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.
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.
|Maile, Laura A; Gollahon, Katherine; Wai, Christine et al. (2014) Blocking ?V?3 integrin ligand occupancy inhibits the progression of albuminuria in diabetic rats. J Diabetes Res 2014:421827|
|Maile, Laura A; Busby, Walker H; Gollahon, Katherine A et al. (2014) Blocking ligand occupancy of the ?V?3 integrin inhibits the development of nephropathy in diabetic pigs. Endocrinology 155:4665-75|
|Clemmons, David; Maile, Laura; Xi, Gang et al. (2011) Igf-I signaling in response to hyperglycemia and the development of diabetic complications. Curr Diabetes Rev 7:235-45|
|Maile, Laura A; Busby, Walker H; Nichols, Timothy C et al. (2010) A monoclonal antibody against alphaVbeta3 integrin inhibits development of atherosclerotic lesions in diabetic pigs. Sci Transl Med 2:18ra11|