SBIR-STTR Award

Organ transplantation is one of the great success stories of modern medicine. However, the supply of suitable organs lags far behind the need and many patients die while waitlisted for organs. Current practice involves "donor preconditioning" to stabilize organs and tissues for transplant in attempts to increase the supply of donor organs acceptable for transplantation. A key goal of preconditioning is to reduce the damage inflicted by ischemia-reperfusion injury (IRI) that occurs when the ischemic/ hypoxic organ is connected to the recipient's circulation. This storm of reactive oxygen species, inflammatory mediators and prothombotic factors threatens to kill the new organ and wreaks havoc upon the recipient as well. Current work indicates that enhancing the low levels of nitric oxide (NO) produced by eNOS and nNOS can dramatically improve IRI and transplant outcomes. The founders of Vasculox, Inc have discovered a receptor, CD47, that continually opposes the action of beneficial NO in all vascular cells. Knocking out CD47 in mice or blocking CD47 with a monoclonal antibody (mAb) results in enhanced tissue perfusion in a number of surgical ischemia models and protection from IRI in both liver and hindlimb models. In this phase one proposal, we seek to perform proof of concept studies in a well-characterized rat model of liver transplantation, testing the efficacy of anti-CD47 mAb treatment of the donor liver to protect it from IRI. Here we will compare the best case scenario in which anti-CD47 mAb is administered to the donor rat and maintained throughout liver harvest, cold storage and reperfusion, with the worst case of no CD47 blockade. In our protocol, the heart-beating donor rat will be treated with anti-CD47 mAb, the liver harvested and flushed with anti-CD47 mAb, subjected to cold storage ischemia and then rewarmed in the presence of mAb under controlled conditions of machine reperfusion. After a fixed interval of reperfusion, levels of released soluble liver enzymes and inflammatory mediators will be assayed, tissue taken for assays of ATP and cyclic nucleotides and the liver fixed for quantitative cytologic analysis and histochemical staining for the distribution of the anti-CD47 mAb and apoptosis (TUNEL and cleaved caspase). A simplified nonheart-beating donor model will also be performed when a period of warm ischemia is interposed between mAb administration and organ harvest. The data obtained in this phase I project will establish proof of concept for anti-CD47 mAb therapy in this liver transplant model. Phase II will address the "real world" situation of transplantation of treated or control livers into living recipients that can then be treated (or not) with the anti-CD47 mAb. While we have chosen to focus here on the liver model, we anticipate that anti-CD47 therapy will be of benefit in the transplantation of other organs (heart, kidney, lung, etc) as well as in surgeries involving organ resection for trauma and cancer and, potentially, in myocardial infarction and stroke.

Public Health Relevance:
The founders of Vasculox Inc, have discovered a regulatory receptor, CD47, that inhibits nitric oxide signaling in all vascular tissues. Nitric oxide provides many beneficial effects in the vascular system, including limiting ischemia-reperfusion injury. Therefore blocking CD47 and removing nitric oxide inhibition improves ischemia-reperfusion injury and holds promise as a means to improve the condition of organs destined for transplant and to control the damage to the recipient caused by ischemia reperfusion injury. Vasculox is developing a monoclonal antibody that targets CD47 and in this project aims to test the efficacy of such an antibody in a rat model of organ harvest, transport and reperfusion.

Public Health Relevance Statement:
The founders of Vasculox Inc, have discovered a regulatory receptor, CD47, that inhibits nitric oxide signaling in all vascular tissues. Nitric oxide provides many beneficial effects in the vascular system, including limiting ischemia-reperfusion injury. Therefore blocking CD47 and removing nitric oxide inhibition improves ischemia-reperfusion injury and holds promise as a means to improve the condition of organs destined for transplant and to control the damage to the recipient caused by ischemia reperfusion injury. Vasculox is developing a monoclonal antibody that targets CD47 and in this project aims to test the efficacy of such an antibody in a rat model of organ harvest, transport and reperfusion.

