Sara Carmen is a senior scientist and Lutz Jermutus is the head of the technology development team within the

display technology group at CAT (Cambridge Antibody Technology). The team is dedicated to exploring innovative solutions in library design and selection strategies, as well as phage and ribosome display.

Concepts in antibody phage display

Sara Carmen and Lutz Jermutus
Date received (in revised form): 15th April 2002

Abstract
This paper introduces the reader to antibody phage display and its use in combinatorial biochemistry. The focus is on overviewing phage display formats, library design and selection technology, which are the prerequisites for the successful isolation of specic antibody fragments against a diverse set of target antigens.
Abbreviations CDR, complementarity determining region VH , variable heavy chain VL , variable light chain scFv, single chain variable fragment g3p, gene 3 protein

Keywords: phage display, antibody, library, valency, selection

N1, rst N-terminal domain of g3p N2, second N-terminal domain of g3p CT, C-terminal domain of g3p IG, intergenic region RT, reverse transcription
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INTRODUCTION TO PHAGE DISPLAY

Animal immunisation followed by hybridoma technology has been used to generate monoclonal antibodies against a variety of antigens. Over the past ten years, advances in molecular biology have allowed for the use of Escherichia coli to produce recombinant antibodies. By restricting the size to either a Fab, a Fv or a linker-stabilised single chain Fv (scFv) (Figure 1) such antibody fragments can not only be expressed in bacterial cells but also displayed by fusion to phage coat proteins.1 The phage display concept was rst introduced for short peptide fragments in 1985.2 Fragments of the EcoRI endonuclease, displayed as a polypeptide fusion (phenotype) to the gene 3 protein (g3p), were encoded on the DNA molecule (genotype) encapsulated within the phage particle. The linkage of genotype to phenotype is the fundamental aspect of phage display. Subsequently, phage display of functional antibody fragments was shown3 when the VH and VL fragments of the anti-lysozyme antibody (D1.3) were introduced, with a linker, into a phage vector at the Nterminus of g3p. Since then, large scFv,

Sara Carmen, Display Technology Development, Cambridge Antibody Technology, The Science Park, Melbourn, Cambridgeshire, SG8 6JJ, UK Tel: +44(0)1763 269 284 Fax: +44 (0)1763 263 413 E-mail: sara.carmen@ cambridgeantibody.com

Fab and peptide repertoires have been generated using a variety of phage display formats. One of the major advantages of phage display technology of antibody fragments compared with standard hybridoma technology is that the generation of specic scFv/Fab fragments to a particular antigen can be performed within a couple of weeks. The starting point is usually an antibody library, of either naive or immune origin, comprising a population of, ideally, 109 1011 clones. After usually two to, maximally, three rounds of selection, the population is enriched for a high percentage of antibody fragments specic for the target antigen. The display of human antibody fragments is of particular interest since any therapeutic derived from these antibody fragments is believed to have a minimal risk of an immune response in patients. Both scFv and Fab formats have been used successfully in antibody libraries displayed on phage. Fab fragments are usually more stable than Fv fragments and have less potential to dimerise than scFvs.4 In addition to the VH and VL segments, they also possess the constant regions (CH and CL ) of the heavy and light chains. They are reputed to be displayed at lower

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A.

B.

C.

D.

Figure 1: Antibody structure and derived fragments. (A) IgG, 160 kilodaltons (kD); (B) Fab fragment, 50 kD; (C) Fv fragment, 30 kD; (D) scFv with linker, 30 kDa

The oxidising periplasm of E. coli favours disulphide bond formation

frequency on the surface of phage, thus alleviating the avidity effects associated with some well-expressed scFvs. Synthetic and naive Fab libraries have been used successfully for the generation of antibody fragments to a variety of antigens.58 Expression of the scFv fragment has a less toxic effect on the cell than the larger Fab molecules.4 This results in a better yield and diversity in scFv libraries. The remainder of this paper will, therefore, focus primarily on scFv libraries since they are generally the more popular choice.9 Expression in E. coli ensures that sufcient quantities of scFv, for screening and characterisation, can be produced with relative ease. Many secreted eukaryotic proteins such as antibodies require disulphide bonds for stability, and the oxidising environment of the E. coli periplasm, where lamentous phage assembles, provides the appropriate conditions for antibody folding. Human antibodies against human proteins can be isolated from diverse human antibody libraries. Moreover, antibodies to toxic molecules such as doxorubicin10 can be obtained, a task difcult with immunisation/hybridoma techniques.

