Clinical Application of Phage Display Technologies
The Basis For The Development of Targeted Therapeutics
BY: Dr. Mekhled Al-Zaza
E-Mail: mekhled@authorsden.com
An unprecedented boom in the number and variety of technologies used to discover and develop new drugs has marked the last decade. Many of these new technologies (As Phage Display Technologies) arose from work surrounding the Human Genome Project
Much of the impact of genomics on drug development thus far has been focused on the identification and validation of biological targets. While much of this research on targets is based only on comparisons of the biology of health and disease, sooner or later it becomes critical to integrate the activity of chemical compounds with the body.
There are two different ways in which chemistry comes into play-- in the form of chemical probes or as compounds being evaluated as potential leads or drugs. The use of chemical probes to elucidate biology is the basis of chemical genomics. A large series of compounds are individually introduced into cells, with the aim of identifying a cell that then undergoes a specific phenotypic change. By identifying the compound introduced into that cell, and then finding which gene or protein was bound by the chemical probe, the researcher succeeds in finding both a genetic link to a change in phenotype and a chemical probe that can cause that change to occur. Genomics and proteomics can also be used in compound evaluation, by providing molecular details about the effect of a compound on the body. This approach may highlight mechanisms of action or toxicity, both of which can be critical for further compound optimization.
With the human genome project nearing completion, we are now faced with the unprecedented challenge of unraveling the function of a very large number of genes especially those associated with disease indications. For many of the targets emerging from genomic research, there is little or no information on their biochemical activity (1).
For many of the targets emerging from genomic research, there is little or no information on their biochemical activity. Using phage display technology, Karo Bio isolates peptides directed to functional sites on target proteins (2). The peptides can be expressed inside cells and shown to affect biological functions, thereby validating even targets of unknown function for drug discovery. The peptides can also be used to format HTS-compatible assays of chemical compound libraries to produce leads for clinical development.
The possibility of using an exponentially replicating phage to detect proteins is explored. Phage that bind to proteins through specific ligands or other means may provide an amplification system that could be of help in characterizing proteins from single cells or trace proteins from tissues. In this application the gels or blots containing separated proteins are screened with phage that can bind to them. Following phage binding the gels or blots are washed and then replicate plated on a host bacterial plate so that the resulting specific phage plaques indicating the presence of specific proteins can be visualized. This approach could theoretically detect a single protein molecule, and preliminary experiments indicate that it may be possible to approach this level of detection (3).
The design of protein therapeutics involves extensive knowledge of the structural and functional properties of proteins and peptides, how they interact with nucleic acids and proteins, form complexes, and ultimately impact pathways involved in disease. Optimizing protein drugs through protein medicinal chemistry involves improving the stability of peptides/proteins and their delivery, while addressing issues around immunogenicity, toxicity.
Directed evolution, guided by the structure and function of phage coat proteins, was used to discover a series of new phage display platforms (4). These studies illuminated the insidious nature of viruses, whose coat proteins are surprisingly resilient to extensive mutagenesis. Shotgun scanning uses combinatorial libraries of alanine-substituted proteins to rapidly map the functional epitopes of receptor-ligand interactions.
Displaying cell-targeting ligands on the phage coat to create phage particles capable of specifically binding, entering, and transuding mammalian cells can alter bacteriophage tropism. With appropriate cell treatments, we have obtained EGF-targeted phage transudation efficiencies of up to 45% in human tumor cell lines. We have selected novel ligands for human tumor cells from diverse libraries of targeted phage particles through the reiterative selection of phage from transduced (GFP-positive) cells. Our recent data suggest that the targeted phage particles themselves will be useful as vectors for gene therapy (5).
Large-scale characterization of genomic targets requires high throughput technologies that can generate research tools for validation of these targets as drug able Phage display technologies offer the ability to generate and select ligands against targets, and engineer drug-like properties into candidates that show high affinity and specificity.
