SBIR-STTR Award

The long-term objective of the proposed study is to develop a monoclonal antibody that inhibits the progression of diabetic retinopathy. Insulin-like growth factor-I (IGF-I) has been implicated in both endothelial dysfunction as well as the proliferative phase of the disease. However directly inhibiting the IGF-I receptor may be lead to neurotoxicity. Following exposure to hyperglycemia, endothelial cells utilize both the PI-3 and MAP kinase pathways to activate changes in endothelial cell permeability and angiogenesis. However unlike cells exposed to normal glucose these pathways are activated in response to IGF-I by an alternative mechanism that requires tyrosine phosphorylation of the transmembrane protein, SHPS-1. SHPS-1 is phosphorylated directly by the IGF-I receptor leading to recruitment of a complex of signaling proteins. For this complex to be activated in response to hyperglycemia plus IGF-I, it must associate with a membrane protein termed integrin associated protein (IAP). Our preliminary studies have shown that in early retinopathy, capillary leakage and leukostasis can be inhibited by disrupting IAP/SHPS-1 association. Additionally IAP knockout mice do not develop increased capillary permeability in spite of longstanding diabetes therefore it appears that IAP/SHPS-1 association is required in vivo for retinopathy progression. The purpose of these studies will be to determine if disrupting IAP/SHPS-1 results in failure of the SHPS-1 complex to assemble normally on endothelial cells and activate the appropriate downstream signaling molecules that mediate endothelial dysfunction. We will determine if a polyclonal antibody direct against amino acids 71-80 in rat IAP has efficacy in vivo. The purified antibody will be injected into diabetic rats and its effects on capillary leakage, leukostasis and endothelial cell apoptosis determined. Subsequently a monoclonal antibody directed against amino acids 71-80 in human IAP will be developed and tested to determine if it inhibits IGF-I stimulated signaling as well as changes in endothelial tube formation and cell permeability. To obtain a monoclonal antibody that can be administered to humans safely it will be necessary to humanize the murine form. A clone secreting the murine antibody will be isolated and cDNAs corresponding to heavy and light chains will be amplified using rtPCR then the cDNAs will be sequenced. These studies should definitively test the hypothesis that inhibiting IGF-I actions by inhibiting SHPS-1/IAP association in endothelial cells in culture and in capillaries in rats inhibis pathophysiologic changes that occur in diabetic retinopathy. If the results of these studies show that the antibody is effective, during phase II studies a humanized form of the monoclonal antibody will be prepared, purified and its effects determined. The long term objective would be to administer this antibody to diabetic patients with early signs of retinopathy to determine if it has efficacy in inhibiting both the early events and preventing late stage manifestations of this disease. Since diabetic retinopathy is the leading cause of blindness in working age adults, there is a need for new therapies that are directed toward treatment of this complication.

Public Health Relevance:
This proposal is directed toward the development of a monoclonal antibody as a drug to be administered to patients with diabetic retinopathy. Diabetic retinopathy is the leading cause of blindness in working age adults (25-74 years). As such it represents a significant public health problem. Drugs that are currently available that inhibit thi problem are effective in approximately 1/3 of patients. The drug to be developed functions by a mechanism that is different than those drugs and therefore has the potential to improve the prognosis of this disease in 2/3 of the affected patients. The work in this proposal will determine if this approach is feasible using animal models of this disease state.