M13 phage biology

Filamentous phage assemblies in the E. Coli periplasm

M13 is a lamentous bacteriophage. The native particle is a thin, cylindrical shape, usually 900 nm long and 67 nm in diameter.11,12 It contains a single-stranded DNA genome (6,407 base pairs in length) which encodes 11 genes,13 ve of which

are coat proteins. The major coat protein is the gene 8 protein (g8p) which is present in almost 2,700 copies and responsible for encapsulating the phage DNA (Figure 2). The distal end of the phage particle is capped by ve copies each of g7p and g9p. At the proximal end, four to ve copies each of g6p and g3p are present. M13 bacteriophage infects only male bacteria, ie those E. coli cells that bear the F-plasmid which encodes the F-pilus. Infection is mediated by the interaction between g3p of the phage and the F-pilus. Filamentous phage have the characteristic that once they have infected their host cell they do not lyse the cell, but instead are able to replicate and are released from the cell membrane while the host cell continues to grow and divide, in contrast to the lytic phages T4 and T7. The structure and function of g3p have been well studied. The protein is responsible for phage infection and for release of the phage particle following assembly.14,15 It has two N-terminal domains (N1 and N2) and a C-terminal domain (CT, Figure 3). N1, which has been shown to be essential for infectivity,16 interacts with the TolA protein of the TolQRA complex located across the periplasmic space between the inner and outer E. coli membranes. The primary interaction of phage with an E. coli cell is mediated by N2 binding the F-pilus and it is this association that is thought to bring N1 into close proximity with TolA. It is not yet understood how TolA contributes to phage infection; however, it is known that the F-pilus retracts and that the M13 genome is injected into the cytoplasm. CT is required for the termination of phage assembly in the periplasm and release of the phage from the cell membrane. Once the cells have been infected and phage protein production commences, the cells are no longer able to be infected. The presence of only very small amounts of the g3p of f1 lamentous phage has been shown to be associated with a resistance of the cell to infection from lamentous

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X IG ori II V VII IX VIII IG IV III

6.4 kb

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I XI

VI

Figure 2: The structure of M13 phage particle. The phage particle is of cylindrical shape (900 nm long) and only 67 nm in diameter. Non-structural proteins are unshaded. All coat proteins are shaded according to their corresponding gene in the genome. The intergenic regions are marked by IG. G8p is the major coat protein and is present in about 2,700 copies. The minor coat proteins g3p, g6p, g7p and g9p are present in approximately ve copies each

All coat proteins can be used for phage display

phage. Its presence in the E. coli membrane disrupts the membrane integrity, causing a number of effects including defective F-pili.17 The ve coat proteins have all been

used as fusion proteins for phage display.18,19 Genomic or cDNA libraries are favoured as C-terminal fusions to g8p since expression constraints due to frameshifts and stop codons are avoided.20 C-terminal polypeptide fusions to g3p have also been demonstrated.21 Since g3p is the most popular fusion protein, this paper will concentrate on display of antibody fragments on g3p in a phagemid system.

