Display technologies: application for the discovery of drug and gene delivery agents

Abstract

Recognition of molecular diversity of cell surface proteomes in disease is essential for the development of targeted therapies. Progress in targeted therapeutics requires establishing effective approaches for high-throughput identification of agents specific for clinically relevant cell surface markers. Over the past decade, a number of platform strategies have been developed to screen polypeptide libraries for ligands targeting receptors selectively expressed in the context of various cell surface proteomes. Streamlined procedures for identification of ligand-receptor pairs that could serve as targets in disease diagnosis, profiling, imaging and therapy have relied on the display technologies, in which polypeptides with desired binding profiles can be serially selected, in a process called biopanning, based on their physical linkage with the encoding nucleic acid. These technologies include virus/phage display, cell display, ribosomal display, mRNA display and covalent DNA display (CDT), with phage display being by far the most utilized. The scope of this review is the recent advancements in the display technologies with a particular emphasis on molecular mapping of cell surface proteomes with peptide phage display. Prospective applications of targeted compounds derived from display libraries in the discovery of targeted drugs and gene therapy vectors are discussed.

Fig. 1

Schema of available display technologies. All display platforms are based on the ability to physically link the polypeptide produced by a library clone to its corresponding genotype. This allows one to recover the DNA encoding the clone selected based on the desired polypeptide phenotype, such as binding to the target. In phage/virus display, linkage between the gene and the encoded polypeptide is achieved by expression of the polypeptide as a fusion with a coat protein from DNA packaged in the same particle. In cell display, linkage between the gene and the encoded polypeptide is achieved by expression of the polypeptide as a fusion with a cell surface molecule from DNA, which the cell receives in order to be transformed. In ribosome display, linkage between the gene and the encoded polypeptide is achieved by stabilization of complexes between the ribosome, mRNA and the encoded polypeptide upon termination of elongation with a permissive marker, such as chloramphenicol or low temperature. In mRNA display, linkage between the gene and the encoded polypeptide is achieved by a puromycin molecule covalently bonding the mRNA 3′ and the translated polypeptide upon the ribosome stalling at the junction of mRNA and an engineered single-stranded DNA linker. In covalent DNA display, linkage between the gene and the encoded polypeptide is achieved by covalent bond that forms between the DNA-binding protein P2A (produced as a fusion with polypeptide) with the DNA encoding the fusion. Figure is not depicted in scale.

Fig. 2

A comprehensive strategy for systematic characterization of ligand/receptor proteome functional on the surface of NCI-60 cancer cell lines by using ligand-mimicking peptides isolated from combinatorial phage display libraries. Identification of cell surface receptors through their soluble ligands enriches for receptors likely to be “targetable” (meaning, can be ligand-directed).

Fig. 3

Schema of an in vivo display library selection. The library of displayed polypeptides is intravenously injected into an animal and let circulate to allow binding of polypeptides to differentially expressed receptors. After that, the blood vessels are perfused to remove unbound library and then the bound ligands are eluted from the extracted tissue. The library clones encoding the ligands are amplified and injected into an animal for a subsequent round of in vivo selection.

Fig. 4

Scheme of a strategy for serial synchronous in vivo display library selection. In each round, the statistical significance (p-value) of recovery frequency is compared between tissue-homing peptides (black line) and nonspecific control peptides (background color lines). In each round, recovery frequencies are compared between tissue-homing peptides (black line) and nonspecific control peptides (background color lines). Progressively increased frequencies of peptides with every subsequent round of selection reflect the enrichment of peptides preferentially homing to the target organ.