SBIR-STTR Award

The long term objective of the proposed studies is to develop a monoclonal antibody that can be administered safely to patients with diabetes to determine if it can inhibit atherosclerosis. Insulin-like growth factor-l (IGF-I) has been implicated in initiating the proliferative phase of atherosclerotic lesion development but inhibiting IGF-I receptor activity may lead to toxicity. IGF-I functions cooperatively with the aV?3 integrin to stimulate smooth muscle celll(SMC) division and migration which are related to the development of atherosclerosis. Therefore the purpose of these studies will be to determine if targeting the aV?3 integrin disrupts IGF-I stimulated atherosclerotic lesion progression. A specific monoclonal antibody directed against the cysteine loop (amino acids 177-183) of the 03 subunit of the aV|33 integrin will be prepared and tested to determine if it inhibits IGF-I stimulated SMC growth and migration in vitro and if this response is due disruption of IGF-I receptor linked signaling mechanisms that lead to stimulation of cell growth. To determine if this antibody has efficacy in vivo Fab fragments of the antibody will be prepared and will be infused into the arteries of pigs that are developing atherosclerotic lesions. A control antibody will be infused into one of the lesion areas to rule out the possibility of nonspecific effects due to autoimmunization. To obtain the structural information that will be necessary to prepare a humanized form of the antibody the clone of cells secreting the antibody will be isolated and the cDNAs corresponding to the heavy and light chains will be amplified using RTPCR. The cDNAs will be isolated, cloned and their sequences determined. The antibody structure will be used to design humanized form of the antibody in phase II. These studies should definitively test the hypothesis that inhibiting IGF-I actions by inhibiting ligand occupancy of aV?33 in blood vessel walls inhibits atherosclerotic lesion progression. If these results show that this approach is feasible a humanized version of this monoclonal would be prepared, purified and tested for toxicity. The long term objective would be to administer this antibody to diabetic patients with atherosclerosis who have wide spread diffuse disease that is not amenable to current therapies such as drug eluting stents. Since atherosclerosis is the cause of death in 80% of diabetics and there is no currently available therapy that specifically treats atherosclerosis in diabetics there is a major need for new therapies that are directed toward the treatment of this complication

The long term objective of the proposed studies is to develop a monoclonal antibody that can be administered safely to patients with diabetes that will inhibit atherosclerosis. Insulin-like growth factor-I has been implicated in the initiation of the proliferative phase of atherosclerotic lesion development but inhibiting IGF-I receptor activity may lead to toxicity. IGF-I functions cooperatively with the ?V?3 integrin to stimulate smooth muscle division and migration which are related to the development of atherosclerosis. The purpose of these studies will be to determine if targeting the ?V?3 integrin disrupts IGF-I stimulated atherosclerotic lesion progression in pigs with diabetes. A secondary purpose will be to humanize the available mouse monoclonal antibody so that it can be administered safely to patients without inducing an immunogenic response. The current monoclonal antibody directed against the cysteine loop (amino acids 177-183 of the ? subunit of the ?V?3 integrin) will be converted to a Fab2 fragment and purified. This Fab2 fragment will then be administered to pigs with diabetes for three months to determine if it inhibits the progressive enlargement of atherosclerotic lesions. Control pigs will receive an irrelevant antibody. At the end of the treatment period, the carotid, femoral and coronary arteries will be analyzed to determine if antibody has inhibited the rate of atherosclerotic lesion progression. Additionally ultrasound of the femoral arteries will be used to determine if the antibody can induce lesion regression. Tissues will be removed and analyzed biochemically to determine that ?V?3 activation by the hyperglycemic stimulus has been inhibited and that IGF-I signaling is inhibited. Tissues will also be tested to determine whether autoimmunization has occurred. In a second part of the study a humanized antibody will be prepared using the complementarity determining regions of the mouse antibody. These regions will grafted onto human framework sequences that are obtained from library of known human sequences. The sequence will then be scanned for regions of potential immunogenicity and peptides prepared and tested directly to determine if they have T-cell reactive epitopes. If immunogenic regions are present they will be modified by mutagenesis and tested to determine if immunogenicity has been removed. Further assessment of the 3 dimensional structure of the antibody will be undertaken to determine those residues that are within 10 angstroms of the CDR antigen binding site. These residues will also be modified to reduce their interaction with the binding domain. The final substituted humanized antibody will then be prepared using PCR and the full length gene product expressed. The purified product will then be tested for its ability to inhibit IGF-I stimulated smooth muscle cell proliferation and migration and clones will be analyzed to select those with the highest levels of expression that can be used in GMP manufacturing. The long term objective will be to prepare large scale quantities of the antibody for administration to human subjects. The treatment goal would be to administer to diabetic patients with atherosclerosis who have wide spread disease that is not amenable to current therapies. Since atherosclerosis is the cause of death in 80% of diabetes and there is no currently available therapy that specifically treats this problem, there is a major need for new therapies that are directed toward treatment of this complication.

Public Health Relevance:
This project has very high relevance to public health. Atherosclerosis is the most common cause of death in patients with diabetes. It also leads to disability due to heart attacks, strokes and amputations. This project is seeking to develop a drug that will specifically inhibit the progression of atherosclerosis in patients with diabetes and that has the potential to prevent these complications.