PHAGE DISPLAY FORMATS

Figure 3: The modular structure of g3p. The N1 domain interacts with the TolA protein in the E. coli membrane. The N2 domain binds the Fpilus and the CT is necessary for termination of phage assembly. The glycine-rich linkers, G1 and G2, are thought to confer exibility to the domains, facilitating the infection process. Antibody fragments can be displayed at the N-terminus of g3p. Alternatively, they can be displayed as an N-terminal fusion to CT

Early phage display formats involved the fusion of peptides to the N-terminus of g3p or g8p in the M13 phage vector. The polypeptide or antibody fragment was displayed in a multivalent format, since all copies of the coat protein are translated as fusion proteins,22,23 although it was not

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Phagemid vectors facilitate construction of large libraries

possible to display larger polypeptides or proteins without affecting the function of g8p. Six residue insertions are usually tolerated, but, when the insert is increased to eight residues, only 40 per cent of phage are infectious and, at 16 residues, this number drops to less than one per cent.24 Larger fusion proteins to g3p are tolerated, but, in a multivalent display format, such fusions may cause a decrease in infectivity2 by sterically hindering the interaction of N2 and the F-pilus. These problems are overcome by the use of phagemids. Phagemids are plasmids ($4.6 kilobases) which encode a signal sequence, the phage coat protein and an antibiotic resistance marker. The antibody fragment/polypeptide is cloned upstream of the g3p/g8p coat protein sequence and expression is controlled by the use of a promoter such as lacZ. The relatively small size of these vectors means that they have higher transformation efciencies than phage vectors,25 hence facilitating the construction of large repertoires or libraries of peptide or antibody fragments. The incorporation of an amber stop codon between the displayed protein and the phage coat protein permits fusion protein expression in suppressor strains of

E. coli such as TG1.26 Non-suppressor strains, such as HB2151,27 will not incorporate a glutamine at the amber codon, thereby resulting in production of only the antibody/polypeptide moiety. A phagemid cannot produce infective phage particles alone. A helper phage such as M13KO7 or VCSM13 is required. The helper phage provides the genes which are essential for phage replication and assembly, including a wild-type copy of the coat protein used for display. Cells already containing the phagemid vector are superinfected with the helper phage. Glucose in the growth media represses the lacZ promoter, preventing expression of g3p-fusion, which would inhibit superinfection. Once the helper phage genome is incorporated into the cell, the glucose is removed and phage production commences. The M13KO7 genome possesses a modied intergenic region (IG),28 causing it to be replicated and packaged less efciently than the phagemid which carries the wild-type M13 IG. This ensures that the genotype (phagemid) and phenotype (g3pscFv fusion) are linked in a single (phage) molecule, which is the key feature of phage display (Figure 4). The antibody fragment can also be fused to a truncated

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Figure 4: Sequence of events depicting phage infection, assembly and secretion using a phagemid system. (1) Phage displaying scFv infects cell. Single-stranded phagemid DNA is injected into cytoplasm. (2) Helper phage superinfect providing genomic information encoding remaining phage proteins for phage replication and assembly. (3) ScFvg3p fusion, from the phagemid vector, and all other structural proteins (including wild-type g3p) from the helper phage vector, are directed to the periplasm. (4) Phage assembly occurs in the E. coli periplasm. Phage containing DNA encoding scFv sequence (genotype) and displaying scFv (phenotype) are secreted from cell membrane

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CT domain in a phagemid system since there will always be copies of wild-type g3p from the helper phage for infection. Selectively infective phage (SIP) technology (reviewed in Jung et al.29 ) exploits the modular nature of g3p and can be used for identifying proteinligand interactions. The ligand is coupled to N1N2 either by the use of an expression vector or, if it is sufciently small (eg an organic molecule), it can be chemically coupled. The receptor/scFv is fused to the CT domain of g3p as part of the phage particle. It is the interaction between the ligand and the receptor which restores the domains of g3p, thereby rendering the phage infectious (Figure 5). The main difference between this technology and standard phage display is that the selection and infection

