Generation of Antibodies by Phage Display - Literature review Example

Generation of Antibodies by Phage Display Name Institution Literature review 1.5.1 The phage Display Technology One of the most important developments of 1980s in medical field was the development of the phage display technology. Initially, the technique was limited to the selection of peptides. However, development of the technique made it possible for display of the antibodies fragment on phage. The technology is grounded on genetic engineering of coat proteins .of filamentous bacteriophages of E. coli such as M13 or FD. During the phage propagation, elongated phage particles assemble through polymerization of the major gene VIII coat protein, before being capped by the terminal gene III protein (Villa 2008). The technology was first described by George P. Smith in the year 1985. Genetics fusions with both coat proteins are possible, resulting to either low-level display (geneIII, 1-5 copies)2 or high-level gene VIII, ≥1000 copies) (Villa 2008). The technology differ with hybridoma technology in the sense that it allows the selection of antibodies against an almost unlimited arrays of biological and non-biological targets, including self-antigens, while completely avoiding use of experimental animals (Villa 2008). The technology allows presentation of large protein and peptide libraries on the surface of the filamentous phage. The presentation makes it possible to select proteins, peptides and antibodies, with high affinity and specificity to almost any target. An exogenous peptide sequences is introduced into a location in the genome of the phage capsid proteins. The encoded peptides are displayed on the phage surface as a fusion product with one of the phage coat protein (Villa 2008). Phage Display Technology has a variety of applications such as therapeutic target validation, vaccine development, drug design and epitope mapping among others. The technology is also used to pick inhibitors for the active and allosteric sites of G-protein binding enzymes, modulatory peptides, receptors, agonists and antagonists (Wirsching Riester and Schwienhorst 2003). It has greatly enhanced the development and discovery of antibody drugs. Analysts continue to explore more options in which the technology can be applied. 1.5.2 Filamentous Phage Filamentous phage apper as thin (6-10nm) and long filamentous particles containing circular single stranded DNA. The actual length of the particle is determined by the length of the DNA it has encapsulated. The filamentous phage belongs to a group of related viruses which only infect gram-negative bacteria via specific adsorption to the tips of bacterial structures called F-pilli. The F-pili are normally involved in the transmission of the F plasmid DNA, or chromosomal DNA containing the intergrated plasmid DNA, from one bacterium to another. Unlike other bacterial viruses, the Filamentous phage does not kill its hosts, but establish a relationship in which new virions are continually released. Because of this non-lytic mode of release, it is possible to grow high-titre cultures of the virus. Also, the growth curve of the filamentous phage shows a rapid increase of virus after a short latent period. The first progeny phage particles are released about 15 minutes after infection and the rate of production is exponential for the first 60 minutes, after which it becomes linear as the bacteria enter stationary phage. The rate of production implies that roughly 1000 phage per bacterium are produced within the first hour after infection (Villa 2008). 1.5.2.1 Filamentous Phage Structure and life cycle Filamentous Phages comprise of long thin filaments resembling the bacterial surface structure referred to as pili (Okayama & Berg 2008). The phage is built by helical arrangement of many copies of a single subunit, and their morphology. These phages which include the most common F1, Fb and M13 phages are single-stranded DNA (Okayama & Berg 2008). It consist of a long lop of ssDNA that is coated with multiple copies of the major coat protein pVIII (O’Neil and Hoess 2013). The formed tube is usually 1µm long and 7nm in diameter. However, since the coat is made of multiple subunits of pVIII, it can be lengthened to accommodate more ssDNA. Additionally, there are four minor coat proteins on the surface of the phage. The filamentous phages are capable of infecting only male bacteria that carry the F sex pillus. The infection is initiated by binding of the coat protein Piii to the sex pillus which results to retraction of