T4 Phage Antibody Display to Develop Antibodies for New, Emerging or Engineered Threats

Executive Summary

Submitted By

Dr. Zhaojun (James) Ren and Dr. Derhsing Lai

At the present time, there is no solution to address the challenges associated with the new, emerging and engineered bio threat at the first encounter. Therefore it will be very important that we can develop the counter measure or antibody to neutralize the bio-threat as fast as possible. We propose the T4 phage antibody display technologies to develop an antibody library that can screen millions (10⁶) to billions (10⁹) of constructs and develop the antibody against the new, emerging and engineered bio-threat within a very short time (less than one week) to fulfill the goal of a shorter developing time and a low cost.

As early as 1997 the dual proteins or peptides T4 phage surface display system with intact domains at high copy number were originally developed by us [1]. Since then, phage display of protein and peptide molecular libraries have rapidly become a vital tool in studies aimed at identifying molecules that bind to a specific target and improving key features of existing molecules with respect to e.g. ultra-fine specificity, affinity and reaction rate characteristics.

In this study plans to construct the large phageT4–antibody libraries that contain high-affinity binders to almost any target protein, and to develop new methods for high-throughput selection of antibody–antigen interactions. This is a most robust procedure in vitro that closely mimics in vivo selection of antibodies in the human immune system. By far the most widely used system is the filamentous Ff phage (M13) display system that is small and restricted in some aspects. Phase I study of us will use the new T4 phage display system to perform:

• Used for immunological diagnosis and vaccine development / production
• Used for peptides/proteins library and antibody library constructions and screening T4 phage-antibody capture and engineering technology [30,31].
• T4 phage display can combine with RF ID Chip platform in the next Phase of proposal.

Introduction

Phage display is a powerful technique for identifying peptides or proteins that have desirable binding properties. The T4 phage display system will make a significant impact in various phage display systems for because: Phage T4 display system makes it possible to construct macromolecular complexes that can simultaneously display two proteins or peptides with different lengths and properties in hundreds to thousands of identical copies of designated protein antigens, domains, or enzymatic activities on a single macromolecular particle.

It is anticipated that such complexes will find widespread applications in vaccine development, protein-protein binding interactions, biotechnology and in drug discovery research. The powerful research values are especially included in protein /antibody libraries construction, that greatly facilitate protein / antibodies optimization, identification of structurally constrained peptide, target protein and its gene discovery, select cytokine receptor agonists, and for drug receptor agonists / inhibitors ligand binding site / active domain identification. In biotechnology, assembling massively multimeric copies of rare, expensive, or unstable antibody / enzymatic activities onto these macro-molecular platforms should make it possible to generate novel biotechnological reagents and techniques [12].

In recent years, the use of surface-display vectors for displaying polypeptides on the surface of bacteriophage, combined with in vitro selection technologies, has transformed the way in which we generate and manipulate protein domains such as antigens, antibodies and enzymes [30]. Phage T4 display is based on expressing recombinant proteins or peptides fused to T4 phage coat dispensable protein Hoc and Soc. the genetic information encoding for the displayed molecule is physically linked to its product via the displaying particle.

Using this technology, we are now able to design repertoires of ligands from scratch and use the power of affinity selection to select those protein domains or repositories having the desired biological properties from a large excess of irrelevant T4 phage particles. With T4 phage display, customer-made proteins (fused antigen peptides, antibodies, enzymes, DNA-binding proteins) may be synthesized and selected to acquire the desired affinity of binding and specificity for in vitro and in vivo diagnosis, various antigenic determinants, heterologous enzymes, single-chain antibodies, and combinatorial peptide libraries, in the future for immunotherapy of human disease.

This explains the basis of T4 phage bipartite display and investigates of the new phenomenon compared with plasmid, Ff phage expression and display. The T4 phage two sites simultaneously display technique is proposes to make contributions to biotechnological applications. This technique focuses mainly on T4-antibody library constructions and screening, and involves the simultaneous selection of VL and VH domains, respectively, where T4 phage display would have the greatest impact on antibody engineering, and vaccine development.

Recombinant antibodies have been displayed on bacteriophage [14], lytic phage [15], bacteria [16], yeast [17], ribosomes [18] provide invaluable data for both basic research and medical diagnostics. Antibodies have opened up new possibilities for high-throughput profiling of protein expression. The past years have seen a number of advances in library design [31]. Phage–antibody libraries have so far yielded antibodies to several hundred targets, including cell-surface markers, peptide hormones, other human proteins, and carbohydrates [30]. We are confident with T4 phage system to conduct a lot of work.