process are coupled. Since interaction leads to infection, there is no need for elution. A variation of this technology termed Cys display has recently been presented.30 Here, the g3p and the antibody fragment to be displayed are both expressed separately in the cell, although both with a terminal Cys. In the oxidising periplasm, g3p and scFv can interact, allowing for the formation of a g3pSSscFv fusion which can later be eluted by reducing agents such as DTT. No selection results have so far been reported with this technology, but there is the possibility of unpaired cysteine residues interfering with disulphide bond formation in the scFv, resulting in misfolding and reduced yields.31 Competition from g3p of the helper
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Figure 5: Formats for scFv display and vectors used. (A) Wild-type phage displaying multiple scFvs at the N-terminus of the N1 domain. (B) and (C) scFv displayed at the N-terminus of N1 or CT, respectively. Both these formats incorporate copies of wild-type g3p. (D) SIP technology. scFv is displayed at the end of CT domain of wild-type g3p in the phage vector. Infectivity is dependent on the scFv interacting with a ligand attached to the N-terminal domains. (E) Hyperphage. The helper phage genome lacks a g3p so all copies of g3p are derived from the phagemid vector system, resulting in multivalent phage

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Most phage do not display a fusion protein

Library sizes have continued to increase

phage and proteolytic degradation of the g3p-fusion usually result in phage populations where as little as ten per cent of phage display a fusion protein.32 This is an important consideration when trying to represent large repertoires of antibody fragments, since at least ten times more phage are required to reach the theoretical diversity. Use of a gene 3-less helper phage helps to restore fusion protein display levels.33 Hyperphage is a modied helper phage which displays the g3p phenotype but which contains a phage vector that lacks the gene 3 sequence.34 Production of such hyperphage particles necessitates exogenous g3p by culturing in the E. coli strain DH5/pIII. These cells possess a lacZ-regulated gene 3 sequence on their chromosome. The resulting infective hyperphage particles are superinfected into cells containing a phagemid, and, during phage production, the only g3p produced is the scFv-fusion from the phagemid vector. This results in multivalent display, a phenomenon also resulting from the use of phage vectors. The potential advantage of this system is the improved chance of nding a clone of interest from large populations, due to a lower background from phage displaying only the helper phage g3p. Alternatively, the g3p could be supplied from cells containing a plasmid encoding the gene 3 sequence regulated by the phage shock promoter (psp).35 This operon is induced following infection by lamentous phage, resulting in production of g3p which can complement a helper phage with a deleted gene 3 sequence.

CONSTRUCTION OF PHAGE ANTIBODY LIBRARIES Naive antibody libraries

Nave libraries contain natural CDRs Synthetic libraries contain articial CDRs

whereas nave libraries have the advantage that they can theoretically be used for an unlimited range of antigens. Construction of these libraries involves relatively straightforward molecular biology techniques such as RT of mRNA, followed by polymerase chain reaction (PCR) with germline-specic primers to amplify the VH and VL gene segments from the cDNA template, and restrictionbased cloning to incorporate the rearranged antibody segments into an appropriate phagemid display vector. Finally, the vectors are transformed into E. coli cells to generate the antibody repertoire. The rst nave scFv phagemid library was constructed using peripheral blood lymphocytes (PBLs) and was predicted to have a diversity of .107 . Antibody fragments isolated from this library demonstrated micromolar afnities.37 About ve years later, a scFv library of .1010 recombinants, derived from tonsil and PBLs, was described.10 This library allowed for the selection of scFvs with afnities in the subnanomolar range. These results support theoretical predictions38 that the size of the library is thought to be proportional to the afnities of the isolated antibodies. The limiting factor for generating large library sizes is the transformation step; even if this is optimised, library sizes in the range of 108 1011 can be accomplished only after numerous electroporations. Recently, protocol improvements, primarily relating to DNA purication and choice of strain, have been described that allow the generation of library sizes of 1010 in a single electroporation step.39

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Synthetic antibody libraries

Construction of synthetic libraries involves rearranging VH and VL gene segments in vitro and introducing articial complementarity determining region (CDRs) of varying loop lengths using PCR and randomised oligonucleotide primers. One of the earliest synthetic repertoires used a diverse repertoire of human VH gene segments paired with a constant V3 (DPL16) light chain