the pilus and internalization of the phage (O’Neil and Hoess 2013). The infected bacteria cannot produce sex pilli which prevent infection of bacteria of more than one phage. A complex process takes place inside the cell where the phage is replicated. The replication is done in a controlled manner and infection with filamentous phage does not lead to death or lysis of the infected bacteria (Hong and Clayman 2000). The infected bacteria continue to produce phage on a continual basis. Assembling of the phage particles does not take place inside the cell. Instead, it occurs as the phage is extruded through the bacterial wall (Ruoslahti, 2002). The pIII and pVIII coat proteins are made with signal peptides that are further used in the production of the perisplasm of the Gram-negative E. coli (Hammer Takacs and Sinigaglia, 2008). The other coat proteins contain hydrophobia sequences that lead to association with the inner membrane of the bacteria. Periplasm is an oxidizing environment allowing formation of disulphide bonds (Hammer Takacs and Sinigaglia, 2008). The site is a good site for the production of recombinant molecules including the antibody fragments. The phage ssDNA proceeds to the cytoplasmic face of the bacterial inner membrane by one of the phage control proteins (pV) and as it passes through the periplasm, it sheds pV and picks up the various coat proteins before it is released as complete virion into the medium (Hammer Takacs and Sinigaglia, 2008). 1.5.2.2 Phagemid Cloning Vectors Phagemid cloning vector is a hybrid cloning vector that is formed from Plasmids and filamentous phage M13. The vector can grow into a plasmid and also can be packaged as a single-stranded DNA in viral capsids (Arap Pasqualini & Ruoslahti 2009). The bacterial host containing the phagemid is subjected to infection in order to enable single-stranded DNA replication and packaging of the phegemid DNA into phage particles. The process is done with presence of helper phage which provides the viral components in the vector (Arap Pasqualini & Ruoslahti 2009). 1.5.3 Antibody Fragments There are many fragments of antibody which are produced through the genetic or chemical mechanisms. The chemical fragmentation involves use of reducing agents to break bonds within the hinge region. This is followed by digestion of the antibody with proteases including papain and pepsin (Wirsching, Riester & Schwienhorst 2003). Genetic fragmentation, on the other hand, involve creation of a multitude of fragment containing molecules, each with unique binding and functional characteristics (Wirsching, Riester & Schwienhorst 2003) Some of the most common fragments of an antibody include F (ab’) 2, Fab’ and Fv. All these fragments are antigen-binding fragments generated from the variable region of IgG and IgM. The fragments take different valency, and Fc content. Other than the binding antigen, there is an Fc content fragment. Fc fragments are generated from heavy chain constant region of an immunoglobulin (Russel M., 2010). (a) F(ab)’2 Fragments F(ab’)2 (11000 daltons) fragments comprise of two antigen-binding regions which are hinged through disulfides (Russel M., 2010). (b) Fab’ Fragments Fab’ (55,000 daltons) fragments are obtained through chemical reduction of F(ab’) fragments. Each fragment comprises a free sulfhydryl groups which are either be alkylated or utilized in conjugation with an enzyme, toxin or other proteins (Russel M., 2010). Since it derived from F(ab’) they sometimes contain a small portion of Fc. (c) Fab Fragments Fab (50000 daltons) fragments are produced from IgM and IgG, consisting of the CL, VH, CH1 and VL regions that are licked by an intramolecular disulfide bond (Russel M., 2010). (d) Fv Fragments Fv (25,000 daltons) are produced from IgM and IgG that contains a complete antigen-binding site. They are the smallest fragment produced from the IgM and IgM. They are similar to the Fab in regard to the binding properties and characteristics. They have three-dimensional binding characteristics and binding properties like the Fab Fragment (Greenwood, Hunter & Perham 2006). The VL and the VH chains of these fragments are bound together by non-covalent interactions. Since the chain tends to break upon dilution, a method has been devised to cross-link the chain through peptide, Intermolecular and glutaraldehyde linkers (Greenwood Hunter & Perham 2006). (e) Fc Fragments Fc (50000 daltons) fragments comprises of CH3 and CH2 regions and part of the hinge region. The hinge regions are bound together by use of disulfides and non-covalent interactions. Like the other fragment of antibody, Fc fragments are produced from fragmentation of IgM and IgM. They are, however, produced from heavy chain constant region of an immunoglobulin (Greenwood, Hunter & Perham 2006). Unlike the other fragments, Fc fragments do not bind antigen. However, it is responsible for the effector functions of antibodies such as complement fixation (Greenwood, Hunter & Perham 2006). 