Phage Display and Biotechnological Applications

Biotechnology Advances 19 (2001) 1 33
Research review paper
Biotechnological applications of phage and cell display

Itai Benhar*
Department of Molecular Microbiology and Biotechnology, The George S. Wise Faculty of Life Sciences, Green Building, Room 202, Tel-Aviv University, Ramat Aviv 69978, Israel
Abstract In recent years, the use of surface-display vectors for displaying polypeptides on the surface of bacteriophage and bacteria, combined with in vitro selection technologies, has transformed the way in which we generate and manipulate ligands, such as enzymes, antibodies and peptides. Phage display is based on expressing recombinant proteins or peptides fused to a phage coat protein. Bacterial display is based on expressing recombinant proteins fused to sorting signals that direct their incorporation on the cell surface. In both systems, the genetic information encoding for the displayed molecule is physically linked to its product via the displaying particle. Using these two complementary technologies, we are now able to design repertoires of ligands from scratch and use the power of affinity selection to select those ligands having the desired (biological) properties from a large excess of irrelevant ones. With phage display, tailor-made proteins (fused peptides, antibodies, enzymes, DNA-binding proteins) may be synthesized and selected to acquire the desired catalytic properties or affinity of binding and specificity for in vitro and in vivo diagnosis, for immunotherapy of human disease or for biocatalysis. Bacterial surface display has found a range of applications in the expression of various antigenic determinants, heterologous enzymes, single-chain antibodies, and combinatorial peptide libraries. This review explains the basis of phage and bacterial surface display and discusses the contributions made by these two leading technologies to biotechnological applications. This review focuses mainly on three areas where phage and cell display have had the greatest impact, namely, antibody engineering, enzyme technology and vaccine development. D 2001 Elsevier Science Inc. All rights reserved.
Keywords: Antibody engineering; Bacterial surface display; Enzyme evolution; Immobilized enzymes; Phage display; Vaccine development
* Tel.: +972-3-6407511; fax: +972-3-6409407. E-mail address: benhar@post.tau.ac.il (I. Benhar). 0734-9750/01/$ see front matter D 2001 Elsevier Science Inc. All rights reserved. PII: S 0 7 3 4 - 9 7 5 0 ( 0 0 ) 0 0 0 5 4 - 9
I. Benhar / Biotechnology Advances 19 (2001) 133
1. Introduction As a result of ongoing genome projects, a large-scale effort to measure and analyze protein protein interactions using experimental methods is under way. These include biochemical techniques such as co-immunoprecipitation or crosslinking (Silver and Hunt, 1993), molecular biological techniques such as the two-hybrid system (Fashena et al., 2000) or the phage- and cell-display systems (reviewed herein), and genetic techniques such as mutant isolation and analysis (Gargus, 1987). Therefore, determining functions of novel gene products identified in genome sequencing efforts has become an important scientific challenge. Identifying proteins that interact with novel gene products can help in deducing their activity, regulation, and potential role in disease. Traditional biochemical analyses of enzyme structure and function and elucidation of receptor or antibody recognition processes are based typically on the utilization of efficient model systems. These are characterized by practical considerations, such as: enrichment of the molecules being studied, a source of tissue or cells that can be obtained in abundance with reasonable ease and the stability of the components to biochemical manipulations. The reductionist's approach is to systematically weed out the molecules of interest until purity. The advent of molecular genetics has enhanced the study of proteinligand interactions both by providing means to produce rare molecules in large quantities via gene-expression systems and by focusing on specific regions or residues of the proteins using site-directed mutagenesis. However, the most commonly used technologies for identifying proteinligand interactions, such as the yeast two-hybrid system (Fashena et al., 2000) and expression library screening (Maser and Calvet, 1995; McCarrey and Williams, 1994) are labor-intensive and often not amenable to high-throughput evaluation of multiple gene products. Two leading methodologies have been added to those mentioned above that provide new horizons for the study of proteinprotein interactions. These technologies are based on the production and presentation of enormous collections of peptides or proteins to be screened by corresponding `binders.' This enables the identification of binding pairs or the selection of molecules by virtue of their function. The approaches are based on the concept of generating a complete set of all possible combinations of the peptides or proteins of interest, collectively referred to as `combinatorial display.' The respective approaches involve the display of (poly)peptides on the surface of bacteria and the combinatorial display of synthetic peptides, organic structures and recombinant proteins on bacteriophage. 2. Phage display The generation of new drugs has long involved the search amongst hundreds of thousands of components using well-defined in vitro screening tests, the output of which was chosen to mimic as closely as possible the desired in vivo activity of the new drug. Today, new library methodologies offer many alternative and at least as powerful routes, by combining the generation of billions of components with a fast screening or selection procedure to identify the most interesting lead candidates.
