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.

Gla-domain mediated targeting of externalized phosphatidylserine for intracellular delivery.

Phosphatidylserine (PS) is a negatively charged phospholipid normally localized to the inner leaflet of the plasma membrane of cells but is externalized onto the cell surface during apoptosis as well as in malignant and infected cells. Consequently, PS may comprise an important molecular target in diagnostics, imaging, and targeted delivery of therapeutic agents. While an array of PS-binding molecules exist, their utility has been limited by their inability to internalize diagnostic or therapeutic payloads.

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.

Phosphatidylserine-Exposing Annexin A1-Positive Extracellular Vesicles: Potential Cancer Biomarkers.

Under physiological conditions, phosphatidylserine (PS) predominantly localizes to the cytosolic leaflet of the plasma membrane of cells. During apoptosis, PS is exposed on the cell surface and serves as an "eat-me" signal for macrophages to prevent releasing self-immunogenic cellular components from dying cells which could potentially lead to autoimmunity. However, increasing evidence indicates that viable cells can also expose PS on their surface.

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.

Tandem-Cleavage Linkers Improve the In Vivo Stability and Tolerability of Antibody-Drug Conjugates.

Although peptide motifs represent the majority of cleavable linkers used in clinical-stage antibody-drug conjugates (ADCs), the sequences are often sensitive to cleavage by extracellular enzymes, such as elastase, which leads to systemic release of the cytotoxic payload. This action reduces the therapeutic index by causing off-target toxicities that can be dose-limiting. For example, a common side-effect of ADCs made using peptide-cleavable linkers is myelosuppression, including neutropenia.

A Novel HER2-targeted Antibody-drug Conjugate Offers the Possibility of Clinical Dosing at Trastuzumab-equivalent Exposure Levels.

Trastuzumab and the related ADC, ado-trastuzumab emtansine (T-DM1), both target HER2-overexpressing cells. Together, these drugs have treatment indications in both early-stage and metastatic settings for HER2 breast cancer. T-DM1 retains the antibody functionalities of trastuzumab and adds the potency of a cytotoxic maytansine payload.

Targeted inhibition of activated protein C by a non-active-site inhibitory antibody to treat hemophilia.

Activated protein C (APC) is a plasma serine protease with antithrombotic and cytoprotective functions. Based on the hypothesis that specific inhibition of APC's anticoagulant but not its cytoprotective activity can be beneficial for hemophilia therapy, 2 types of inhibitory monoclonal antibodies (mAbs) are tested: A type I active-site binding mAb and a type II mAb binding to an exosite on APC (required for anticoagulant activity) as shown by X-ray crystallography. Both mAbs increase thrombin generation and promote plasma clotting.

Long-Acting IL-33 Mobilizes High-Quality Hematopoietic Stem and Progenitor Cells More Efficiently Than Granulocyte Colony-Stimulating Factor or AMD3100.

Mobilization of hematopoietic stem and progenitor cells (HSPCs) has become increasingly important for hematopoietic cell transplantation. Current mobilization approaches are insufficient because they fail to mobilize sufficient numbers of cells in a significant fraction of patients and are biased toward myeloid immune reconstitution. A novel, single drug mobilization agent that allows a more balanced (myeloid and lymphoid) reconstitution would therefore be highly favorable to improve transplantation outcome.

Maytansine-bearing antibody-drug conjugates induce in vitro hallmarks of immunogenic cell death selectively in antigen-positive target cells.

Oncology treatment has been revolutionized by the introduction of immune checkpoint inhibitor drugs, which enable 20-40% of patients to generate anti-tumor immune responses. Combination treatment approaches with chemotherapeutic drugs may enable responses in the remaining patient cohorts. In this regard, a handful of drugs are promising due to their ability to induce immunogenic cell death in target cells.

Preclinical Safety Studies of Enadenotucirev, a Chimeric Group B Human-Specific Oncolytic Adenovirus.

Enadenotucirev is an oncolytic group B adenovirus identified by a process of bio-selection for the ability to selectively propagate in and rapidly kill carcinoma cells. It is resistant to inactivation by human blood components, potentially enabling intravenous dosing in patients with metastatic cancer. However, there are no known permissive animal models described for group B adenoviruses that could facilitate a conventional approach to preclinical safety studies.

OvAd1, a Novel, Potent, and Selective Chimeric Oncolytic Virus Developed for Ovarian Cancer by 3D-Directed Evolution.

Effective therapeutics for ovarian cancer continue to be urgently needed, particularly for chemotherapy-resistant cases. Here we present both a 3D-Matrigel culture-based expansion of our directed evolution method for generation of oncolytic virotherapies and two promising ovarian-cancer targeted oncolytic viruses, OvAd1 and OvAd2. OvAd1 was developed using Matrigel cell cultures, whereas OvAd2 was developed in parallel using traditional monolayer tissue culture methods.

Armed therapeutic viruses - a disruptive therapy on the horizon of cancer immunotherapy.

For the past 150 years cancer immunotherapy has been largely a theoretical hope that recently has begun to show potential as a highly impactful treatment for various cancers. In particular, the identification and targeting of immune checkpoints have given rise to exciting data suggesting that this strategy has the potential to activate sustained antitumor immunity. It is likely that this approach, like other anti-cancer strategies before it, will benefit from co-administration with an additional therapeutic and that it is this combination therapy that may generate the greatest clinical outcome for the patient.

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.

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.

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.

Mx1 and IP-10: biomarkers to measure IFN-beta activity in mice following gene-based delivery.

Recombinant interferon-beta (IFN-beta) protein is used successfully for the treatment of multiple sclerosis (MS). Gene therapy might be an alternative approach to overcome drawbacks occurring with IFN-beta protein therapy. A critical issue in developing a new approach is detection of biologically active IFN-beta in preclinical models.

A prodrug strategy using ONYX-015-based replicating adenoviruses to deliver rabbit carboxylesterase to tumor cells for conversion of CPT-11 to SN-38.

ONYX-015 has been used successfully in the clinic as a cancer therapeutic in combination with chemotherapy. The combination of ONYX-015 and chemotherapy appears to be more efficacious than either regimen alone. In this study, we try to enhance this combination by "arming" ONYX-015 with a therapeutic transgene, an approach more commonly used with nonreplicating viruses in the context of gene therapy.

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).