1.5.4 Construction and Screening of phage-displayed antibody libraries In order to display antibodies, proper construction of the phage library is necessary. Phage library displays the antibody fragment on the surface of the phage. There are several types of phage display libraries such as protein libraries, peptide libraries and antibody libraries. The type of the phage display library to be constructed depends on the application in which the library is intended. There are those that are intended for medical research while others are constructed to serve scientific purpose. However, the most common type of libraries is scFv Phage Display Libraries (Villa 2008). 1.5.4.1 Construction of ScFv Phage Display Libraries Currently, antibody engineering depends on the ability to screen large combinatorial libraries of antibody genes (Sidhu 2005). In order to isolate a highly specific antibody for many biomedical uses; the library of 105-1011 clones must be screened (Villa, 2008). The attempt to design and produce high throughput screening methods started in mid 1990s. The attempts produced little fruits until the development of various display systems. The use of better display systems made it possible to establish a direct physical link between the protein encoded by a gene, a gene, and molecule recognized by this protein (Villa 2008). This development in the display systems has considerably improved screening of the large recombinant protein libraries. Currently, the most common used method of screening of protein and peptides in the antibody is phage display. 1.5.4.2 Types of Antibody Repertoires The antibody repertoire refers to the entire set of antibodies produced with respect to a particular tissue. Most of the antibodies are natural occurring while some are artificially made. The natural ones are classified as immune and Naïve repertoires while the artificial one is referred as synthetic repertoires. 1.5.4.2.1 Naïve Repertoires: “Single Pot Libraries” Naïve repertoires are generated by tapping the natural primary unselected immune repertoire through cloning of Abs that recognizes a variety of Ags (In-Ahrens 2005). It involves amplifying the rearranged V genes with the polymerase chain reaction from B-cell mRNAs encoding Immunoglobulin M that is taken from non-immunized donors. The procedure enables recovery of Abs before the encountered AG and unscreened for tolerance by the immune system. A naïve repertoires is a major source of Abs against toxic Ags and nonimmunogenic (Hong and Clayman 2000). 1.5.4.2.2 Immune Repertoires Immune repertoire refers to the sum of total of functionality diverse B and T-cells in the circulatory system (In-Ahrens 2005). In the phage display, immune libraries are obtained only against the set of antigens to which an immune response was induced. They are normally to study immune response of animals. An immune phage antibody repertoire is enriched with antigen-specific antibodies some of which have been affinity matured by the immune system. The repertoires yield to high affinity than obtained from hybridomas (In-Ahrens 2005). Naïve repertoires Naïve libraries which generate the naïve repertoires can be created from collections aV-genes from the IgM/IgM mRNA of B cells of the non-ummunised human donors, isolated from peripheral blood lymphocytes, bone marrow, tonsils or spleen cells or from animal and human sources, e.g mice. IgM and/ or IgG variable regions for amplified and cloned into the vectors designed for selection and screening (Hong and Clayman 2000). It is important for naïve library to be as large as possible. The number of at least 108 individual clones is necessary to prepare of a representative sample of binding repertoire. An ideal naïve repertoire library contain a representative sample of the primary repertoire of immune system, although it does not contain a representative sample of the primary repertoire of immune system, although it does not contain the great proportion of antibodies with somatic hypermutations produced by natural immunization (Villa 2008). Immune repertoires are generated in immune antibody libraries. Its pool of antibodies is enriched with antigen-specific antibody and a significant portion of these antibodies have undergone affinity maturation by the immune system. The libraries use immunized animals to isolate –genes from the immune the IgG mRNA of B-cells. Under some circumstances the recombinant antibody selected from the immune antibodies obtained from hybridomas technology (Clackson & Lowman 2004). The advantage of these repertoires is that the preparation of the libraries is easier than naïve repertoires libraries. However, it is time requirement is long for animal immunization. 