Public Health Relevance:
Relevance This project has very high relevance to public health. Atherosclerosis is the most common cause of death in patients with diabetes. It also leads to disability due to heart attacks, strokes and amputations. This project is seeking to develop a drug that will specifically inhibit the progression of atherosclerosis in patients with diabetes and that has the potential to prevent these complications.

Public Health Relevance Statement:
Relevance This project has very high relevance to public health. Atherosclerosis is the most common cause of death in patients with diabetes. It also leads to disability due to heart attacks, strokes and amputations. This project is seeking to develop a drug that will specifically inhibit the progression of atherosclerosis in patients with diabetes and that has the potential to prevent these complications.

Project Terms:
3-D structure; 3-dimensional structure; 3D structure; Affinity; Amino Acids; Amputation; Antibodies; Antigenic Determinants; Apoplexy; Arterial Fatty Streak; Atheroma; Atheromatous; Atheromatous degeneration; Atheromatous plaque; Atheroscleroses; Atherosclerosis; Atherosclerotic Cardiovascular Disease; Binding; Binding (Molecular Function); Binding Determinants; Binding Sites; Biochemical; Blood Vessels; Body Tissues; CDR; CHO Cells; Cardiac artery; Cardiac infarction; Cause of Death; Cell Communication and Signaling; Cell Growth in Number; Cell Line; Cell Lines, Strains; Cell Multiplication; Cell Proliferation; Cell Signaling; CellLine; Cellular Proliferation; Cerebral Stroke; Cerebrovascular Apoplexy; Cerebrovascular Stroke; Cerebrovascular accident; Chinese Hamster Ovary Cell; Combining Site; Complementarity Determining Regions; Complication; Complimentarity Determining Region; Computer Simulation; Computerized Models; Coronary artery; Culture Media, Serum-Free; Cysteine; Development; Diabetes Mellitus; Diagnosis, Ultrasound; Disease; Disorder; Drugs; Echography; Echotomography; Epitopes; Extracellular Matrix, Integrins; Family suidae; Femoral Artery; Framework Regions; Genetics-Mutagenesis; Goals; Half-Cystine; Heart artery; Human; Human, General; Hyperglycemia; Hypervariable Loop; Hypervariable Regions; IGF-1; IGF-1 Receptor; IGF-I; IGF-I-SmC; IGF1; IGF1R; Immunoglobulin Hypervariable Region; Injection of therapeutic agent; Injections; Insulin-Like Growth Factor 1; Insulin-Like Growth Factor 1 Receptor; Insulin-Like Growth Factor I; Insulin-Like Somatomedin Peptide I; Insulin-Like-Growth Factor I Receptor; Integrins; Intracellular Communication and Signaling; L-Cysteine; Lead; Leiomyocyte; Length; Lesion; Libraries; Mammals, Mice; Man (Taxonomy); Man, Modern; Mathematical Model Simulation; Mathematical Models and Simulations; Medical Imaging, Ultrasound; Medication; Methods; Mice; Moab, Clinical Treatment; Modeling; Models, Computer; Molecular Biology, Mutagenesis; Molecular Interaction; Monoclonal Antibodies; Murine; Mus; Muscle, Involuntary; Muscle, Smooth; Mutagenesis; Myocardial Infarct; Myocardial Infarction; Myocytes, Smooth Muscle; Patients; Pb element; Peptides; Pharmaceutic Preparations; Pharmaceutical Preparations; Phase; Pigs; Protein-Free Media; Proteins; Public Health; Reactive Site; Receptor, IGF Type 1; Receptor, IGF-I; Receptor, Insulin-Like Growth Factor Type 1; Scanning; Serum-Free Culture Media; Serum-Free Media; Signal Transduction; Signal Transduction Systems; Signaling; Simulation, Computer based; Smooth Muscle Cells; Smooth Muscle Myocytes; Smooth Muscle Tissue Cell; Smooth muscle (tissue); Somatomedin C; Stimulus; Streaks, Arterial Fatty; Stroke; Structure of femoral artery; Suidae; Swine; T-Cells; T-Lymphocyte; Testing; Thymus-Dependent Lymphocytes; Tissues; Toxic effect; Toxicities; Treatment Period; Ultrasonic Imaging; Ultrasonogram; Ultrasonography; Ultrasound Test; Ultrasound, Medical; Vascular Accident, Brain; aminoacid; antigen binding; atheromatosis; atherosclerosis plaque; atherosclerotic lesions; atherosclerotic plaque; atherosclerotic vascular disease; biological signal transduction; brain attack; cardiac infarct; cerebral vascular accident; computational modeling; computational models; computational simulation; computer based models; computerized modeling; computerized simulation; coronary attack; coronary infarct; coronary infarction; cultured cell line; diabetes; diabetic; diabetic patient; diagnostic ultrasound; disability; disease/disorder; drug/agent; gene product; heart attack; heart infarct; heart infarction; heavy metal Pb; heavy metal lead; human subject; humanized antibody; hyperglycemic; immunogenic; immunogenicity; in silico; inhibiting antibody; migration; novel; porcine; prevent; preventing; public health medicine (field); public health relevance; response; sonogram; sonography; sound measurement; stroke; suid; synthetic peptide; three dimensional structure; thymus derived lymphocyte; treatment days; treatment duration; ultrasound; ultrasound imaging; ultrasound scanning; vascular; virtual simulation; vulnerable plaque