Phage display has proven to be a very powerful technique for obtaining libraries containing millions or even billions of different peptides or proteins. Phage display (Smith, 1985) has been used for affinity screening of combinatorial peptide libraries to identify ligands for peptide receptors, define epitopes for monoclonal antibodies, select enzyme substrates (Kay et al., 1996), and screen cloned antibody repertoires (Griffiths and Duncan, 1998). Regulated associations between select proteins are the basis of cellular signal transduction pathways, and defining a signal transduction pathway can, in many cases, be reduced to tracing a chain of proteinprotein interactions. One of the most successful applications of phage display has been the isolation of monoclonal antibodies, and fragments thereof, using large phage antibody libraries (Winter et al., 1994). Indeed, in the last few years, very efficient techniques have been developed to design and build large libraries of antibody fragments, and ingenious selection procedures have been established to derive antibodies with the desired characteristics. Much progress has been made in this rapidly developing field and many possible applications of phage technology have been developed (Hoogenboom et al., 1998). These include the creation and screening of libraries to discover novel therapeutic targets and methods for selection of biologically active ligands. The most widely used library methodology is based on the use of filamentous phage (Smith, 1985), a bacteriophage that infects male Escherichia coli. Display systems based on other phage were also developed, including display on phage l heads (Sternberg and Hoess, 1995; Mikawa et al., 1996; Santini et al., 1998) and tail (Maruyama et al., 1994; Kuwabara et al., 1997), P4 capsid (Lindqvist and Naderi, 1995), T7 capsid (Houshmand et al., 1999; Yamamoto et al., 1999), T4 capsid (Jiang et al., 1997; Ren and Black, 1998; Mullaney and Black, 1996, 1998), and MS2 coat (Mastico et al., 1993; Heal et al., 1999). The phage-display systems and the molecules that were displayed using them are listed in Table 1. Filamentous phage display is based on cloning DNA fragments encoding millions of variants of certain ligands (e.g. peptides, proteins or fragments thereof) into the phage genome, fused to the gene encoding one of the phage coat proteins (usually pIII, but also pIV, PVI or pVIII, Table 1). Upon expression, the coat protein fusion is incorporated into new phage particles that are assembled in the periplasmic space of the bacterium. Expression of the gene fusion product and its subsequent incorporation into the mature phage coat results in the ligand being presented on the phage surface, while its genetic material resides within the phage particle. This connection between genotype and phenotype allows the enrichment of specific phage, e.g. using selection on an immobilized target. Phage that display a relevant ligand are retained by virtue of their binding to the target, while nonadherent phages are washed away. Bound phage can be recovered from the surface, used to reinfect bacteria and reproduced for further enrichment, and eventually for analysis of binding. The success of ligand phage display hinges on the combination of this display and enrichment method, with the synthesis of large combinatorial repertoires on phage. Phage selection is not limited to the isolation of antibodies or short peptides as described above. As listed in Table 1, this approach has also been instrumental in studies and manipulation of a variety of other biologically active molecules, e.g. cytokines (Gram et al., 1993; Saggio et al., 1995; Buchli et al., 1997), receptors (Lowman et al., 1991; Scarselli et al., 1993), enzymes (McCafferty et al., 1991; Soumillion et al., 1994; Pedersen et al., 1998;
4 Table 1 Phage display systems Phage Coat protein M13 pIII (minor)
Displayed molecules Peptides
References
M13 M13
M13 l
P4 T7 T4
MS2
Numerous reports. Recent reviews: Daniels and Lane, 1996; Lowman, 1997; Rodi and Makowski, 1999; Cabilly, 1999 Antibodies Numerous reports. Recent reviews: Winter et al., 1994; Burton and Barbas, 1994; Rader and Barbas, 1997; Hoogenboom, 1997; Hoogenboom et al., 1998; Griffiths and Duncan, 1998 Cytokines Gram et al., 1993; Saggio et al., 1995; Buchli et al., 1997 Receptors Lowman et al., 1991; Scarselli et al., 1993 Enzymes McCafferty et al., 1991; Soumillion et al., 1994; Pedersen et al., 1998; Demartis et al., 1999; Forrer et al., 1999 Enzyme inhibitors Roberts et al., 1992; Pannekoek et al., 1993; van Meijer et al., 1996; Huang et al., 1998 Catalytic antibodies Janda et al., 1994; Sastry et al., 1995; Baca et al., 1997; Fujii et al., 1998 DNA-binding Rebar and Pabo, 1994; Jamieson et al., 1994; proteins Choo and Klug, 1994; Choo et al., 1997 Cellulose-binding Smith et al., 1998; Berdichevsky et al., 1999 proteins pVI Enzyme inhibitors Jespers et al., 1995 Enzymes Hufton et al., 1999 cDNA libraries Hufton et al., 1999 pVIII (major) Peptides Numerous reports. Recent reviews: Felici et al., 1995; Ladner, 1995; Cortese et al., 1996; Lowman, 1997; Wilson and Finlay, 1998 Antibodies Kang et al., 1991; Wang et al., 1997 Enzymes Corey et al., 1993 Enzyme inhibitors Markland et al., 1991, 1996 pVII/pIX Antibodies Gao et al., 1999 D (Head protein) Peptides Sternberg and Hoess, 1995 IgG-binding protein Sternberg and Hoess, 1995 Enzymes Mikawa et al., 1996 Protein A Mikawa et al., 1996 cDNA libraries Santini et al., 1998 pV (Tail protein) Peptides Maruyama et al., 1994; Kuwabara et al., 1997 Enzymes Maruyama et al., 1994 Psu capsid protein Peptides Lindqvist and Naderi, 1995 10B capsid protein Peptides Houshmand et al., 1999 cDNA libraries Yamamoto et al., 1999 Hoc capsid protein Peptides Jiang et al., 1997 Antibodies Ren and Black, 1998 Soc capsid protein Peptides Jiang et al., 1997 Antibodies Ren and Black, 1998 Internal protein III Enzymes Mullaney and Black, 1996, 1998 Green fluorescent Mullaney and Black, 1996 protein Coat protein Peptides Mastico et al., 1993; Heal et al., 1999
Demartis et al., 1999; Forrer et al., 1999), substrates (Matthews and Wells, 1993; Ohkubo et al., 1999) enzyme inhibitors (Roberts et al., 1992; Pannekoek et al., 1993; van Meijer et al., 1996; Huang et al., 1998), catalytic antibodies (Sastry et al., 1995; Janda et al., 1994; Baca et al., 1997; Fujii et al., 1998) DNA-binding proteins (Rebar and Pabo, 1994; Jamieson et al., 1994; Choo and Klug, 1994; Choo et al., 1997), receptor domains for constructing binding molecules with novel properties (reviewed in Nygren and Uhlen, 1997), and cellulosebinding domains (Smith et al., 1998; Berdichevsky et al., 1999). Scaffolds different from antibodies have been reported to form suitable binding ligands for many types of molecules. There are ample examples of host scaffolds that contain sufficient permissive regions to accommodate a reasonable numbers of substitutions, which may be used to generate a library of localized variability. Alternative scaffolds reported to date include b-sheet proteins (Tramontano et al., 1994), a-helical bundle proteins (Ku and Schultz, 1995), combinations of these two (Tramontano et al., 1994), a separate group of highly constrained protease inhibitors (Markland et al., 1996; Tramontano et al., 1994; Rottgen and Collins, 1995), and, most recently, Green fluorescent protein (GFP) (Abedi et al., 1998) and cellulose-binding domains (Smith et al., 1998). Since secreted as well as cytoplasmic and nuclear proteins have been displayed on phage, the use of phage display is often the first strategy to define permissive sites for randomization and to generate ligand-binding variants (Choo et al., 1997). Alternatively, the use of lambda phage (Mikawa et al., 1996), bacterial cell display (Georgiou et al., 1997), and eukaryotic cell-display methods have been reported. With regard to functional selection methods, choosing other types of molecules besides antibodies is validated by the fact that antibody expression and folding may be impaired in the subcellular location where the desired functional activity is required. It would be advantageous to engineer the antibody for intracellular expression, for example by building stable disulfide-free antibodies (Proba et al., 1997), or to use libraries of scaffolds that are naturally produced in the targeted cell organelle, provided that effective and sufficient structural diversity may be obtained. 3. Bacterial surface display The targeting and anchoring of heterologous proteins to the outer surface of yeast and mammalian cells (Schreuder et al., 1991) have been utilized for various applications. However, this review is focused on bacterial surface-display systems that are becoming an increasingly important research area (Georgiou et al., 1997; Hofnung, 1991; Little et al., 1993; Francisco and Georgiou, 1994; Fischetti et al., 1996). Surface display of heterologous proteins in bacteria has been employed as a tool for basic and applied research in microbiology, molecular biology, vaccinology and biotechnology. The first examples of heterologous surface display were reported a decade ago, when short gene fragments were inserted into the genes for the E. coli outer membrane proteins LamB, OmpA and PhoE, and the gene fusion products were found to be accessible on the outer surface of the recombinant bacteria (Charbit et al., 1988; Agterberg et al., 1990a,b; Tommassen et al., 1994; Janssen and Tommassen, 1994). Those display systems, however, were not suitable in most cases for the display of large proteins (Tommassen et al., 1994).
