Discovery of target-selective ligands for diagnostic and therapeutic applications for cancer

An effective cancer treatment depends on two things. One is the ability to detect cancer early and the other is a therapy that can eliminate most or all cancer cells with least damage to normal tissue. The work describes here relates to the development and use of methods for discovery of cancer specific ligands. Such ligands can be peptides that bind selectively to a certain target or a peptide substrate that is catalyzed by a particular protease. These ligands can be used for development of a diagnostic probe or to design a targeted 'prodrug' therapy. First part of the work is about an approach for synthesis and screening of a 500,000 molecule 'dimeric' peptide library on beads. This approach was developed and used to identify molecules that can bind selectively to epithelial cancer cells for incorporation into an in vivo implantable probe that can capture early circulating cancer cells. Such an implantable probe will have the advantage of sampling whole or large volumes of blood as compared to current ex vivo methods that use a small volume. To demonstrate the feasibility of this idea in vitro binding experiments using BIAcore were done. These ligands were immobilized on an inert surface of polyethlylene glycol and it was shown that they could selectively capture cancer cells under shear stress conditions.; Our second aim was to find a small peptide which can bind specifically to PSMA so that it can be used for delivery of drugs or for imaging cancer. To start with, we cloned the extracellular domain of PSMA with a His-tag and generated a stable PSMA secreting drosophila cell line. Then; with the use of recombinant PSMA we screened 'in solution' an M13 phage displayed library that consisted of 109 random peptides. Bound phages were captured using magnetically labeled Anti-His tag. A consensus sequence was obtained. One peptide sequence represented more than 30% of the sequenced phages. This peptide was synthesized as a monomer and a dimer with Biotin or FITC.; The third part of work is about development and use of a proteomics based approach to identify substrates for FAP or Seprase. FAP is an excellent target for cancer therapy because it is expressed in most of epithelial cancers, with little or almost no expression in any normal tissue. FAP is a type II transmembrane serine protease and in vitro it has been demonstrated to have both dipeptidyl peptidase activity capable of cleaving N-terminal dipeptides from polypeptides as well as collagenolytic activity capable of degrading gelatin and type I collagen. A number of efforts are already being made to design FAP targeted drugs based on antibodies and small molecule inhibitors. (Abstract shortened by UMI.)...