612. Effect of Adenovirus dl520 as Replicating Agent in Combination with Fractionated Radiotherapy in a Glioma Xenograft Model

Abstract

Top of pageAbstract Phage display libraries have provided an extraordinarily versatile technology to facilitate the isolation of peptides, growth factors, single chain antibodies, and enzymes with desired binding specificities or enzymatic activities. However, overall diversity of peptides in phage display libraries can be significantly limited by Escherichia coli protein folding and processing machinery, which result in sequence censorship. To achieve an optimal diversity of displayed eukaryotic peptides, the library should be produced in eukaryotic cells using a eukaryotic display platform. Previously, we have demonstrated that polypeptides (peptide, growth factor, single chain antibodies) of various sizes could be efficiently displayed on the envelope glycoproteins (SU) of a eukaryotic virus, avian leukosis virus (ALV). The displayed polypeptides could efficiently attach to cognate receptors without interfering with viral attachment and entry into susceptible cells. Furthermore, we constructed a model library of randomized eight amino acid linear peptides using the ALV eukaryotic display platform and screened the library for specific epitopes using immobilized antibodies. A virus library with approximately 2 × 106 different members was generated from a plasmid library of approximately 5 × 106 diversity. The nucleotide sequences of the randomized 24 nucleotide/eight amino acid regions of plasmid and virus libraries showed no significant sequence censorship. Different populations of peptide epitopes were selected from the virus library when different monoclonal antibodies were used as the target. Most of the phage, yeast and retrovirus peptide display libraries have been built in linear and cyclic form and peptides have been selected from these libraries against targets that recognize a short continuous sequence. However, most molecular recognition events are mediated by folded proteins with binding sites created from different exposed regions of the polypeptide chains. In the present study we have displayed different groups of protein molecules and scaffolds on SU of ALV. The displayed molecules were a homotrimer cytokine (macrophage migration inhibitory factor), different knottin protein scaffolds with disulphide-bonds i. e. ω-conotoxin (GVIA), and cellulose binding domain (CBD). We also took advantage of expression in a eukaryotic system and displayed a glycosylated form of CBD on SU of ALV. Moreover, we also displayed green fluorescent protein (GFP) on the SU of ALV. GFP arises from a novel three-dimensional fold called β-can formed by11 β-strands wrapping into a cylinder. Viruses with chimeric envelopes replicated at similar rates and to approximate similar titers as wild-type virus. Both wild-type and chimeric viruses incorporated similar levels of envelope glycoproteins in viral particles and the displayed domains were stably incorporated into chimeric viruses. We are now in process of constructing a large ALV display library by randomizing seven amino acids in CBD that will be used to isolate novel cell-targeting polypeptides.