1.5.4.2.3 Synthetic Repertoires Synthetic repertoires are generated by mutational modification of the CDR sequence of selected frameworks coding antibody variable domains. CDRs are modified by predetermined level of randomization. These repertoires are then cloned into an antibody domain framework and subcloned to a phage display vectors to generate a library of about 107—1010 clones (Villa 2008). There are also synthetic repertoires which are produced by selecting one or more antibody frameworks within the CDR loops. The CDR regions can be completely or partially designed randomized oligonucleotides. The types of the synthetic repertoires can be constructed either with restricted V-gene usage or by all of the known V-gene segments of the species. High diversity in composition and length is found particularly in the CDR3 H3, which significantly contributes to antigen-binding, making this particular CDR an attractive target for randomization. Efficient cloning system and a combination of the dual antibody cloning strategy allows construction of very large repertoires with about 109-11 individual clones (Villa 2008). The main disadvantage between these repertoires over the naïve repertoires is that the contents, local variability and overall diversity can be controlled and defined. 1.5.5 Selection of Antibody Libraries: Bio-panning The phage antibody selections involve the sequential enrichment of specific binding phage from a large excess of non-binding clones (Villa 2008)). This is attained through multiple rounds of phage from a large excess of non-binding to the target washing to remove non-specific phage and elution to the retrieve specific binding phage. Any procedure that separates clones that bind from those that do not can be used as selection methods. Consequently, many different selection methods are available for the separation of the antibodies. The most used methods include selection using immobilized antigens, in vivo selection, selection using antigens and selection in cells. 1.5.5.1 Selection using immobilized antigens In this method, the avidity effect that is produced by the immolilization of the antigen on the phage surface enables selection of ligands with weak affinity (Barbas, 2011). In this method, an immunotube is coated with 2 mL of antigen with PBS at moderate temperature for an overnight (Barbas 2011). A tube is washed three times with PDS and blocks the tube for a period of one hour with the 4mL of 2 percent MPBS at 380 C. The third step involves washing the tube for another three times with PBS and adding 1mL of 1012-1013 phage antibodies that is diluted with 1 mL of 2 percent MPBS and incubate for 30 min on a rotating turntable followed by an additional two hours standing (Barbas 2011).The tube is once again watched with PBS/Tween and twice with PBS by pouring buffer in and out of the tube. This is followed by eluding the bound phage by adding 1.0 mL of 100 Mm triethlamine, capping the tube, and rotating it on a turntable for 8 min (Barbas 2011). The eluent is transferred to a new and neutralized immediately by adding 0.5 mL of 0.1 M Tris-HCL immediately. One-half of the eluent is added to 10 mL of exponentially growing E. coli TGI and the culture is incubate without shaking at 370C for half an hour. In order to titer the eluted phage, 1µL and 0.1µL of the culture is plated on 100 mm TYE/ amp/ glu plates. Plating these two amounts of culture covers an out-put titer of 104-107 at a given number of colonies. Since the titer tends to increase, it is necessary to plate additional dilutions during the later rounds of selection (Villa 2008). The remainder of the culture is centrifuged at 2800g for 15 min, resuspended in 0.5 mL of 2X TYmedia, plated on two 150 mm TYE/amp/glu plates, and incubated at 370 C for a period of overnight (Villa 2008). In the following day, a 3mL of 2XTY/ glu media is added to each plate and scraped the bacteria from the plate with a bent glass rod. A glycercol stocks are made by mixing the 1.4mL of bacteria and 0.6mL of 50 percent glycerol. This stock is saved at a temperature of -700C. The next step involves the preparation of phagemid particles for the next round of selection. The phagemid is prepared from the glycerol stocks by infection with helper phage. The selection process is repeated from the step 1 for a total of 2-4 rounds. 