The genomic information coding for antibody variable domains is usually derived from B cells of either nave (nonimmunised) or immunised donors.36 An antibody repertoire from immunisation is generally restricted to generating antibodies against the antigen of the original immunogenic response,

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Antibodies that form functional proteins in a reducing environment are rare

Large library sizes can alternatively be created in vivo recombination

sequence. A randomised VH CDR3 ranging from four to 12 residues was introduced40 which resembles the natural loop length diversity more closely than an earlier synthetic repertoire which had loop lengths of either ve or eight residues.41 A library of .108 recombinants was generated from which antibodies to a variety of human antigens, as well as a diverse collection of nonhuman proteins, were isolated. Most antibody fragments usually require an oxidising environment for folding, although it is possible to isolate antibodies that are able to fold into a functional and stable antibody domain structure in a reducing environment. These antibodies are relatively rare and might be restricted to certain germlines. They may possess an above average thermodynamic stability and should be particularly useful for intracellular applications. A previously isolated scFv (F8), shown to be functionally expressed in both bacterial and plant cytoplasm, was used as a scaffold framework.42 Specic residues in the CDR3s of the heavy and light chain were randomised and a library (diversity of 5 3 107 ) was generated. Antibodies of submicromolar afnity, which can be functionally expressed in the cytoplasm, have been successfully isolated to a variety of targets, such as proteins from plant viruses, as well as more common protein targets such as lysozyme. Some of the more recently constructed synthetic repertoires have opted to restrict the number of frameworks used. The HuCAL library43 is a fully synthetic antibody library which has yielded antibodies with nanomolar (nM) afnities to a number of antigens. The framework diversity is limited to seven heavy and seven light chain consensus sequences. Based on sequence analysis of human variable antibody domain genes, the authors determined that the majority of canonical structures are represented by these consensus sequences. For each antibody germline family, scFv sequences were optimised for expression by

avoiding rare codons in E. coli. All six CDRs are anked by restriction sites to facilitate afnity maturation.

In vivo recombined antibody libraries

To get around the earlier limits imposed by the transformation step, some libraries have been constructed using in vivo recombination or combinatorial infection to generate highly diverse repertoires of either synthetic or naive antibody fragments. A synthetic Fab library was constructed using two vectors,5 each with a different antibiotic selectable marker. One was a plasmid derived from pUC19 and the second was a phage vector (fdDOG), and the heavy and light chain antibody sequences were cloned into these vectors, respectively. The vectors were then incorporated into the same cell by a simple phage rescue strategy and cells were subsequently co-infected by the chloramphenicol-resistant bacteriophage P1. P1 provides the Cre recombinase which recognises the loxP sites anking the heavy and light chain antibody sequences and recombines them within the cell. The library was predicted to contain 6.4 3 1010 recombinants, the recombinants being detected on the basis of their resistance to all three antibiotics. Recently, a single vector system also using Cre recombinase has been described.44 A phagemid vector containing both VH and VL segments was transfected into cells and the phage population produced from this primary library was then allowed to infect bacteria at a high multiplicity of infection (ratio of phage to cells), so ensuring that more than one vector is incorporated per cell. The VH and VL segments were then exchanged between vectors generating multiple new VH /VL combinations. Using this method, a naive scFv library with a diversity of 3 3 1011 was created. This library is potentially more stable than those previously constructed using in vivo recombination because of the use of the smaller phagemid vector.

