Use of Bacteriophages to Prevent, Diagnose, and Treat Diseases

Adhya, Sankar

Abstract

The purpose of this project is to create a new method of drug delivery to cancer cells through a combination of internal and external protein display on bacteriophage T4 and utilizing mammalian endocytosis pathways. Phage T4 has been used extensively for protein display. The two accessory capsid proteins, Hoc and Soc that are non-essential for capsid formation. One can fuse other non-phage proteins to either Hoc or Soc or both in order to display the fusion protein on the phage head surface. This is achieved by growing mutants of T4 negative for Hoc or Soc in bacterial host that expresses from plasmid(s) HOC and SOC fused to single chain antibody genes that have been shown to bind to specific cancer cells for display, causing attachment of fused antibody to head particle during phage maturation16. Hoc can display at either its N or C terminus, while SOC can display at its N and C termini concurrently, allowing for large numbers of displayed proteins, or variations in the proteins being displayed. The mature head of T4 also contains, among others, internal proteins called IPI, IPII, and IPIII. The latter three proteins can be fused to contain foreign proteins through linker sequence, which is cleaved by protein gp21through the cleavage sequence, termed CTS making the two cleaved products free. This discovery has allowed a variety of proteins from 12-160kD to be fused to the CTS. The process takes place inside the phage head article. The internal proteins are all released into the phage host during DNA injection. The foreign proteins have demonstrable biological activity after injection into the bacteria. Endocytosis. A number of endocytosis pathways in mammalian cells have been elucidated to date. They are generally categorized as clathirin or caveolae dependent pathways, macropinocytosis, ICAM-1 (intercellular adhesion molecule -1) pathway, etc. Of these, the ICAM-1 pathway involves endocytosis with formation of relatively large vesicles (up to 500nm), movement to late endosomes and recycling compartments, as well as lysosomal trafficking. The variety in the pathway allows for degradation of the internalized cargo or recycling of internalized receptors and signaling proteins back to the cell membrane. Cancer cells look different from normal cells in overexpression of membrane associated proteins, including ICAM-1 receptor (as an indicator of metastatic activity). The overexpression of these molecules allows antibodies against the surface proteins and other affinity-based proteins to target cancer specifically, wiping out malignant cells while leaving healthy cells relatively untouched.