Phage Display Selection and Identification of Novel Pancreatic Cancer Targeting Peptides

Abstract

Pancreatic ductal adenocarcinoma (PDA) is currently the fourth leading cause of cancer deaths in the U.S. It has been recently estimated that the disease causes approximately 30,000 deaths per year in the U.S., and that pancreatic cancer patients exhibit a 5‐year survival rate of only 3%. The high lethality of pancreatic cancer is due to the fact that most cases are diagnosed at advanced metastatic stages. While there are diagnostic methods in use clinically, most are inadequate, potentially leading to delayed diagnosis and further spreading of the cancer prior to treatment. Due to insufficient diagnostic techniques, it is imperative to develop new methods, which may facilitate early diagnosis. One such modality employs the use of peptides with high binding affinity and specificity to a pancreatic cancer biomarker.Bacteriophage (phage) display technology is a high throughput method for discovering peptides that confer specific binding properties to a single molecular target. The peptides are typically displayed on the distal end of coat protein III of the Fd filamentous phage. Here we report using phage display technology to select peptides with specific binding to human pancreatic cancer cells. To do so, a naïve fUSE5 15‐mer library (kind gift from Dr. George Smith) was negatively selected against normal pancreatic ductal cells (hTERT‐hPNE). Unbound cells were collected, amplified by infection of E. coli K91BK overnight, and isolated using polyethylene glycol (PEG), NaCl precipitation and centrifugation. Amplified phage were then subjected to four rounds of positive selection against pancreatic cancer cells (Mia‐Paca‐2) grown to 80–90% confluency. Between each round of selection, phage were collected, amplified in E. coli K91BK and isolated as described above. Stringency was increased in the fourth round of selection by performing the selection against cells grown to 60% confluency. After the final round of selection, phage DNA was extracted and polymerase chain reaction (PCR) amplicons were identified by paired‐end (2×150bp) next generation sequencing (MiSeq) followed by translation and sorting of the sequences (Genewiz, NJ). The “top” 10 peptides identified by this method will next be screened in cell binding assays to determine individual binding constants. Peptides that exhibit high binding affinity and specificity for Mia‐Paca‐2 cells may subsequently be employed as probes in the detection of pancreatic cancer.Support or Funding InformationWestern Illinois University Research Council GrantThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.