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Quality control of antibody libraries

As larger libraries are possible with improved technology, the task of assessing diversity remains difcult. BstNI ngerprinting is a quick way of checking for diversity since the scFv sequence can be amplied directly from colonies by PCR. The product is then digested using the BstNI enzyme and different sequences will give different band patterns on an agarose gel. Sequencing is a preferable method since it also provides information on clones that have frameshifts or stop codons and demonstrates additional diversity which may not be obtained from a BstNI restriction digest; however, the sample size required to assess library sizes above 109 is well beyond most laboratory sequencing capabilities. Furthermore, information attained by sequence diversity may not equate to the functional diversity of the library. A clone can contain a complete and in-frame VH VL sequence, but it may not produce functional protein because the antibody fragment may be aggregated, misfolded or toxic to the cell. Aggregated antibody may result in bald phage ie phage that display only g3p from the helper phage vector. As the vast majority of phage are either monovalent or bald, large populations of phage, much greater than the library size, are required for efcient representation of the library diversity. The best measure to assess library quality is how well the library performs in selections. Populations of medium to high afnity (nM range) antibodies should be isolated directly from a quality library for a highly diverse panel of antigens. Ways to improve display levels, other than those mentioned earlier, may include co-expression of scFv fragments with the E. coli Skp and/or FkpA periplasmic proteins. These chaperones have been shown to improve both soluble and displayed scFv levels.4547 Alternatively, clones that display g3p from the helper phage, due to absence of g3-fusion protein, can be effectively removed using a modied helper phage.

Library quality can be assessed by its performance in selections

A protease cleavage site introduced into the gene 3 sequence of the helper phage genome between N2 and CT resulted in the KM13 helper phage,48 which was then used to rescue a phagemid library. Clones that expressed misfolded scFvs and, as a consequence, packaged only helper phage g3p, were rendered noninfectious by incubation with trypsin. Trypsin can also be used effectively as an elution reagent49,50 which also removes bald clones via their protease cleavage site. It can also be used sequentially following, for example, competitive elution with the original antigen.51 Using this method, a dramatic reduction in background from non-specic phage was observed compared with standard elution methods. This can probably be explained by effective removal of all frameshifted, or poorly expressing, clones from the population, when previously they had been seen to have a growth advantage.52,53

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Co-expression of chaperones can improve display levels

SELECTION TECHNOLOGIES
While a lot of work has concentrated on the generation of larger and more rational or designed libraries, other researchers have concentrated their efforts on the selection process itself. The selection protocol can be divided up into ve main steps: (i) coating of antigen; (ii) block; (iii) incubation of phage with antigen; (iv) washing to remove non-specic phage; and (v) elution (Figure 6AD). Surfaces can be coated with antigen in a variety of ways, the most common being direct adsorption to a plastic surface where it is non-covalently associated via electrostatic and Van-der-Waals interactions. If it is a short peptide or a small organic molecule, it is more commonly conjugated to a carrier protein such as BSA (bovine serum albumin) or KLH (keyhole limpet haemocyanin) before immobilisation onto the plastic surface, as it will not remain bound on its own or, if bound, will be sterically difcult to access by the phage library. Antigen can be biotinylated for selections in solution. The phageantibodyantigen

Small molecules are rst conjugated to a carrier molecule

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Figure 6: The phage display/selection cycle. (A) Antigen is immobilised onto plastic. (B) Library/outputs are incubated with the antigen surface. (C) Unbound phage are removed and the surface is washed. (D) Phage are eluted. (E) E. coli cells are infected with eluted phage. (F) Cells are plated onto selective agar plates. (G) Selection output is amplied. (H) Helper phage superinfect cells containing phagemid vector. (I) Phage replication and assembly provides population of enriched phage for next round of selection