NIH Spending Category:
Biotechnology; Cardiovascular; Digestive Diseases; Heart Disease; Heart Disease - Coronary Heart Disease; Liver Disease; Organ Transplantation; Stroke; Transplantation

Project Terms:
Abscission; Active Oxygen; Address; Animal Model; Animal Models and Related Studies; Anti-Inflammatories; Anti-Inflammatory Agents; Anti-inflammatory; Antibodies; Antigenic Surface Determinant Protein OA3 Gene; Antiinflammatories; Antiinflammatory Agents; Apoplexy; Apoptosis; Apoptosis Pathway; Assay; Bile; Bile Juice; Bile fluid; Binding; Binding (Molecular Function); Bioassay; Bioavailability; Biologic Assays; Biologic Availability; Biological Assay; Biological Availability; Biological Preservation; Bizzozero's corpuscle/cell; Bleeding; Blood Circulation; Blood Platelets; Blood Pressure; Blood Vessels; Blood flow; Bloodstream; Body Tissues; Brain; CD47; CD47 Antigen (Rh-Related Antigen, Integrin-Associated Signal Transducer) Gene; CD47 Glycoprotein Gene; CD47 gene; Cancers; Cardiac infarction; Cell Communication and Signaling; Cell Death, Programmed; Cell Signaling; Cell-Death Protease; Cells; Cerebral Stroke; Cerebrovascular Apoplexy; Cerebrovascular Stroke; Cerebrovascular accident; Circulation; Circulatory Collapse; Cleaved cell; Clinic; Clinical, Transplantation, Organ; Common Rat Strains; Cryofixation; Cryopreservation; Cyclic Nucleotides; Data; Deetjeen's body; Dysfunction; EC 1.14.13.39; EDRF Synthase; Encephalon; Encephalons; Endogenous Nitrate Vasodilator; Endothelium; Endothelium-Derived Growth Factor Synthase; Endothelium-Derived Relaxing Factor; Enzymes; Excision; Extirpation; Flushing; Flushings; Functional disorder; Genetic; Goals; Graft Material; Grafting Procedure; Grafting, Liver; Guanylyl Cyclase-Activating Factor Synthase; Harvest; Hayem's elementary corpuscle; Heart; Hemorrhage; Hindlimb; Hypoxia; Hypoxic; IAP Gene; ICE-like protease; INFLM; In Situ Nick-End Labeling; Infection; Inflammation; Inflammation Mediators; Integrin-Associated Protein Gene; Intracellular Communication and Signaling; Ischemia; Ischemia-Reperfusion Injury; Kidney; Killings; Knock-out; Knockout; L-Arginine,NADPH[{..}]oxygen oxidoreductase (nitric-oxide-forming); Length of Life; Leukocyte Surface Antigen CD47 Gene; Life; Ligands; Liver; Liver Transplant; Longevity; Lung; MER6 Gene; Malignant Neoplasms; Malignant Tumor; Mammals, Mice; Mammals, Rats; Marrow platelet; Mechanics; Mice; Moab, Clinical Treatment; Modeling; Modern Medicine; Molecular Interaction; Monoclonal Antibodies; Mononitrogen Monoxide; Murine; Mus; Myocardial Infarct; Myocardial Infarction; NADPH-Diaphorase; NIH; NO Synthase; National Institutes of Health; National Institutes of Health (U.S.); Nervous System, Brain; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide, Endothelium-Derived; Nitric-Oxide Synthetase; Nitrogen Monoxide; Nitrogen Protoxide; Nitrogen oxide; OA3 Gene; Operation; Operative Procedures; Operative Surgical Procedures; Organ; Organ Donor; Organ Harvestings; Organ Transplantation; Organ Transplants; Organ Transplants, Including Bone Marrow for DCT; Outcome; Oxidative Burst; Oxygen Deficiency; Oxygen Radicals; Patients; Perfusion; Phase; Physiologic Availability; Physiopathology; Platelet Activation; Platelets; Preservation, Biologic; Preservation, Biological; Pro-Oxidants; Process; Production; Protocol; Protocols documentation; Rat; Rattus; Reactive Oxygen Species; Receptor Protein; Removal; Reperfusion Damage; Reperfusion Injury; Reperfusion Therapy; Respiratory Burst; Respiratory System, Lung; Reticuloendothelial System, Platelets; Rewarming; Shock; Signal Transduction; Signal Transduction Systems; Signaling; Site; Solid; Staining method; Stainings; Stains; Stroke; Surface Antigen Identified by Monoclonal Antibody 1D8 Gene; Surgical; Surgical Interventions; Surgical Procedure; Surgical Removal; System; System, LOINC Axis 4; THBS1; TSP; TSP-1; TSP1; TUNEL; Thrombocytes; Thrombosis; Thrombospondin 1; Tissues; Transplantation; Transplantation Surgery; Transplantation of liver; Transplantation, Hepatic; Transplanted tissue; Trauma; United States National Institutes of Health; Urinary System, Kidney; Vascular Accident, Brain; Vascular System; Vascular blood supply; Vascular constriction (function); Vasoconstriction; Warm Ischemia; Work; bioavailability of drug; biological signal transduction; blood loss; blood supply; body system, hepatic; brain attack; cardiac infarct; caspase; cerebral vascular accident; circulatory shock; cleaved; cold preservation; cold storage; coronary attack; coronary infarct; coronary infarction; cystein protease; cystein proteinase; cysteine endopeptidase; cytokine; efficacy testing; endothelial cell derived relaxing factor; fighting; heart attack; heart infarct; heart infarction; hemodynamics; iNO; improved; inhaled nitric oxide; leukocyte oxidative burst; life span; lifespan; liver transplantation; malignancy; model organism; mouse model; neoplasm/cancer; new approaches; novel approaches; novel strategies; novel strategy; organ allograft; organ graft; organ system, hepatic; organ xenograft; pathophysiology; preconditioning; preservation; public health relevance; pulmonary; receptor; renal; reperfusion; resection; respiratory burst (leukocyte); stroke; success; surgery; terminal nick end labeling; thrombocyte/platelet; transplant; vascular; vascular supply; wound