6 Table 2 Bacterial display systems Bacterium
Membrane protein
Displayed molecules Peptides Peptides Metal binding proteins Antibodies Protein A Peptides Peptides Peptides Peptides Peptides Enzymes Antibodies Receptors Toxin domain Cellulose binding proteins Peptides Toxin domain Peptides Enzymes HIV GP120 Peptides Peptides Peptides Toxin domain Peptides Peptides Peptides Peptides Protein A Enzymes Peptides Receptors Antibodies Enzymes Peptides
References Charbit et al., 1988; Leclerc et al., 1991; Bradbury et al., 1993; Sousa et al., 1996; Persic et al., 1999 Pistor and Hobom, 1988; Agterberg et al., 1990a,b Kotrba et al., 1999 Fuchs et al., 1991, 1996; Dhillon et al., 1999 Steidler et al., 1993 Tommassen et al., 1994; Janssen and Tommassen, 1994 Chang and Lo, 2000; Chang et al., 1999 Hedegaard and Klemm, 1989 Lu et al., 1995 Pallesen et al., 1995 Francisco et al., 1992 Francisco and Georgiou, 1994; Francisco et al., 1993b; Chen et al., 1996 Benhar et al., 2000 Benhar et al., 2000 Francisco et al., 1993a Maurer et al., 1997 Maurer et al., 1997 Malmborg et al., 1997 Jung et al., 1998a,b Kwak et al., 1999 Sewani et al., 1998 Xu and Lee, 1999 Wu et al., 1989; Newton et al., 1989 Klauser et al., 1990 Schorr et al., 1991 Bingle et al., 1997 Pozzi et al., 1992 Oggioni et al., 1999 Schneewind et al., 1992, 1993 Schneewind et al., 1992, 1993 Hansson et al., 1992 Hansson et al., 1992 Gunneriusson et al., 1996, 1999 Strauss and Gotz, 1996 Mesnage et al., 1999
A. Gram-negative bacteria E. coli LamB OmpA
Pal Pap PhoE TraT Type 1 fimbriae FliC FimH Lpp-OmpA
AIDA-I F pilin Pseudomonas Inp ExtB OmpC Flagellin Neisseria IgAb E. coli OmpA RsaA
Salmonella Caulobacter
B. Gram-positive bacteria Streptococcus M1 protein M6 protein Staphylococcus Synthetic anchor Protein A FnBPB S-layer protein EA1
B. anthracis
Since then, not only outer membrane proteins but also lipoproteins, fimbria proteins and flagellar proteins, as well as dedicated systems with coupled translocation and surface anchoring, have been employed to achieve heterologous surface display on gram-negative bacteria (Hofnung, 1991; Little et al., 1993; Francisco and Georgiou, 1994), for example E. coli and Salmonella spp. In parallel, systems have been described for heterologous surface display on gram-positive bacteria such as staphylococci (Hansson et al., 1992), streptococci (Pozzi et al., 1992; Hanski et al., 1992) and mycobacteria (Stover et al., 1993). The bacterial display systems and the molecules that were displayed using them are listed in Table 2. The most common application of bacterial surface display has been in the development of live bacterial vaccine-delivery systems. In this context, the cell-surface display of heterologous antigenic determinants has been considered advantageous for the induction of antigenspecific antibody responses when using live recombinant cells for immunization (Stover et al., 1993; Georgiou et al., 1997; Nguyen et al., 1995). Bacterial surface display has also been applied in generating whole-cell bioadsorbents for environmental purposes (Sousa et al., 1996), novel microbial biocatalysts [surface display of active enzymes on E. coli (Francisco et al., 1992, 1993a,b) and staphylococci (Strauss and Gotz, 1996)], diagnostic tools [bacteria with surface-displayed antibody fragments (Fuchs et al., 1991; Francisco et al., 1992)] and for the display of entire peptide libraries (Lu et al., 1995) as an alternative to the rapidly developing phage-display technology. It should, however, be specified that combinatorial display is commonly applied using phage systems, and rarely using bacterial display systems. The latter are usually dedicated to the display of functionally defined molecules. 4. Surface display in gram-negative bacteria Display systems have also been developed for the expression of a number of heterologous proteins on the surface of gram-negative bacteria. Most often, foreign gene products have been fused to outer membrane proteins, such as the maltoporin LamB (Charbit et al., 1988), the phosphate-inducible porin PhoE (Tommassen et al., 1994), the outer membrane protein OmpA (Agterberg et al., 1990a,b), and to lipoproteins such as the major lipoprotein Lpp, the TraT lipoprotein and the peptidoglycan-associated lipoprotein PAL (Fuchs et al., 1991, 1996; Dhillon et al., 1999; Chang and Lo, 2000; Chang et al., 1999). Proteins from the filamentous structures present on gram-negative bacteria have also been employed for surface-expression purposes. These include fimbria proteins such as the FimA protein (Hedegaard and Klemm, 1989) and the FimH adhesin of type I fimbriae (Pallesen et al., 1995), the flagellar protein flagellin (Wu et al., 1989; Newton et al., 1989) and pili proteins such as the PapA pilus subunit (Steidler et al., 1993) and F Pilin (Malmborg et al., 1997). Certain display systems have been based on proteins that have specific mechanisms for translocation and surface anchoring. The lipoprotein pullulanase from Klebsiella pneumoniae has been employed for surface-display purposes in E. coli (Kornacker and Pugsley, 1990). Using this system, the target protein becomes transiently anchored to the outer membrane by its N-terminal fatty acid moiety and is subsequently released into the culture medium. The b-domain of the Neisseria gonorrhoeae IgA protease precursor (Igab) and the E. coli AIDA-I have also been employed for surface exposure of various heterologous proteins. When used as a C-terminal-
fusion partner, the Igab and AIDA-l mediate attachment of hybrid proteins on the outer surface of Salmonella typhimurium and E. coli strains without the OmpT protease (Maurer et al., 1997; Klauser et al., 1990). One particularly interesting system is based on the combined features of an Lpp-OmpAH chimera (Francisco and Georgiou, 1994; Francisco et al., 1992, 1993a,b; Stathopoulos et al., 1996; Georgiou et al., 1997; Daugherty et al., 1998). The Lpp-OmpAH chimera is comprised of the signal sequence and first nine amino acids of Lpp, fused to a region encompassing either three or five transmembrane helices of OmpA. This Lpp-OmpAH display system has been shown to give efficient translocation and surface anchoring of the fused gene products, resulting in a high number of chimeric surface proteins present in an accessible form on E. coli cells. 