The long-term objective of this project study is to develop a monoclonal antibody that inhibits the progression of diabetic retinopathy. Insulin-like growth factor-I (IGF-I) has been implicated in both endothelial dysfunction as well as the proliferative phase of the disease but IGF-I concentrations in retina are not increased. During hyperglycemia IGF-I activates both the PI-3 and MAP kinase pathways in endothelial cells by an aberrant mechanism that requires tyrosine phosphorylation of the transmembrane protein, SHPS-1 and this leads to enhanced cellular sensitivity to IGF-I. For SHPS-1 to be phosphorylated its extracellular domain must bind to another membrane protein, integrin associated protein (IAP). Under normoglycemic conditions the region of IAP that binds to SHPS-1 is cleaved but during hyperglycemia cleavage is inhibited. The antibody being developed disrupts IAP/SHPS-1 association thereby inhibiting this aberrant signaling pathway. Our phase1 studies showed that the antibody inhibited retinal capillary leakage, formation of acellular capillaries and pericyte ghosts. Therefore it appears that IAP/SHPS-1 association is required for retinopathy progression. The studies in aim 1 will determine if disrupting IAP/SHPS-1 results in inhibition of neovascularization. A polyclonal antibody directed against amino acids 71-80 in rat IAP will be injected into vitreous of hypoxemic rats and its effects on retinal neovascularization determined. These studies will also determine if the antibody can arrest the progression of established changes in capillary leakage and retinal thickening in that have occurred in diabetic rats. The studies in aim 2 will pursue the most important objective, humanization of the mouse monoclonal antibody that was prepared during phase 1. Murine amino acids that could potentially cause immunogenicity will be substituted with human residues in order to prevent autoimmunization. The humanized form will be tested to confirm that immunogenicity is been eliminated and that its affinity has not been reduced. The cDNAs encoding the antibody will be transfected into CHO cells and a high producing cell line developed. The purified protein will be tested to determine if it inhibits IGF-I stimulated signaling and changes in endothelial tube formation and permeabili- ty. If its efficacy is retained it will be administered intraocularly to monkeys to determine the effective concentration in vitreous that inhibits retinal endothelial dysfunction. These studies should definitively test the hypothesis that inhibiting IGF-I actions by inhibiting SHPS-1/IAP association in retina cells will inhibit the pathophysiologic changes that occur in diabetic retinopathy. Their successful completion will represent major progress toward the long term objective of this project; the development of an antibody that can be administered to patients with diabetic retinopathy that inhibits both the early events and prevents late stage manifestations of this disease. Since diabetic retinopathy is the leading cause of blindness in working age adults, there is a need for new therapies that are directed toward treatment of this complication.

Public Health Relevance Statement:


Public Health Relevance:
This proposal is directed toward the development of a humanized monoclonal antibody as a drug to be administered to patients with diabetic retinopathy. Diabetic retinopathy is the leading cause of blindness in working age adults (25-74 years). As such it represents a significant public health problem. Drugs that are currently available to treat this problem are effective in approximately 1/3 of patients. The drug to be developed functions by a mechanism that is different from those drugs and therefore it has the potential to improve the prognosis of this disease in 2/3 of the affected patients. The work that i proposed will humanize the antibody and determine if it has efficacy in a primate model of this disease state.

Project Terms:
Adult; Affect; Affinity; Age; Amino Acid Substitution; Amino Acids; Antibodies; Antibody Formation; Attenuated; Binding (Molecular Function); Biochemical; Biological Models; Blindness; Blood capillaries; capillary; Capillary Permeability; CD47 Antigen; Cell Line; Cells; Characteristics; Chinese Hamster Ovary Cell; Cleaved cell; Complementary DNA; Complex; Complication; Computer Simulation; Data; Development; diabetic rat; Diabetic Retinopathy; Disease; Disease model; Disease Progression; Dose-Rate; Dropout; Endothelial Cells; endothelial dysfunction; Endothelium; Epitopes; Evaluation; Event; Exposure to; expression vector; Extracellular Domain; Extravasation; Human; humanized antibody; humanized monoclonal antibodies; Hyperglycemia; Hypoxemia; Hypoxia; immunogenic; immunogenicity; Immunoglobulin Fragments; improved; inhibiting antibody; Injection of therapeutic agent; Insulin-Like Growth Factor I; Integral Membrane Protein; Mediating; Membrane Proteins; Methods; Mitogen-Activated Protein Kinase 3; Modeling; Monkeys; Monoclonal Antibodies; Mus; neovascularization; novel; outcome forecast; Pathway interactions; Patients; Peptides; Pericytes; Permeability; Pharmaceutical Preparations; Phase; polyclonal antibody; prevent; Primates; Process; Protein Binding; Protein Biosynthesis; Proteins; public health medicine (field); public health relevance; Rattus; Research; response; Retina; Retinal; Retinal Diseases; Retinal Neovascularization; Signal Pathway; Signal Transduction; Signaling Protein; Staging; Structure; synthetic peptide; Testing; Thick; Time; Toxic effect; Toxicology; Tube; Tyrosine Phosphorylation; Work