Pure antigen in native conformation improves phage output specicity

Reversibly bound antigen may reduce the number of specic antibody hits

complexes are subsequently captured using streptavidin-coated paramagnetic beads. Selections on cell surfaces have also been successfully demonstrated,5459 as well as on protein bands from nitrocellulose membranes following twodimensional (2D) electrophoresis.50,60,61 Antigen purity is important since contaminants presented to a nave antibody library are very likely to generate antibodies to proteins other than the target antigen. The proportion of protein that is immobilised while retaining its native structure and how often each epitope is presented and accessible are further considerations. While antigen is usually coated onto solid phase by passive adsorption, this process may cause denaturation of the molecules.62,63 This,

in turn, can sometimes favour nonspecic phage or antibody binding,64 since the denatured form will present exposed hydrophobic residues, causing a sticky surface. Factors such as the chemistry of antigen immobilisation, the blocking agent used and the density of immobilised antigen can inuence how well the antibody fragments can access a given epitope (Figure 7).64 The main purpose of the blocking agent is to coat any exposed areas of the solid surface that are not already coated with an antigen molecule. During the blocking phase, antigen that is reversibly bound may well desorp.64 It is essential to rinse thoroughly after blocking since residual blocking agent containing desorped antigen may compete for

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Figure 7: Accessibility of epitope. The amount of accessible epitope can be signicantly lower than the antigen concentration. Antigen is represented by shaded circles, with the epitope being represented in black. White circles represent the blocking agent. The plastic surface is represented by a thin black line. Adapted from 64 Zhuang et al.

Quality of round one output is critical to selection success

Small changes in selection protocol can inuence output diversity

specic antibody fragments after addition of the library. Zhuang et al.64 recommend the use of streptavidin-coated beads and a biotinylated antigen since, unlike passive absorption, this process does not favour denaturation of the antigen and, perhaps more importantly, immobilisation/ coating is nearly covalent due to the tight interaction of biotin and streptavidin. Incubation with the library can be performed at various temperatures ranging from 48C to 378C. Some protocols shake/agitate the selection tubes/plates to improve the chances of specic phage encountering antigen. Phage displaying scFv that are not able to bind the antigen will either remain in solution or may bind non-specically. The washing step is thought to be crucial for the removal of non-specic phage. Many repeated quick rinses may favour the removal of non-specic and low afnity clones since their fast off-rates will cause the majority of them to be in solution when the wash buffer is discarded. Repeated washes are thought to have a dilution effect as more and more non-specic clones are removed. There are many elution reagents to choose from, ranging from those which are pH dependent (triethylamine, glycine/HCl) to those with enzymatic

mechanisms (trypsin and chymotrypsin) or competitive elution with soluble antigen. The nature of the antigen can inuence how well specically bound phage is eluted from the antigen. For instance, a protein that has an overall positive charge may favour elution of specic phage at a low pH such as glycine/HCl at pH 2.2. It is crucial to try to maximise the sequence diversity accessed from the total repertoire at round one since it is this subset that is used for further selection rounds and its diversity will inuence the success of selection. At round one, the number of clones that will not bind the antigen far exceeds the number that do. Limiting antigen concentration or lack of exposure of certain epitopes may result in the loss of specic clones, and potent clones present at low level may be missed. During subsequent selection rounds, growth advantages may result in the over-representation of some clones thus, adopting more than one selection strategy increases the chance of obtaining diverse round one output populations. Selections comparing immunotube and multipin surfaces on ten to 11 antigens65 resulted in the isolation of 124 different antibodies in total, each strategy producing very different panels of

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Selections on cell surface are more complex

antibodies with very few clones common to both strategies. Small variations such as the surface area and the concentration of antigen and phage were sufcient to cause these differences in output diversity. Three alternative selection strategies were adopted (B. M. Edwards et al., unpublished data) to isolate antibodies from a 1011 library to BLyS (B lymphocyte stimulator). The antigen was (i) immobilised on immunotubes; (ii) biotinylated and coupled to streptavidincoated plates; or (iii) biotinylated and used in soluble selection. The selection resulted in 2,730 scFvs which specically recognised the BLyS antigen; of these, 1,287 were sequence unique and most were only isolated once. By using diverse selection strategies, the authors maximised sequence diversity. Cell surface selections can be problematic since a number of antigens in addition to the target protein may be displayed on the cell surface. Edwards et al.59 used seven different selection strategies for cell surface selections on human adipose tissue. Out of 109 unique antibodies, shown specically to bind adipocyte plasma membrane, 87 were isolated from just one of the strategies. These few examples support the theory that the choice of selection methods may inuence output numbers and diversity. Selection methodologies should be tailored to t each antigen class. More recently developed selection techniques include the use of antigen prepared by western blotting on nitrocellulose membranes as a target for selections.50,60,61 2D gel electrophoresis was used for the production of protein proles from patients with different disease states. Isolation of phage antibodies to proteins on these blots is likely to prove extremely useful in the further characterisation of these diseases. Antigen can also be displayed as a fusion to a LppOmpA hybrid on the surface of the E. coli cells.66 This allows a novel selection approach called delayed infectivity panning (DIP). The antigen on the bacterial cell surface is exposed to a

