-001-a short-acting factor VIIa with enhanced prohemostatic activity.

Background: Traumatic injury and the associated acute bleeding are leading causes of death in people aged 1 to 44 years. Acute bleeding in pathological and surgical settings also represents a significant burden to the society. Yet there are no approved hemostatic drugs currently available.

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).

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.

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

Background: 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.

Allogeneic somatic cell therapy: process development challenges and future opportunities.

Cell-replacement therapy has emerged during the past decade as a potential solution for many diseases. However, for this promise to be fulfilled, numerous process development challenges specific to these products need to be overcome. This editorial overview highlights some key observations derived from research on an allogeneic somatic cell therapy product for the treatment of Parkinson's disease.

Enhanced protein production using HBV X protein (HBx), and synergy when used in combination with XBP1s in BHK21 cells.

The demand of therapeutic protein production from mammalian cells has expanded greatly since the first biologic was approved in 1982. It remains a major challenge to exploit the exocytic pathway and increase cell viability during the production process. Hepatitis B virus X protein (HBx) is a multifunctional viral transcription activator that regulates a variety of cellular events including transcription, cell cycle and proliferation, survival, and apoptosis.

Exploiting diversity: genetic approaches to creating highly potent and efficacious oncolytic viruses.

Oncolytic viruses possess several key attributes that make them a highly attractive treatment for cancer. They exhibit clinically validated synergy with chemotherapy and an ability to selectively destroy tumor cells to the exclusion of normal cells. Oncolytic viruses can replicate and, therefore, amplify their dose in a tumor-dependent manner.

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.

A comparative study examining the cytotoxicity of inducible gene expression system ligands in different cell types.

Inducible gene expression systems are being used in many in vitro and in vivo applications for target discovery, target validation and as components in exploratory therapeutic agents. Ideally, the ligands, which activate the systems, are benign so that the effects can be strictly attributed to the induced protein. As a first step to defining the potential effects of these inducers, we tested three of them, doxycycline, muristerone A and mifepristone (for tet-, ecdysone- and progesterone antagonist-inducible systems respectively), for toxicity across a panel of normal cells and cancer cell lines.

Development and validation of a robust and versatile one-plasmid regulated gene expression system.

We have developed a one-plasmid regulated gene expression system, pBRES, based on a mifepristone (MFP)-inducible two-plasmid system. The various expression elements of the pBRES system (promoters, 5' and 3' untranslated regions (UTRs), introns, target gene, and polyA sequences) are bounded by restriction enzyme sites so that each module can be conveniently replaced by alternate DNA elements in order to tailor the system for particular tissues, organs, or conditions. There are four possible orientations of the two expression units relative to each other, and insertion of a variety of expression elements and target genes into the four different orientations revealed orientation- and gene-dependent effects on induced and uninduced levels of gene expression.

Identification of novel insertion sites in the Ad5 genome that utilize the Ad splicing machinery for therapeutic gene expression.

Therapeutic transgene expression from oncolytic viruses represents one approach to increasing the effectiveness of these agents as cancer therapeutics. In the case of the oncolytic adenovirus (Ad), however, the genomic packaging capacity is constrained. To address this, we explored whether a transposon-based system could identify sites in the viral genome where endogenous Ad promoters could drive transgene expression via splicing and still maintain the replication capacity of the virus.

Development of a transposon-based approach for identifying novel transgene insertion sites within the replicating adenovirus.

Therapeutic gene delivery from an oncolytic adenovirus (Ad) is one approach to enhancing the potency of Ad-based virotherapies for cancer. To identify therapeutic transgene insertion sites compatible with the replicating virus, a methodology that broadly scans the viral genome is needed. To address this we modified a transposon (Tn7)-based in vitro transposition system to take advantage of its nonprejudiced scanning ability to identify insertion sites compatible with viral replication.

Genetically based therapeutics for cancer: similarities and contrasts with traditional drug discovery and development.

The field of molecular therapeutics is in its infancy and represents a promising and novel avenue for targeted cancer treatments. Like the small-molecule and antibody therapeutics before them, however, the genetic-based therapies will face significant research and development challenges in their maturation toward an approved cancer therapy. To facilitate this process, we outline and examine in this review the drug development process, briefly summarizing the research and development paradigms that have accompanied the recent successes of the small-molecule and antibody-based cancer therapeutics.

Multigene expression from a replicating adenovirus using native viral promoters.

We have developed a novel therapeutic gene delivery system for oncolytic adenoviruses that takes advantage of the endogenous gene expression machinery (promoters, splicing, polyadenylation signals) of the E3 transcription unit for gene delivery. In this work, we use two sites in the E3 region (6.7 K/gp19K and ADP sites) to demonstrate that (1) multiple therapeutic genes (MCP-3, TNFalpha) can be expressed from a single replicating Ad, (2) timing of expression of these therapeutic genes mimics that of the E3 region genes they replaced, (3) expression of the remaining genes in the complex E3 transcription unit is maintained, and (4) the multigene-expressing virus retains replication competence and ability to induce classical adenovirus cytopathic effects that parallel those of the parental adenovirus (ONYX-320).

Armed therapeutic viruses: strategies and challenges to arming oncolytic viruses with therapeutic genes.

Oncolytic viruses are attractive therapeutics for cancer because they selectively amplify, through replication and spread, the input dose of virus in the target tumor. To date, clinical trials have demonstrated marked safety but have not realized their theoretical efficacy potential. In this review, we consider the potential of armed therapeutic viruses, whose lytic potential is enhanced by genetically engineered therapeutic transgene expression from the virus, as potential vehicles to increase the potency of these agents.