1.5.5.2 Selection using antigens in solution Other than selection of antibodies on immobilized antigen, the selection can be done in a solution. The selection of antibodies in a solution is very appropriate in dealing with problems related to antigens that change conformation when coated onto solid surfaces. In addition, selection of antibodies in a solution is easier than in an immobilized solution. In order to select phage antibodies in solution antigen is biotinylated using any kits sold by immunochemical reagent companies (Mathys 2011). The biotinylated antigen is incubated where the antigen and bound phage antibodies are separated from unbound phage antibodiesusing avidin magnetic beads and streptavidin (Mathys 2011). The method involves biolinlating the antigen using a kit as recommended by the producers. For the case of diothiothereitol, NHS-SS biotin is used for biotinlation (Barbas 2011). A 1.5-mL microcentrifuge tube is blocked with 2 percentage MPBS at room temperature for an hour then washing the tube once with PBS (Chames 2012). This is followed by blocking 100µL of streptavidin-magnetic beads with 1 mL of 2 percent MPBS at room temperature for an hour in a 1.5 Ml Microcentrifuge tube (Proetzel & Ebersbach 2012). After blocking, the beads are collected by pulling one side with the magnetic tube holder. The buffer is discarded. The library of streptavidin binders is pre-depleted by incubating 1012-13 phage (1013 for a first-round selection, 1012 for subsequent rounds) in 2 percent MPBS with 100 µL of streptavidin-magnetic beads for an hour in a volume of 1mL (Proetzel & Ebersbach 2012). The beads are pulled to one side with a magnetic tube holder and the phage antibodies are collected and the beads are discarded. 1 mL of pre-depleted phage antibodies are added to the blocked tube and then biotinylated antigen is added (Proetzel & Ebersbach 2012). 100µL of the blocked streptavidin-magnetic beads are added to the tube and incubated on the rotator at room temperature for 15 min. The tube is placed in the magnetic rack for 30s. This makes the beads to migrate towards the magnet. The tubes are aspirated leaving the beads on the side of the microcentrifuge tube. The aspiration is done with a 200-µL pipet tip on a pasteur pipet attached to a vacuum source. The beads are washed with PBS/Tween, followed by twice with 2 percent MPBS. The beads are then transferred after every second wash to a fresh 1.5-mL to enhance the efficient washing (Proetzel & Ebersbach 2012). ImL of 100mM triethylamine is added to elute the phage. The tube is capped and rotated end-over-end for about ten minutes. The beads are drawn to one sides of the tube with the magnet and the solution is transferred to an Eppendorf tube that is containing 500 µL of 1M Tris-Hcl, pH 7.4 ((Proetzel & Ebersbach 2012).A number of selections are done which can be determined by performing an ESISA for antigen binding using the polyclonal phage prepared from every round of selection (Proetzel & Ebersbach 2012). 1.5.5.3 Selection on cells Selection on cells is a special case where antigen forms Son cell surfaces. Direct panning on cell surfaces carrying the antigen are carried out on adherent cells grown in monolayers, or in suspension. Antigens are normally in low concentration on the cell surfaces and cell sorting and substitution are practically hard. Due to this limitation, the selection is not commonly used as it results to discrepancies with the expected results. 1.5.5.4 In vivo selection In this selection, phage libraries are injected intravenously into animal and the tissues are collected and examined for phage bound to tissue-specific endothelial cell markers (Hong and Clayman 2000). The technique, which was first used Pasqualini, was initially used to isolate peptides that are home to cerebral vascular endothelium and renal in vivo. The technique has later been used for identification of receptors-ligand pairs in various body organs such as uterus, testiness, adrenal gland, lungs and kidney. The technique has a number of advantages. First, peptides displayed on the phage particles are identified and tested functionality and must overcome natural mechanisms of degradation. Second, peptides recognizing unspecific molecules are depleted from circulation. Third, in vivo selection proved to be able to identify receptors expressed selectively on tumor endothelium. The receptors may serve as molecular targets for the development of diagnostic techniques and targeted therapies. 