5. Surface display in gram-positive bacteria Gram-positive bacteria have also been considered suitable for bacterial surface-display purposes (Hansson et al., 1992; Pozzi et al., 1992; Fischetti et al., 1996; Georgiou et al., 1997). A range of proteins, including antigenic determinants, heterologous enzymes and single-chain Fv (scFv) antibodies, have been targeted and anchored to the cell surface of gram-positive bacteria. Several gram-positive bacterial hosts have been investigated in this context, but until recently the research has focused on nonpathogenic staphylococci used in food-fermentation processes (Hansson et al., 1992; Pozzi et al., 1992; Hanski et al., 1992; Samuelson et al., 1995). Schneewind et al. (1992, 1993, 1995) have elucidated the mechanisms of cell-surface targeting and subsequent anchoring of surface proteins on staphylococcal cells several years after heterologous surface display had been achieved on staphylococci and streptococci. They found that Staphylococcus aureus protein A was sorted to the cell surface by its C-terminal surface-anchoring region. That region consists of a charged repetitive region, postulated to interact with the peptidoglycan cell wall. The Ctermini of numerous gram-positive bacterial surface receptors are highly homologous, suggesting that this or a similar mechanism is utilized for their targeting to the cell surface (Pozzi et al., 1992; Schneewind et al., 1993, 1995). The surface-display systems developed for Staphylococcus xylosus (Hansson et al., 1992) and Staphylococcus carnosus (Samuelson et al., 1995) both take advantage of the cell-surface-anchoring regions of protein A. The Staphylococcus gordinii surface-display system (Pozzi et al., 1992) uses the similar C-terminal region of the M6 protein of Streptococcus pyogenes to achieve surface exposure of various chimeric surface proteins. While the staphylococcal systems utilize gene expression from plasmid vectors, the streptococcal system is based on incorporation of the target genes into the streptococcal chromosome by homologous recombination (Hansson et al., 1992). Recently, heterologous surface expression was achieved in Bacillus anthracis by integrating into the chromosome a translational fusion harboring the DNA fragments encoding the cell wall-targeting domain of the S-layer protein EA1 tetanus toxin fragment C. This construct was expressed under the control of the promoter of the S-layer component gene (Mesnage et al., 1999).
The major applications of bacterial surface display (see below) are antibody and enzyme display, and vaccine development. In addition, other less common applications include peptide display for mapping epitopes of monoclonal antibodies. In one example, a random peptide library in a conformationally constrained thioredoxin region was introduced into the flagellin gene of E. coli and thus surface-exposed on the E. coli flagellum (Lu et al., 1995). The epitopes for three different immobilized monoclonal antibodies were mapped by the selection of bacterial clones and the identified consensus epitopes had several amino acids in common with motifs found in the original antigens used to generate the monoclonal antibodies. A very different application of bacterial surface display was recently suggested by Sousa et al. (1996), who displayed polyhistidyl peptides on the surface of E. coli using the LamB system. E. coli cells with surface-exposed histidine tags adsorbed approximately 11 times more Cd2 + ions than control cells. Such bacteria could perhaps be used for bioadsorption of heavy metal ions, potentially valuable for environmental (bioremediation) applications. Powerful techniques to monitor and characterize the exposed target proteins being displayed are crucial for the full exploitation of any display technology. Traditional techniques for studying (recombinant) surface-displayed proteins include immunofluorescence and immunogold methods. Although these methods reveal whether a heterologous protein is produced in an accessible form or not, they fail to give reliable quantitative information about the number of exposed chimeric surface proteins per bacterial cell. Immunofluorescent staining can give misleading results by positive staining of partially lysed cells and should thus be accompanied by additional assays. Flow cytometry, in the form of fluorescence-activated cell sorting (FACS), has been used as the method of choice to characterize surface display on bacteria (Fuchs et al., 1991; Stover et al., 1993; Francisco et al., 1993b; Nguyen et al., 1995; Samuelson et al., 1995). 6. Surface display of antibodies Phage display is by far the major tool for the isolation and engineering of recombinant antibodies, while bacterial display plays a minor role in that field. Antibodies in the form of recombinant antibody fragments were the first proteins to be successfully displayed on the surface of phage (McCafferty et al., 1990). This was achieved by fusing the coding sequence of the antibody variable (V) regions encoding for a single-chain Fv (scFv) fragment to the amino terminus of the phage gene III, coding for the phage minor coat protein pIII. Initial attempts to display FabH fragments fused to pVIII, the phage major coat protein, were also successful (Gram et al., 1992). However, the pVIII site, although very popular for peptide phage display, is not suitable for the efficient display of large polypeptides such as antibodies. For this reason, most antibody phage-display systems utilize the pIII site. Antibodies were first displayed using a phage vector, based on the genome of fd-tet (Zacher et al., 1980) and its gene III as fusion partner. In this vector, the genes coding for antibody scFv fragments were cloned in-frame with gene III and downstream of the gene III signal sequence, which normally directs the export of the phage-coat protein to the periplasm. Here, the antibody VH and VL domains may fold correctly, both stabilized by an
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intramolecular disulfide bridge, and pair to form a functional scFv (Skerra and Pluckthun, 1988; Better et al., 1988). Initially, phage vectors that carried all the genetic information required for the phage life cycle were used (McCafferty et al., 1990; Clackson et al., 1991). Later, phagemids became the popular type of vector for display. Phagemids are small plasmid vectors that carry gene III with appropriate cloning sites (for the insertion of cloned antibody genes) and the phage intergenic region (for rolling-circle replication and packaging) (Bass et al., 1990; Hoogenboom et al., 1991; Garrard et al., 1991). In phagemids, the scFv may be fused at the Nterminus of an intact gene III (McCafferty et al., 1990; Hoogenboom et al., 1991) or at the Nterminus of a truncated gene III, lacking the first two N-terminal domains (Bass et al., 1990; Barbas et al., 1991). Phagemids have high transformation efficiencies and are therefore ideally suited for generating very large repertoires. They may also be formatted for direct secretion of the unfused antibody fragment, without subcloning (Hoogenboom et al., 1991). Many phagemids utilize the lac promoter to control the expression of the antibodypIII fusion product (Hoogenboom et al., 1991; Barbas et al., 1991). For display of the antibody pIII product, glucose, acting in catabolite repression of the lac promoter, is removed or depleted, leading to the expression of sufficient fusion product to generate monovalent phage particles. Whenever expression-mediated toxicity is an issue [which is the case for some, mostly hybridoma-derived, antibody fragments (Thompson et al., 1996)], tighter regulation of expression levels may be required. The use of a lac promoter with an additional transcriptional terminator (Krebber et al., 1996), the tightly regulated tet promoter (Zahn et al., 1999) or of the phage shock promoter (psp) (Rakonjac et al., 1997) may allow display of relatively toxic products and reduce expression-mediated library biases. The phagemid DNA encoding the antibodypIII fusion are preferentially packaged into phage particles using a helper phage such as M13KO7 or VCS-M13, which supplies all structural proteins. Since the helper phage genome encodes wild-type pIII, typically over 90% of rescued phage display no antibody at all and the vast majority of the rescued phage particles that do display the fusion product will only contain a single copy. More efficient, multivalent display may be preferable when selecting very