population of phage displaying antibody fragments. Non-specic infection is prevented by growing the cells at 168C, which prevents formation of the F-pilus. Once non-specic phage have been removed, the temperature is raised to 378C, which facilitates F-pilus formation and, thereby, permits infection.

FUTURE
As phage display technology moves into its second decade, a number of phage display-derived antibodies are now in advanced clinical trials. Library sizes are getting larger and library design is more tailored to the needs and later application of antibody fragments. Much more is understood about antibody structure and important contact residues involved in antibodyantigen interactions.67 The expression of eukaryotic proteins in E. coli has improved with the use of frameworks that do not have detrimental effects on the host cell. Production of secondary libraries for afnity maturation is greatly facilitated by restriction sites anking whole V gene segments, permitting heavy and light chain shufing,68 or anking specic CDRs, allowing hypervariable regions to be shufed within the frameworks using randomised CDR cassettes.43 Furthermore, automation of phage display enables high throughput selection of specic antibodies and renders the process further compatible with hybridoma technology.69 Alternative display technologies, such as ribosome display, have been optimised (for review, see Amstutz et al.70 ). Libraries with a diversity of 1014 are possible using this cell-free system. It may be possible to isolate slightly different populations of scFv fragments since constraints on diversity from expression in E. coli are avoided with this in vitro technology and afnity maturation can occur simultaneously with selections. A new display-less technology has been shown to isolate antibodies with subnanomolar afnities; termed periplasmic expression and cytometric screening (PECS), this technology directs soluble scFv to the

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periplasmic space.71 Fluorescently labelled ligand (up to 10 kilodaltons) is then incubated with the cell population. Specic binding of the ligand with the scFv in the periplasmic space results in an increased uorescence which allows positive cells to be separated from the remaining population using uorescenceactivated cell sorting (FACS). While technologies will further improve to allow for faster, more automated selection of higher-afnity binders in fewer selection cycles, the complexity of antigens will gradually increase. This will provide further opportunities for the continued development of creative approaches to library design and selection strategies, as exemplied by the advent of the rst in vivo selection in humans.72

Parren, P. W. H. I., Lundkvist, A., Burton, D. R. and Bjorling, E. (2002), Neutralising human Fabs fragments against measles virus recovered by phage display, J. Virol., Vol. 76, pp. 251258. 9. Holt, L. J., Enever, C., de Wildt, R. M. T. and Tomlinson, I. M. (2000), The use of recombinant antibodies in proteomics, Curr. Opin. Biotechnol., Vol. 11, pp. 445449.

10. Vaughan, T. J., Williams, A. J., Pritchard, K. et al. (1996), Human antibodies with subnanomolar afnities isolated from a large nonimmunised phage display library, Nat. Biotech., Vol. 14, pp. 309314. 11. Makowski, L. and Russel, M. (1997), Structure and assembly of lamentous bacteriophages, in Kay, B., Winter, G. and McCafferty, J., Eds., Structural Biology of Viruses, Oxford University Press, New York; pp. 352380. 12. Webster, R. E. (1996), Biology of the lamentous bacteriophage, in Phage Display of Peptides and Proteins, Academic Press Inc., San Diego, CA., pp. 120.
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