1.5.5.5 Remarks The section has reviewed various selection methods used in the selection of antibody libraries. The most common methods include selection using immobilized antigens, selection using antigens in solution, in vivo selection and selections on cells. The procedure involved in each method has been described as well as their advantage over the others. Selection using antigen is the most preferred over the other due to its advantages. In vivo selection is becoming increasing popular due to its applications in the medical and research institutions. 1.5.6 In vitro Diagnostic Applications of Phage Display In vivo finds a wide application in the study of antibodies and proteins. Some of the applications include isolation of high-affinity antibodies, mapping antibody and generating immunogens. In the generating immunogens, small segments of different proteins were displayed on M13 virus particles to elicit antibodies against the coat proteins of parasites and viruses (Barbas 2011). The immunological responses to injected M13 phage are T-cell dependent and do not require adjuvant. In regard to isolation of high-affinity antibodies, phage is useful in cases where monoclonal antibodies cannot be obtained by classical hybridoma techniques, such as antibodies against non-immunogenic or toxic antigens (Barbas 2011). Lastly, in vivo is applied in mapping of antibody epitopes. Particles of DNA that encode parts of the protein antigen are fused to a gene encoding one of the capsid proteins. Phage particles can be tested with the antibody to determine which display fragment react with the antibody (Barbas 2011). 1.5.7 Recent Innovations in Display Technology Display technology has found a wide application in the modern world. Some of the applications include selective infective phage, isolation of allergens by phage display and tumor targeting. Enrichment for phage displaying high-affinity molecules over non-specific binders is not only the most difficult but also the most complex tasks in the phage display technology. In the selection infective phage, the N-terminal domains of pIII are replaced by the gene for a protein or peptide resulting to the production of noninfective phage particles. An adapter molecules consisting of the ligard coupled covalently to the N-terminal domains provide the missing N-terminal domains which is necessary for phage infectivity (Barbas 2011). Infectivity is restored when noninfective phage and adapter molecules are mixed only to phage particles displaying peptides that are capable of binding the ligand with the later providing the missing N-terminal domains of pIII to the phage. The method eliminates the need for physical separation of specific ad unspecific binders and consequently provides an efficient and rapid procedure for the selection of the high-affinity interactions (Barbas 2011). Tumor targeting peptide ligand is used for delivery of cytotoxic chemotherapy, proapoptonic peptides and cytokines to receptors in the angiogenic vasculature showing marked therapeutic efficacy in tumor-bearing mouse models. Tumor targeting peptide ligands can also deliver imaging agent to tumor vasculature (Barbas 2011). Phage particles with amino acids 2 to 4 on every wild-type pVIII coat protein replaced with random octamers are referred as landscape phage (Barbas 2011). The substitution results to a fixed peptide framework that allows phage to have properties dependent on the introduced variable peptides. Moreover, additional properties may arise owing to the global architecture of the phage particle. Clones that can bind to dioxin, streptavidin, avidin, and beta-galactosidase with nanomolar affinity were selected from landscape phage libraries against immobilized targets and were named substitute-antibody filaments (Azzazy & Highsmith 2007). Due to their novel service peptides, these filaments may have binding advantages over their immunoglobulin counterparts. References Arap, W, Pasqualini R & Ruoslahti E, 2009, Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model. Science 279:377-380. Azzazy H, & Highsmith W, 2007, Phage display technology: Clinical applications and recent innovations. ClinBiochem 35:425-445. Barbas, C., 2011, Phage display: A laboratory manual. 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