large antibody libraries, to guarantee selection when a limited number of phage particles per clone are available. Monovalent display, on the other hand, may be essential when selecting antibodies for higher affinity. Therefore, the use of inducible promoters (Lutz and Bujard, 1997) or the use of a helper phage with gene III deleted (Griffiths et al., 1993), may allow the modulation of the valency of displayed antibodies. Phage antibody selection involves the sequential enrichment of specific binding phage from a large excess of non-binding ones. This, as shown in Fig. 1, is achieved by multiple rounds of phage binding to the target, washing to remove nonspecific phage and elution to retrieve specific binding phage. Any method that separates phage that bind from those that do not, can be used for phage selection, and indeed, many different selection methods have been used. The most popular selection methods include affinity selection (also called biopanning) on immobilized antigen coated onto solid supports, columns or BIAcore sensor chips (Clackson et al., 1991; Marks et al., 1991; Griffiths et al., 1994; Malmborg et al., 1996), selection using biotinylated antigen (Hawkins et al., 1992), panning on fixed prokaryotic cells (Bradbury et
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Fig. 1. The phage affinity-selection (biopanning cycle). Recombinant DNA techniques are used to generate a library consisting of millions of different antibodies, or of variants (mutants) of an existing antibody. The resulting phage library is subjected to several cycles of affinity selection including capture of phage with antigen, washing to remove unbound phage, elution to release antigen-bound phage, and amplification in E. coli. When adequate enrichment has been obtained, individual antigen-specific clones are isolated and characterized.
al., 1993) and on mammalian cells (Cai and Garen, 1996), subtractive selection using sorting procedures (de Kruif et al., 1995), enrichment on tissue sections or pieces of tissue (van Ewijk et al., 1997), and, finally, selections using living animals, as reported for peptide phage libraries (Pasqualini and Ruoslahti, 1996). Phage antibodies bound to antigen may be eluted in different ways: with (one step or gradients of) acidic solutions such as HCl or glycine buffers, with basic solutions like triethylamine, with chaotropic agents, with DTT when biotin is linked to antigen by a disulfide bridge (Marks et al., 1991), by enzymatic cleavage of a protease site engineered between the antibody and gene III, or by competition with excess antigen (Marks et al., 1991; Bradbury et al., 1993; Griffiths et al., 1994; de Kruif et al., 1995). Antibody phage display has been the leading tool in antibody engineering during the last decade. Initially applied to the isolation of antibodies against various target molecules, the
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technology has evolved into an efficient tool for the in vitro manipulation of antibody affinity, specificity and stability. The most successful application of antibody phage display has been the isolation of antibodies from large libraries. For that purpose, three types of libraries were used. First, immune libraries that were generated by cloning antibody genes from B cells (usually from spleens) of immunized donors. This approach takes advantage of the fact that antibodies directed against the immunogen are enriched in such donors, and that they have been subjected to in vivo affinity maturation by the host immune system (Clackson et al., 1991; Burton et al., 1991). A further advantage of immune libraries is the ability to isolate many antibodies that are immunogen-specific from a single library. Immune libraries have been produced from a variety of species, including mouse (Clackson et al., 1991; Chester et al., 1994; Kettleborough et al., 1994; Berdichevsky et al., 1999), human (Barbas et al., 1993; Cai and Garen, 1995), rabbit (Lang et al., 1996; Li et al., 2000a), camelids (Arbabi Ghahroudi et al., 1997; Muyldermans and Lauwereys, 1999), sheep (Li et al., 2000b), and chicken (Davies et al., 1995; Yamanaka et al., 1996). Immunization, however, is not always feasible, due to ethical considerations, possible toxicity or lack of immunogenicity of the antigen. Consequently, non-immune libraries were developed. Non-immune libraries are further divided into naive and synthetic antibody libraries. Naive libraries were constructed using the same methodology that was applied in the construction of immune libraries. The difference was that here B cells from non-immunized donors were used as sources for antibody genes. Several naive libraries have been constructed, and because they are not biased towards any specific antigen, were used to isolate antibodies against multiple antigens (Marks et al., 1991; Gram et al., 1992; Vaughan et al., 1996). With naive libraries, the affinities of isolated antibodies correlated with the library size. Hence it was necessary to construct very large libraries to obtain high-affinity antibodies (Vaughan et al., 1996; de Haard et al., 1999). Synthetic or semisynthetic libraries were the next generation of non-immune libraries. These were constructed artificially by assembling in vitro antibody V genes with synthetic D and J segments. The DJ segments contained information for coding CDR3 loops of random sequence and varying length. The resulting libraries were sometimes referred to as `singlepot' libraries, because each such library was a source of antibodies directed against multiple antigens (Hoogenboom and Winter, 1992; Garrard and Henner, 1993; Nissim et al., 1994; de Kruif et al., 1995). Antibodies selected from phage libraries are not always suitable for direct application as therapeutics or biotechnological reagents. In some cases, manipulation of the antibody affinity, valency, specificity or stability was required. For such cases, phage display was applied, in a manner similar to the process of producing synthetic libraries and selecting the best binders from them. Such secondary libraries contained variants of the initially isolated antibodies, where mutations were introduced either randomly, or following rational design. Mutations were introduced into the antibody genes using one of several methods, including site-directed mutagenesis (Schier et al., 1996; Chowdhury and Pastan, 1999) error-prone PCR (Hawkins et al., 1992), Chain shuffling (Clackson et al., 1991; Marks et al., 1992), DNA shuffling (Crameri et al., 1996), and mutator E. coli strains (Low et al., 1996; Irving et al., 1996). Using such approaches it was possible to obtain antibodies having extremely high affinities, well below 100 pM (Schier et al., 1996; Yang et al., 1995). Advanced phage-