Bimolecular display technologies are tools to construct a large pool of modularly coded biomolecules, and to display them for property selection followed by determination of. The structures via a decoding step. However, functional display of diverse genomic mammalian proteins usually requires native machinery for correct folding and post-transnational modification, not possible with current technologies such as prokaryotic phage display and in vitro RNA-protein fusions. We have overcome this limitation, by developing a display technology platform that creates protein display libraries using native expression systems, including human cells, where each expressed protein is covalently linked to its corresponding cDNA. The sensitivity of detection, accomplished by sequencing of the DNA, is many orders of magnitude better than direct detection of protein using mass spec or 2D gels. We have used this technology to screen small molecule drugs against human protein libraries, rapidly determining mode of action and the mechanism of drug side effects (6). All expressed proteins in a cell can be screened for interaction with a compound in a single experiment using this display technology.
Phage display technologies have proven to be a powerful enabling technology in genomics and drug development. The directed evolution of proteins can be engineered for specific properties and selectivity. They provide an approach for the engineering of human antibodies, as well as protein ligands, and for such diverse applications as arrays, separations, and drug development. And offer the potential for rapid development of validation reagents to a wide variety of cellular marker (7) s. Furthermore, for certain classes of proteins, libraries of antibody fragments provide a rich source of fully human therapeutic precursors. Also serve as a powerful approach to optimizing lead therapeutics for increased potency.
Phage display technology used to select and affinity mature peptides, human antibodies, and single domain antibody molecules with exquisite cancer specificity. For these three different types of molecules, phage libraries have been designed, made, and used, and different affinity maturation strategies were applied. Examples discussed will include the isolation of anti-CEA peptides, anti-MUC-1 and MHC-peptide antibodies, and various single domain variable domain-based antibodies. Such phage-library-selected peptides and proteins with anticancer address are expected to form the basis for more specific cancer diagnosis and treatments (8).
Low-molecular-weight ligands specific for the surface of cancer cells are discovered using phage display and/or small molecule screening. Ligands are then functionalized for use in cancer detection and treatment. Fictionalization includes conjugation to monocrystalline iron oxide nanoparticles (MIONs) and gadolinium for MRI-based detection, to radio metal chelators for radioscintigraphy, and to near-infrared fluorophores for optical imaging (9). Various aspects of the discovery and functionalization process will be discussed.
Phage display technology is using to make antibody libraries from lymphocyte subpopulations from celiac disease (CD) patients (10). CD is an autoimmune pathology related to the ingestion of wheat gluten. CD patients often develop other autoimmune disease such as diabetes, thyroiditis, alopecia, autoimmune hepatitis, and cerebellar ataxia. Phage libraries from CD lymphocytes proved to be a valuable source of antibodies to auto antigens. Characterization and biological properties of these antibodies will be discussed.
To identify HIV-specific epitopes, were screened random peptide libraries (RPL) displayed on phages by using HIV-positive sera. By several criteria, these peptides behaved as antigenic mimics of conformational B-cell epitopes generated in vivo in the course of the natural HIV-1 infection. Consistent with findings, sera of monkeys infected with SHIVs carrying envelopes from different primary isolates, such as DH12, 89.6, and 89.6P, also recognized the pool of HIV-specific epitopes (G. Scala et al., J. Immunol 1999, 162: 6155). When injected in a group of five Rhesus macaques with QS21 adjuvant, a pool of five epitopes induced an antibody response specific for each of the single epitopes, and this response cross-reacted with HIV-1 envelope proteins in three monkeys. The mimotope-immunized animals, together with a control group of four monkeys immunized with wild-type phages and a group of three naïve animals, were challenged iv. With 60 AID50 of SHIV-89.6PD. During three months of observation, monkeys in the control groups experienced high peaks of viremia, an irreversible decline of CD4 T cells, and AIDS-like syndromes (11). The mimotope-immunized monkeys were unprotected from primary infection; however, three out of five animals showed reduced levels of peak viremia with low or undetectable levels thereafter.
In order to look for new antibiotic targets and lead compounds, were developed a method for isolation of random peptides that inhibit essential intracellular processes in bacteria. A library of random peptides expressed as fusions to E. coli thioredoxin (aptamers) was expressed under the tight control of the arabinose-inducible PBAD promoter (12). Selections and screens were applied to the library in order to isolate aptamers that inhibit essential functions, a metabolic pathway, and a signaling pathway. We are currently characterizing some of these
aptamers.
There are a lot of research done in phage display technologies and therapeutic application: Cancer-Targeting Peptides and Antibodies, Ligands for Cancer Detection and Treatment, Phage That Home to Bone Marrow, Libraries from Celiac Disease Patients, HIV-1 Epitope as Candidate AIDS Vaccine, Aptamer-Based Bacterial Inhibition System (13).
