High Throughput Sequencing Enhanced Phage Display for Affinity Ligand Discovery 304 views

Phage display is a popular technique used in numerous applications; it discovers molecules that selectively bind anything from cancer cells to TNT. Its versatility is exemplified in its numerous applications; It is used to discover potential therapeutic targets, as well as develop new therapeutics, map epitopes of protein-protein interactions, and select tissue and cell targeting ligands for targeted imaging and therapy. However, phage display screens are frequently hindered by a high false positive rate. The cause of this issue remained difficult to elucidate and correct because phage display screen results were typically analyzed with low throughput Sanger sequencing. We improved phage display by integrating it with high throughput sequencing, making it possible to quantify the diversity of phage libraries and more efficiently select targeting molecules from the libraries. We also developed new methods to analyze the high throughput data. These improvements will make phage display an even more attractive technology, by making the process quicker and more cost-effective. To confirm that this new approach to phage display is effective, we performed screens on streptavidin and found that our approach easily identified the correct molecules with a much lower false positive rate. After this confirmation, this new technique was utilized in multiple new screens. First, we applied it to pancreatic cancer by performing screens on pancreatic cancer-derived, cancer-associated fibroblasts (CAFs) and discovered targeted peptides that selectively bound the CAFs in pancreatic cancer tumors in vivo. Next, we looked for a targeted delivery option for PPAR agonists, which are efficacious therapeutics for patients with metabolic syndrome, but whose use is limited by their side effects. An innovative phage screen was performed on adipose tissue samples from patients with metabolic syndrome and then clinically relevant, targeted drug-loaded liposomes were developed with peptides discovered in the screen. Imaging the immune response in vivo is an important currently unmet clinical need. We screened T cell surface proteins in pursuit of developing an agent capable of imaging T cells. We found peptides that selectively bind their targets and developed targeted liposomes with the peptides. The design of high throughput sequencing enhanced phage display yielded an effective technique for affinity ligand discovery.

PHD (Doctor of Philosophy)

Phage Display; High Throughput Sequencing; Pancreatic Cancer; Targeted Liposomes

English

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2018-04-26