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selection schemes in combination with secondary antibody libraries were useful in the isolation of antibodies having improved stability and folding properties (Spada et al., 1998; Forrer et al., 1999; Jung et al., 1999). Finally, phage display enabled the isolation of catalytic antibodies, where catalysis or binding to transition-state analogs were used as the selective criteria (Sastry et al., 1995; Janda et al., 1994; Baca et al., 1997; Fujii et al., 1998). The expression of functional antibodies on the surface of E. coli (Fuchs et al., 1991; Little et al., 1993; Francisco et al., 1993b; Maurer et al., 1997) and staphylococci (Gunneriusson et al., 1996, 1999) has led to discussion whether this strategy would be a way to create inexpensive diagnostic tools or alternatives to the rapidly developing phage technology for the selection of peptides or recombinant antibody fragments from large libraries (Little et al., 1993; Georgiou et al., 1997). Most studies of bacterial display of antibodies involved the Lpp-OmpAH system developed by Georgiou and coworkers (Francisco and Georgiou, 1994; Francisco et al., 1993b; Georgiou et al., 1997; Chen et al., 1996) (schematically drawn in Fig. 2). The Lpp-OmpAH system was applied for the development of a quantitative immunoassay that utilizes E. coli bacteria expressing scFv antibody fragments attached to the cell surface (Chen et al., 1996). Scatchard analysis demonstrated that the antibodies on the surface of the cells retained full binding activity and that about 60 000 scFv molecules were displayed per cell. The bacteria were used as the antibody reagent in that assay, and, following incubation with analyte simple centrifugation could separate the antibody-bound from unbound analyte. The Lpp-OmpAH system was also applied for antibody affinity maturation by bacterial surface display of scFv libraries utilizing FACS (Daugherty et al., 1998). This system was employed to isolate clones with high affinity to a fluorescently labeled hapten from libraries
Fig. 2. Schematic representation of the Lpp-OmpAH display system. Surface display on E. coli is facilitated by the Lpp signal peptide (residues 1 9) followed by residues 46 159 of OmpA. The OmpA fragment is composed of five membrane-spanning b-strands, with the C-terminus located outside the cell. A protein linked to the Cterminus of the construct is thus located at the cell surface.
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constructed by randomizing heavy and light-chain residues in the anti-digoxin 26-10 derived scFv-antibody. The use of flow cytometry enabled the detection of rare library members directly in heterogeneous populations and the optimization of selection conditions prior to sorting. A similar approach was used for in vitro scanning saturation mutagenesis of the antidigoxin 26-10 antibody's binding site that allows the high throughput production and characterization of antibody mutants with improved binding affinity (Chen et al., 1999), and to investigate the effects of mutation frequency on the affinity maturation of the displayed antibody (Daugherty et al., 2000). A possible advantage of the bacterial-display systems over the phage-display techniques lies in the fact that bacterial selection can be accomplished through FACS technology, using fluorescently labelled antigens. This avoids crucial steps in phage-display selection procedures such as immobilization of the antigen, elution of bound phage and reinfection of bacteria with eluted phage. Filamentous phages are too small to be compatible with current FACS technology (Francisco and Georgiou, 1994). It has, however, been shown that E. coli cells expressing functional cell-surface-anchored antibody fragments can he separated by FACS (Francisco and Georgiou, 1994; Francisco et al., 1993b; Fuchs et al., 1996; Chen et al., 1999; Daugherty et al., 2000). Moreover, by manipulating the concentration of the fluorescent ligand it is possible to obtain higher and lower affinity antibodies in a single experiment based on signal intensity. Using this approach it was possible to analyze quantitatively the effect of mutation frequency (inserted by error-prone PCR) on the frequency of clones that remain functional and on the likelihood of isolating gain-of-function mutants of a high-affinity single-chain antibodies (Daugherty et al., 2000). The results demonstrated that large protein libraries can be surface displayed on bacteria, that scFv fragments can be functionally expressed on bacterial cells, and that such cells can be efficiently enriched by FACS technology. This approach should promote future use of bacterial display as an alternative or at least a complement to the phage systems. 7. Surface display of enzymes, substrates and inhibitors Both phage and cell display have contributed to the field of enzyme technology. A number of enzymes have been displayed on phage (McCafferly et al., 1991; Corey et al., 1993; Soumillion et al., 1994; Maruyama et al., 1994; Mikawa et al., 1996; Mullaney and Black, 1996; Pedersen et al., 1998; Demartis et al., 1999; Forrer et al., 1999; Hufton et al., 1999; Legendre et al., 2000) and bacteria (Francisco et al., 1992; Schneewind et al., 1992, 1993; Strauss and Gotz, 1996; Jung et al., 1998a, b). Initially attempts were made to apply phage display for the isolation of `improved' enzymes, or for enzymes with altered substrate specificity by selecting for improved binding to transition-state analogs. This approach resulted in modest improvements for catalytic antibodies displayed on phage (Fujii et al., 1998; Baca et al., 1997; Hansson et al., 1997; Arkin and Wells, 1998). However, binding of transition-state analogs does not correlate well with improved catalysis (Fujii et al., 1998; Arkin and Wells, 1998). Specialized selection schemes using reactive substrates (Janda et al., 1994), inhibitors (Light and Lerner, 1995), active-site ligands (Widersten and Mannervik, 1995), and reactive products (Janda et al., 1997) have also been used in attempts to select for
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improved catalysts. The selection from libraries of phage-displayed enzymes based on catalytic activity of the mutants is more complex than selection based on affinity for a ligand, which is how phage display is routinely applied. Several strategies have been proposed to alleviate this problem (Soumillion et al., 1994; Hansson et al., 1997; Atwell and Wells, 1999; Demartis et al., 1999; Jestin et al., 1999). These novel approaches are based on linking the enzymatic reaction product to the surface of the phage that displays the active enzyme. It is thus possible to identify the reaction products (and the phage they are linked to) with an appropriate antibody. Recently, a technique based on phage display for the directed evolution of enzymes of industrial interest was proposed. This technique is based on the use of relevant suicide inhibitors specifically designed to irreversibly bind the active pocket of the enzyme following their cleavage (Janda et al., 1997). By applying phage display for enzyme evolution, it should also be possible to focus selection on particular properties besides catalysis. The robustness of the phage themselves (their tolerance to a wide pH range and buffer conditions) makes it is possible to improve the stability and the catalytic properties of the displayed enzymes by altering library construction and selection conditions. In addition to the display of enzymes, phage were used to display enzyme substrates and enzyme inhibitors. The term `substrate phage' was coined by Matthews and Wells (1993) to describe a prototype approach for identifying novel substrates for enzymes (mostly proteases). A library of fusion proteins was constructed containing an amino-terminal domain used to bind to an affinity support, followed by a randomized protease substrate sequence and the carboxylterminal domain of M13 gene III. Each fusion protein was displayed as a single copy