Application and Technology

Our T4 phage display system consists of two function-types of plasmids and three types of T4 phage derivative vector strains as follows:

Expression Plasmids:

• SOC- fusion expression plasmid pESoc,
• HOC- fusion expression plasmid pTHoc

Integration Plasmids:

• SOC- integration plasmids pRH;
• HOC-integration Plasmid pIH.

T4 Phages surface display vector sub-strains:

• SOC- gene deletion T4 phage T4-SOC;
• HOC- gene deletion T4 phage T4-HOC;
• Both SOC-HOC genes deletion T4 phage T4-SOC-HOC.

SOC and HOC are nonessential external capsid accessory proteins. SOC bind in trimeric arrays on the hexagonal capsid surface lattice, and HOC in the lattice center. Following refolding- processing - maturation of the major protein gp23, SOC binds in equimolar quantity to cleaved gp 23*, i.e. 960 monomer per head and HOC 160 per head [13]. The foreign proteins can be displayed by fusing its gene to SOC and HOC gene on phage genome or binding SOC- and HOC- fused foreign proteins onto the T4 polyhead on protein-level at > 104 copies number per polyhead of <50 aa (amino acid) sized domains with biological activities [1,3,11]. The foreign proteins are arisen from the target genes which are fused and expressed to SOC gene (C- terminal) or HOC gene (N-terminal) via the integration plasmids. The high T4 phage recombination rate and a positive selection marker employing phage lysozyme gene recombination established an easy route for showing of plasmid-manipulated SOC and HOC gene derivatives integrated into the phage genome.

The development of the bipartite T4 SOC- HOC protein display system is attractive, because two different proteins or peptides with different amino acid lengths and properties from different sources can be displayed simultaneously on one T4 particle surface in high copy number (Soc: 960 and Hoc: 160 per phage) in fixed relative spacing [13], and NH2 (Hoc) versus COOH (Soc)-terminal orientation.

Merits: T4 is superior and has great potential. Filamentous phage display has had a major impact on molecular cloning techniques; however, there are a number of limitations. The major advantages of the T4 system over the M 13 system are:

1. The potential for displaying two proteins/peptides simultaneously in multiple copies at fixed distances from each other. Unlike the proteins used in the phage M13 display system, phage T4 HOC and SOC are nonessential for phage infectivity and productivity and are added onto the capsid surface after the completion of capsid assembly. This may better simulate biologically relevant binding surfaces. M13 only N-terminal short peptides are displayed in high copy number from the major coat protein gpVIII, whereas only less than 5 copies of polypeptide can be displayed from a minor coat protein gpIII [12].
   
   
2. Secretion of the fusion does not occur. T4 proteins/peptides will not be eliminated by the secretory apparatus of E.coli. Unlike the M13 and yeast system, the fusion protein to be displayed is secreted through the host membrane, posing toxicity and folding problems for some cloned genes. The T4 SOC and HOC system overcomes these limitations, secretion- related toxicity is avoided in the lytic T4 phage [1,3,10]. Moreover, in icosahedral phage T4 assembly, protein folding takes place in the cytoplasmic milieu and have distinct advantage over the filamentous phages for display proteins.
   
   
3. Most importantly, T4 is able to display proteins with various sizes up to 800 amino acid (aa) length on SOC, more than 1000 aa on HOC [5], and with various sequences in a much less restricted way. One of the most valuable and different features of the SOC and HOC display system is the possibility of binding large protein domains in an ordered array on the phage surface.
   
   
4. Another distinctive feature is the foreign proteins could be displayed both on T4 capsids and polyheads (long planar tubules of the major capsid protein), i.e. not only on mature phage but also on intermediate empty coat, allows potential for structure determination of displayed proteins. Polyheads have an even higher carrying capacity for display [1,3].
   

Advantages over lambda and T7 phage system: The copy number is at least twice as high as the SOC display system is capable of presenting up to ~103 copies per virus particle and more than 104 per polyhead or giant phage particle of V3-sized, and perhaps much larger protein domains [1]. Furthermore, it can simultaneously display two different proteins / peptides, spacing-separately outside T4 envelope surface. T4 is able to display proteins with various sizes and various amino acid chains in a much less restricted stringency. T4 phage particles possess higher antigenicity.

We are confident that by employing this system the results would be a more efficient strategy and would prove to be more convenient, universal and efficient in the field of counter measure of bio-threats.

Reference
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