Agents which are capable of specifically targeting tumor vasculature have potential diagnostic and therapeutic applicability to a wide variety of solid tumors. Growth of any primary tumor or metastasis beyond a very small size is dependent on the formation of new vasculature. Targeting tumor vasculature offers an additional mechanism with which to control primary and metastatic disease. The alphav,beta3 integrin, a cell membrane protein, is relatively conserved in its expression, with few normal tissues expressing this molecule. However, alphav,beta3 is upregulated on proliferating endothelial cells. Endothelial cells proliferate at a very low rate in normal tissue, and the proliferation rate is increased only in the uterus, healing wounds and tumors. Thus the alphav,beta3 integrin is a potential neovasculature target for diagnostic and therapeutic applications. Peptides with a cyclized RGD sequence recognize the alphav,beta3 integrin with high specificity. The objective of this proposal is the determination of a more optimal targeting agent and initial characterization of these novel tumor neovasculature targeting agents. To this end there are three specific aims: 1) to produce and radiolabel a peptide with one or more ACDCRGDCFCG binding motifs; 2) to determine the specificity, avidity and affinity of the monovalent and multivalent peptides in vitro to ascertain which forms are most likely to be useful for diagnostic imaging and therapeutic purposes; and 3) to determine the normal tissue distribution and maximum tumor and normal tissue uptake of the various peptides, as well as the stability and half-life of the peptides in vivo and when bound to tumor vasculature. Peptides will be produced by insertion of plasmids bearing the DNA sequence of the desired RGD peptide into E. coli bacteria. The protein is then collected and purified. Kinetic analysis will be performed using D247, a alphav,beta3 integrin-expressing cell line, and traditional calculations such as the Scatchard analysis. Dual-label techniques using RGD and a DRG peptides labeled with I125 and I131 respectively will be used to determine normal tissue and distribution and tumor uptake.