on filamentous phagemid particles (substrate phage). Phage were then bound to an affinity support and treated with the protease of interest. Phage that displayed good protease substrates were released, whereas phage with substrates that resisted proteolysis remained bound. This approach was valuable in identifying novel substrates or optimizing existing ones (Matthews and Wells, 1993; O'Boyle et al., 1997; Ke et al., 1997a,b; Ohkubo et al., 1999). A direct approach similar to that used for the isolation of antibodies from phage libraries was undertaken for the isolation of enzyme inhibitors. In these studies, engineered variants, based on small proteins derived from natural inhibitors (Roberts et al., 1992; Pannekoek et al., 1993; van Meijer et al., 1996; Huang et al., 1998) or peptide libraries (Ploug et al., 1998), were displayed on phage. Inhibitors were isolated by selecting phage on immobilized enzymes. Potent inhibitors of trypsin (Markland et al., 1991; Roberts et al., 1992), tPA (Pannekoek et al., 1993; van Meijer et al., 1996; Ploug et al., 1998), beta-lactamase (Huang et al., 1998), serine proteases (Jespers et al., 1995), human plasma kallikrein and human thrombin (Markland et al., 1996) were readily isolated using this approach. Enzyme inhibitors that are based on novel protein scaffolds were isolated by Sollazzo and coworkers (Martin et al., 1997; Dimasi et al., 1997). They isolated inhibitors of hepatitis C virus NS3 protease from an antibody `camelized VH' library (Martin et al., 1997) and from a `Minibodi' library (Dimasi et al., 1997). While phage display was used primarily for the molecular evolution of enzymes, substrates and inhibitors, with cell display the emphasis was different. Certain enzymes have been expressed with retained activity on the surface of gram-negative and grampositive bacteria (Table 2), and the potential use of such recombinant bacteria as novel
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microbial biocatalysts has been investigated. E. coli b-lactamase, which is normally a periplasmic enzyme, has been successfully exposed on the outer surface of E. coli using the pullulanase system (Kornacker and Pugsley, 1990) or the Lpp-OmpAH system (Francisco et al., 1992) as has been the Cex exoglucanase of Cellulomonas fimi (Francisco et al., 1993a). Bacteria displaying heterologous enzymes on their surface hold great potential as wholecell biocatalysts. For industrial applications, such surface-engineered cells need to be killed and chemically fixed to prevent disintegration and leakage of the displayed proteins under process conditions. It is also highly desirable to couple the chemically stabilized cells onto a solid support matrix for additional mechanical stability, flexibility in reactor choice, and easy separation from processed medium. Using such an approach, a method designed to prepare an immobilized whole cell biocatalyst from beta-lactamase-displaying E. coli cells was recently described (Freeman et al., 1999). The method is based on crosslinking carried out in the presence of beta-lactamase inhibitors, that protects the active site from chemical modification resulting in up to threefold higher specific activities without affecting the cellstabilizing effect of the glutaraldehyde treatment. However, not all enzymes can be efficiently exposed on the bacterial surface. Alkaline phosphatase, which is normally periplasmic, was found to be retained in the periplasm of E. coli when expressed via the Lpp-OmpAH system (Stathopoulos et al., 1996). The efficacy of using E. coli with surface-displayed heterologous enzymes as novel microbial biocatalysts remains to be seen. Surface expression is undoubtedly an inexpensive way to produce immobilized enzymes, but gram-negative bacteria might suffer from practical drawbacks such as inhibition of growth or cell lysis. The E. coli display systems seem to be limited in the size of proteins they are capable of exporting to the surface (Tommassen et al., 1985; Stathopoulos et al., 1996). Recently, the ice-nucleation protein (Inp), a glycosyl phosphatidylinositolanchored outer membrane protein found in some gram-negative bacteria was used for enzyme display in E. coli. By using Pseudomonas syringae Inp as an anchoring motif, the functional display of Zymomonas mobilis levansucrase (LevU) on the cell surface was obtained (Jung et al., 1998a,b). The cells expressing Inp-LevU were found to retain both the ice-nucleation and whole-cell levansucrase enzyme activities. Viability of the cells was also maintained over 48 h in the stationary phase. When the levansucrase-displayed cells were used as the enzyme source, levan was efficiently synthesized from sucrose with 34% conversion yield, generating glucose as a by-product. Regarding enzyme display on gram-positive bacteria, a lipase from Staphylococcus hyicus and E. coli b-lactamase were expressed on the outer cell surface of S. carnosus with retained activity (Strauss and Gotz, 1996). Approximately 10 000 enzyme molecules were found to be present on each cell and it was suggested that the rigid structure of gram-positive bacteria would make them particularly appropriate as microbial catalysts. More recently, bacterial surface display was applied for enzyme evolution. The E. coli surface-display system based on Inp (Jung et al., 1998a,b) was used to selectively screen for improved variants of carboxymethyl cellulase (CMCase) (Kim et al., 2000). A library of mutated CMCase genes generated by DNA shuffling was fused to the ice nucleation protein gene so that the resulting fusion proteins were displayed on the bacterial cell surface. Some cells displaying mutant proteins grew more rapidly on carboxymethyl cellulose plates than controls, forming heterogeneous colonies. In contrast, cells displaying the parent CMCase
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formed uniform tiny colonies. These variations in growth rate were assumed to result from altered availability of glucose caused by differences in the activity of variant CMCases at the cell surface. Staining assays indicate that large, rapidly growing colonies exhibited increased CMCase activity. Increased CMCase activity was confirmed by assaying the specific activities of cell extracts after the expression of unfused forms of the variant genes in the cytoplasm. The best-evolved CMCases showed about a 5- and 2.2-fold increase in activity in the fused and free forms, respectively. Sequencing of evolved CMCase variant genes showed that most amino acid substitutions occurred within the catalytic domain of the enzyme. These results demonstrate that the bacterial surface display of enzyme libraries may provide a direct way to correlate evolved enzyme activity with cell growth rates. This technique may provide a useful technology platform for directed evolution and high-throughput screening of industrial enzymes, including hydrolases. 