Vasculox, Inc. is developing a humanized anti-CD47 mAb for reducing ischemia-reperfusion injury (IRI) in organ transplantation. In spite of improvements in surgical technique, organ preservation and immunosuppression, IRI remains a serious limitation and is responsible for delayed graft function, initial graft failure and contribtes to poor long-term graft survival, thus representing an area of significant unmet medical need. Increasing nitric oxide (NO) signaling can provide a substantial therapeutic benefit in reducing IRI. The founders of Vasculox discovered that thrombospondin-1 (TSP1) binding to its receptor, CD47, limits NO signaling in all vascular tissues. Blocking TSP-1 binding with an anti-CD47 monoclonal antibody (anti-CD47 mAb) relieves this inhibition of NO signaling and improves outcomes in several animal models of IRI. We have recently demonstrated efficacy of an anti-CD47 mAb to improve liver function in an ex vivo perfusion model and in a rat liver transplant model. Vasculox has characterized a panel of 9 mouse monoclonal mAbs (400 series mAbs) that are unique in reacting broadly across species with human, rodent, dog and pig providing a significant advantage for clinical development. Three of these mAbs reverse the TSP1-CD47 mediated inhibition of NO-stimulated cGMP formation in cultured cells and one of these will be taken forward for humanization. In this Phase II SBIR grant the specific aims are: Aim 1. Humanize the lead CD47 mAb candidate, produce research grade material for preclinical studies. Aim 2A. Establish optimal conditions for efficacy of CD47 mAb in a rat syngeneic liver transplant model including administration and dosing modality (pre-treat organ alone, treat recipient alone or treat both) and adjuvant treatments to enhance the NO signaling pathway (arginase inhibitors and/or L-arginine) Aim 2B. Demonstrate efficacy of the humanized mAb in a large animal (porcine) model of transplant using the optimized treatment modality as determined in Aim 2A. Aim 3. Carry out non-GLP (preliminary) pharmacokinetic and safety studies in the rat and non-human primate using the CD47 humanized mAb to prepare for GLP IND-enabling studies. This phase II proposal addresses several critical path milestones for Vasculox that will allow us to address transplant IRI as well as additional indications for anti-CD47 therapy including IRI arising from surgical procedures, trauma, sickle cell crisis and pulmonary hypertension.

Public Health Relevance:
Ischemia reperfusion injury (IRI) is a complicating factor in many cardiovascular diseases and surgical procedures including organ transplantation. Increasing nitric oxide (NO) signaling can provide a substantial therapeutic benefit in reducing IRI. The founders of Vasculox discovered that a receptor called CD47 limits NO signaling. Blocking CD47 with a monoclonal antibody (CD47mAb) relieves this inhibition of NO signaling and has the potential to enhance organ transplantation and save lives. Vasculox is developing a humanized CD47mAb for reducing IRI in organ transplantation that reacts with many different species. In this proposal we will humanize the lead CD47mAb candidate and determine its optimal dosing modality in rat liver transplantation. We will then confirm its efficacy in pigs, a more human-like species. Finally, we will conduct studies of how long the mAb is available in the circulation and test the CD47mAb for safety in two animal species as required by the FDA. These studies represent critical path milestones for Vasculox and must be accomplished before we can begin clinical trials to improve organ transplantation, allow for more transplantable organs and thereby save more lives.