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. Therefore, a prohemostatic therapeutic intervention should take this thrombotic risk into consideration. OBJECTIVES: CT-001 is a novel factor VIIa (FVIIa) with enhanced activity and desialylated N-glycans for rapid clearance. We assessed CT-001 clearance in multiple species and its ability to reverse APC-mediated coagulopathic blood loss. METHODS: The N-glycans on CT-001 were characterized by liquid chromatography-mass spectrometry. Three species were used to evaluate the pharmacokinetics of the molecule. The potency and efficacy of CT-001 under APC pathway-induced coagulopathic conditions were assessed by coagulation assays and bleeding models. RESULTS: The N-glycosylation sites of CT-001 had high occupancy of desialylated N-glycans. CT-001 exhibited 5 to 16 times higher plasma clearance in human tissue factor knockin mice, rats, and cynomolgus monkeys than wildtype FVIIa. CT-001 corrected the activated partial thromboplastin time and thrombin generation of coagulopathic plasma to normal in in vitro studies. In an APC-mediated saphenous vein bleeding model, 3 mg/kg of CT-001 reduced bleeding time in comparison with wildtype FVIIa. The correction of bleeding by CT-001 was also observed in a coagulopathic tail amputation severe hemorrhage mouse model. The efficacy of CT-001 is independent of the presence of tranexamic acid, and the combination of CT-001 and tranexamic acid does not lead to increased thrombogenicity. CONCLUSION: CT-001 corrected APC pathway-mediated coagulopathic conditions in preclinical studies and could be a potentially safe and effective procoagulant agent for addressing APC-mediated bleeding.

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

ABSTRACT: 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). A next-generation molecule, CT-001, is being developed as a potentially safer and more efficacious rFVIIa-based therapy. We sought to evaluate the peripartum hemostatic status of pregnant women and assess the ex vivo hemostatic activity of rFVIIa and CT-001 in peripartum blood samples. Pregnant women from 2 study sites were enrolled in this prospective observational study. Baseline blood samples were collected up to 3 days before delivery. Postdelivery samples were collected 45 (±15) minutes after delivery. Between the 2 time points, soluble fibrin monomer and D-dimer increased whereas tissue factor, FVIII, FV, and fibrinogen decreased. Interestingly, the postdelivery lag time and time to peak in the thrombin generation assay were shortened, and the peak thrombin generation capacity was maintained despite the reduced levels of coagulation proteins after delivery. Furthermore, both rFVIIa and CT-001 were effective in enhancing clotting activity of postdelivery samples in activated partial thromboplastin time, prothrombin time, thrombin generation, and viscoelastic hemostatic assays, with CT-001 demonstrating greater activity. In conclusion, despite apparent ongoing consumption of coagulation factors at the time of delivery, thrombin output was maintained. Both rFVIIa and CT-001 enhanced the upregulated hemostatic activity in postdelivery samples, and consistent with previous studies comparing CT-001 and rFVIIa in vitro and in in vivo, CT-001 demonstrated greater activity than rFVIIa.

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). Twenty-one nanobodies were identified that selectively recognize APC compared with the protein C zymogen. Overall, 3 clusters of nanobodies were identified based on the competition for APC in biolayer interferometry studies. APC functional assays for anticoagulant activity, histone H3 cleavage, and protease-activated receptor 1 (PAR1) cleavage were used to understand their diversity. These functional assays revealed 13 novel nanobody-induced APC activity profiles via the selective modulation of APC pleiotropic activities, with the potential to regulate specific mechanisms for therapeutic purposes. Within these, 3 nanobodies (LP2, LP8, and LP17) inhibited all 3 APC functions. Four nanobodies (LP1, LP5, LP16, and LP20) inhibited only 2 of the 3 functions. Monofunction inhibition specific to APC anticoagulation activity was observed only by 2 nanobodies (LP9 and LP11). LP11 was also found to shift the ratio of APC cleavage of PAR1 at R46 relative to R41, which results in APC-mediated biased PAR1 signaling and APC cytoprotective effects. Thus, LP11 has an activity profile that could potentially promote hemostasis and cytoprotection in bleedings associated with hemophilia or coagulopathy by selectively modulating APC anticoagulation and PAR1 cleavage profile.

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. METHODS AND METHODS: CT-001 has 4 N-glycans (T106N/N145/V253N/N322) with terminal sialic acid residues removed to promote active clearance via the asialoglycoprotein receptor, and P10Q/K32E substitutions introduced to its gamma-carboxyglutamic acid (Gla) domain for enhanced phospholipid affinity and activity. RESULTS: In mice, CT-001 had a half-life of 5 min and a clearance of 467 mL/h/kg at 3 mg/kg, significantly faster than WT FVIIa (t1/2 = 1.8 h, Cl = 39 mL/h/kg). Interestingly, CT-001 was efficacious in reducing blood loss even with its rapid clearance. In a severe hemorrhage mouse model with tail amputated 5 cm from the tip, 1 mg/kg CT-001 provided efficacy comparable to 3 mg/kg WT FVIIa. The fast clearance of CT-001 resulted in significantly reduced thrombogenicity in comparison to WT FVIIa in a FeCl3-induced carotid artery thrombosis mouse model, and further confirmed in a soluble tissue factor-induced thrombosis model. CONCLUSIONS: The data on CT-001 demonstrate that a short duration of highly active FVIIa procoagulant activity has the potential to be an optimal paradigm for the treatment of acute bleeds.