The definition of a ligand/receptor-based molecular map of human vasculature by using phage display technologies, will translate phage display technologies into clinical applications and turn out to be the basis for the development of targeted medicine(14).
We have done research, which will be useful to develop this direction (mechanism of ejection DNA from of the bacteriophages heads by physical methods: Microcalometr, Viscosimetr)(15) ,(Clinical Application of Phage Display Technologies, The Basis For The Development of Targeted Therapeutics) (16).
Conclusion
We used coat of phage to develop targeted medicine and phage display technology. The translation of phage display technologies into clinical applications is the main problem and task, which faces people who is trying to discover and develop new drugs.
We used The DNA of phage, to study mechanism of ejection DNA from the the bacteriophages heads by physical methods: Microcalometr, Viscosimetr.We noticed the main factors of the DNA condensation and packaging in virus head and after its ejection through the whole with diameter close to ds-DNA, are caused by the surrounded solution “quality” and so called “hydration forces” between ds-DNA parallel packaged segments and more exactly by the deference of this parameters inside and out side the cased of the phage.
As result of the researches, will use of phage display technologies in definition of a ligand/receptor-based molecular map of human vasculature, which have been useful in functional genomic and proteomics. And display methods, which promise to have benefit in the development of therapeutics targeting many different disorders, including cancer, AIDS, autoimmune and other diseases.
References
1. Use of Phage Display Technologies in Developing Genomics-Derived Medicine
Dr. Davinder Gill, Millennium Pharmaceuticals, Inc.
2. Target Validation and Drug Discovery for Genomic Targets Using Phage Display
Dr. Paul Hamilton, Director of Molecular Biology, Karo Bio USA, Inc.
3.Targeting of Phage Display Vectors to Mammalian Cells
Dr. Erkki Koivunen, Group Leader, Department of Biosciences, Division of Biochemistry, University of Helsinki
4. Use of Exponential Amplification of Phage for Detection of Proteins
Dr. Carl R. Merrill, Laboratory of Biochemical Genetics, National Institute of Mental Health, National Institutes of Health
5. Teaching an Old Phage New Tricks: Artificial Coat Proteins and Shotgun Scanning
Dr. Gregory A. Weiss, Department of Chemistry, University of California, Irvine
6. Directed Evolution of Bacteriophage Tropism: Applications in Gene Discovery and Gene Therapy
7. Eukaryotic Protein Display for Analyzing Small Molecule-Proteome Interactions
Dr. Bassil Dahiyat, President and Chief Executive Officer, Xencor
8. 30 Phage Display Libraries from Celiac Disease Patients
Dr. Roberto Marzari, Department of Biology, University of Trieste
9.. Cancer-Targeting Peptides and Antibodies from Phage Display Libraries
Dr. Hennie R. Hoogenboom
10. Low-Molecular-Weight Ligands for Cancer Detection and Treatment
Dr. John V. Frangioni
11. Phage Display Libraries from Celiac Disease Patients
Dr. Roberto Marzari, Department of Biology, University of Trieste
12. Phage-Displayed HIV-1 Epitope as a Candidate AIDS Vaccine
Dr. Giuseppe Scala, Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health; and Department of Biochemistry, University of Naples
13. Aptamer-Based Bacterial Inhibition System
Dr. Jonathan Blum, Instructor in Medicine, Department of Microbiology and Molecular Genetics, Harvard Medical School; and Clinical Assistant, Division of Infectious
14. Mapping Human Vasculature by in Vivo Phage Display
Dr. Renata Pasqualini, Associate Professor of Medicine and Cancer Biology, M.D. Anderson Cancer Center, University of Texas
15.The Thermodynamic Basis of Mechanism of Bacterial Virus Infection Prof.Mrevlishvili G.M.,
Mdzinarashvili T.D., Al-zaza M., Tsindaze L.T., Tushishvili D.G. 15 -the International Conference on " Chemical Thermodynamics", julep 26- August 1,1998, Porto Portugal, C 3-2.
16. Clinical Application of Phage Display Technologies, The Basis For The Development of Targeted
Therapeutics. Dr.Mekhled Al-zaza. Poster abstract “Drug Discovery Technology Europe” ,15-16 March 2005 U.K