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. While clinically proven as an effective pro-coagulant, activated factor VII (FVIIa) use in acute bleeding has been hampered by unwanted thromboembolic events. Enhancing the ability of FVIIa to quickly stop a bleed and clear rapidly from circulation may yield an ideal molecule suitable for use in patients with acute bleeding. OBJECTIVES: To address this need and the current liability of FVIIa, we produced a novel FVIIa molecule (CT-001) with enhanced potency and shortened plasma residence time by cell line engineering and FVIIa protein engineering for superior efficacy for acute bleeding and safety. METHODS: To address safety, CT-001, a FVIIa protein with 4 desialylated N-glycans was generated to promote active recognition and clearance via the asialoglycoprotein receptor. To enhance potency, the gamma-carboxylated domain was modified with P10Q and K32E, which enhanced membrane binding. RESULTS: Together, these changes significantly enhanced potency and clearance while retaining the ability to interact with the key hemostatic checkpoint proteins antithrombin and tissue factor pathway inhibitor. CONCLUSIONS: These results demonstrate that a FVIIa molecule engineered to combine supra-physiological activity and shorter duration of action has the potential to overcome the current limitations of recombinant FVIIa to be a safe and effective approach to the treatment of acute bleeding.

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.

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.

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. 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. FINDINGS: 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. CONCLUSION: 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.

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. As such it may address some of the current production challenges. In this study we demonstrate that HBx can enhance protein production during transient transfection and in stable cell lines. XBP1s is a potent transcription factor and has been demonstrated to enlarge the ER secretion pathway and increase protein production. We explored the possibility of combinational engineering of HBx with XBP1s in BHK21 cells. Our data revealed that combinational engineering of HBx with XBP1s further enhances protein production compared with HBx or XBP1s alone.

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. In addition, they can be genetically manipulated to include additional therapeutic factors to create a multimodal anticancer agent. These characteristics lead to the expectation that oncolytic viruses will serve as an additional tool in the treatment repertoire of clinical oncologists. In their clinical development to date, these agents were safe and well tolerated, but lacked efficacy as monotherapies. In this review, three genetic-based methods to increase the potency and efficacy of oncolytic viruses, in which human adenovirus is utilized as an example of a prototype oncolytic virus, are discussed.

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 >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.

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. In contrast to both muristerone A and mifepristone that showed no significant toxicity on any of the tested cells, we observed that doxycycline induced cell death in selected cancer and primary cell lines. The different susceptibility of cell lines to the ligands commonly used in these inducible systems suggests that it is important to consider the effects of the inducers prior to their use in experimental in vitro cell culture systems.

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. Induced target gene expression from the pBRES system was shown to be comparable to the two-plasmid system and higher than the expression from the cytomegalovirus (CMV) promoter in vivo, while maintaining low uninduced levels of expression. Finally, a pBRES expression cassette was transferred to an adeno-associated virus (AAV) vector and shown to be capable of regulated gene expression in vivo for nearly 1 year.

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. Using GFP as a reporter gene and an E3-deleted Ad genome as a target, we tested three splicing signals. RACE analysis confirmed that gene expression from the GFP-expressing Ads occurs via splicing and traced expression to the Ad major late promoter (MLP). Replacement of the GFP transposon by an equivalent splice acceptor-luciferase expression cassette in the same orientation confirmed that substitute transgenes are also expressed via splicing from the MLP. Interestingly, insertion of the substitute transgene in the opposite orientation also resulted in expression that, in some cases, originated from within the ITR region of the viral genome. In summary, splice acceptor sequences can be used to control transgene expression from endogenous Ad promoters and this represents a genomically economical approach to arming oncolytic Ads.

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. Using this system with a plasmid containing an E3-deleted Ad5, we identified several unique sites for promoter-based expression cassette insertions within the Ad genome. The transposon-based expression cassette is bounded by PmeI restriction endonuclease sites unique to the transposon, making expression cassette substitutions easy to perform. Additional expression cassettes containing different promoters and reporter genes were substituted into two of the newly identified transgene insertion sites. The results suggest that the ease and orientation of expression cassette substitution depend on both the insertion site location and the promoter and gene of the replacement expression cassette. These studies establish the transposon-based system as an efficient approach to scanning the Ad genome and identifying insertion sites compatible with viral replication and represents a powerful tool for the development of armed therapeutic viruses for cancer.

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. Using this background, we compare and contrast the research and development experiences of small-molecule and antibody therapeutics with genetic-based cancer therapeutics, using oncolytic viruses as a defined example of an experimental molecular therapeutic for cancer.

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). This system conserves the DNA packaging capacity of the size-constrained viral genome for therapeutic genes and can potentially be used to link therapeutic transgene expression to tumor-restricted viral replication. Potential clinical implications are discussed.

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. Several classes of therapeutic genes are outlined, and potential synergies and hurdles to their delivery from replicating viruses are discussed.