8. Vaccine development Both phage- and cell-display systems have been used for vaccine development, but with differing approaches. Phage display has been used mostly for the identification of immunogenic epitopes or mimotopes on displayed peptides. These, in turn could become lead molecules for the design of peptide-based vaccines. In only a handful of cases have peptidedisplaying phage themselves been used for immunization. A number of groups used monoclonal of polyclonal antibodies to select peptide libraries. At the dawn of the phage-display era, George Smith, who pioneered the phage-display technology (Smith, 1985), proposed that foreign sequences could be inserted within the minor coat protein, pIII, of filamentous phage. This will result in the creation of a fusion protein that is incorporated into the virion; the virions display the foreign amino acids encoded in the insert on their surface. Phage, bearing specific antigenic determinants from a target gene, can be purified in infectious form from a vast excess of phage that carry other determinants, by virtue of their affinity to the antibody directed against the gene product. Fusion phage can be used as a source of antigen, as a carrierhapten conjugate for obtaining immunological reagents in rabbits, and for B epitope mapping. By generating a fusion-phage library expressing virtually all possible short, amino acid sequences, it may be possible to study epitopes on immunologically important proteins without the use of synthetic peptides and without ever having cloned the genes (Parmley and Smith, 1989). Indeed, de la Cruz et al. (1988) showed that cloning of repeat regions of the circumsporozoite protein gene of Plasmodium falciparum into the pIII gene of a filamentous phage results in the display of the recombinant proteins on the phage surface. The displaying phage were both antigenic and immunogenic when injected to rabbits. Shortly thereafter phage display in the form of combinatorial peptide libraries was used to map linear epitopes of monoclonal antibodies (Scott and Smith, 1990; Cwirla et al., 1990; Devlin et al., 1990; Felici et al., 1991, reviewed in Lane and Stephen, 1993; Daniels and Lane, 1996). The potential of phage display for identification of neutralizing viral epitopes was demonstrated by Perham (di Marzo Veronese et al., 1994) who inserted peptide sequences
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from the V3 loop of gp120 from HIV-1 into the N-terminal region of the major coat protein (pVIII) of filamentous bacteriophage fd. This approach led to their display in multiple copies on the surface of the virion. Peptides displayed in this way were shown to be remarkably effective structural mimics of the natural epitope. They were recognized by human HIV antisera and evoked high titres of antibodies in mice, which cross-reacted with other strains of HIV and were capable of neutralizing the virus. Soon it was realized, however, that peptide sequences selected from phage library do not always correspond to the exact (linear) sequence of the original epitope. It was postulated that such peptides mimic the structural features of the epitope in being recognized by epitopespecific antibodies, and by eliciting upon injection into animals, antibodies capable of recognizing the original epitope. Such peptide mimics were thus named `mimotypes' (Cortese et al., 1994; Folgori et al., 1994; Mennuni et al., 1996; Demangel et al., 1996). Mimotypes were later identified for various nonprotein molecules such as the O-antigen part of the human pathogen Shigella flexneri serotype 5a lypopolysaccharide (Phalipon et al., 1997), the Neisseria meningitidis group B polysaccharide (Moe et al., 1999) and meningococcal group C polysaccharide (Grothaus et al., 2000). While most of the studies were carried out using filamentous phage peptide libraries, other phage systems were later developed for similar purposes. A 36-amino-acid PorA peptide from N. meningitidis was cloned into the display vectors to generate fusions at the N terminus of Hoc or Soc capsid genes of phage T4 (Jiang et al., 1997). The PorA-Hoc and PorA-Soc fusion proteins retained the ability to bind to the capsid surface, and the bound peptide was displayed in an accessible form as shown by its reactivity with specific monoclonal antibodies. Both the PorA-Hoc and PorA-Soc recombinant phages were highly immunogenic in mice and elicited strong antipeptide antibody titers. Once it was established that phage display can be useful for epitope identification, the use of displaying phage as immunogens was considered. A number of groups compared the magnitude of antibody response that was elicited by various immunization protocols. Tested criteria included display valency (monovalent vs. polyvalent display), the presence and nature of added adjuvants, and the route of administration. Willis et al. (1993) displayed the antigenic determinants of the circumsporozoite protein of the malaria parasite, P. falciparum in multiple copies on pVIII of Fd phage. The peptide epitopes in the hybrid phage were found to be strongly immunogenic in four different strains of mice without the use of external adjuvants, and the antibodies were highly specific to the individual epitopes. When tested in nude mice, the immune response was found to be T-cell dependent and to undergo class-switching from IgM to IgG. Proliferation assays of T cells, taken from lymph nodes of BALB/c mice injected with bacteriophage particles in the presence or absence of Freund's complete adjuvant, indicated no difference in the immune response. Meola et al. (1995) tested if mimotopes could be useful in developing a vaccine against the human hepatitis B virus. They compared the humoral immune response of animals immunized either with a recombinant HbsAg vaccine or with mimotopes. Immunogens were prepared by fusing the mimotypes on different carrier molecules (phage coat protein pIII and pVIII, recombinant human H ferritin, HBV core peptide) and by synthesizing multiple antigenic peptides carrying the mimotypes' amino acid sequences. These immunogens were injected
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into mice and rabbits and sera were collected and tested for the presence of HBsAg-specific antibodies. The results confirmed that mimotopes could induce a humoral immune response resembling that induced by the original antigen, and HBsAg mimotopes displayed on phage prove to be the best immunogens, inducing the most repro- ducible and potent immunization. Delmastro et al. (1997) showed that intranasal or intragastric administration of phage into mice induces an immunological response both to the wild type phage proteins and to mimotopes displayed on them. Using mimotopes of human hepatitis B virus surface antigen and of human hepatitis C virus peptides, they showed that the response induced by oral administration is specifically cross-reactive with the original antigen. Bastien et al. (1997) investigated whether a recombinant bacteriophage displaying a disease-specific protective epitope could be experimentally used as a vaccine to confer protection of immunized animals against infection. They genetically engineered a recombinant phage, fd, displaying at its surface a chimeric pIII coat protein (monovalent display) fused to the previously identified protective epitope 173187 from the glycoprotein G of the human respiratory syncytial virus. A selected recombinant fd phage elicited a strong immune response in mice, inducing a high level of circulating RSV-specific antibodies. Mice immunized with the recombinant phage acquired a complete resistance to RSV infection as evidenced by the lack of detectable virus particles in their lungs following intranasal challenge with live RSV. This was the first report of the ability of a phage presenting an immunogenic peptide to prevent infection of immunized animals by a pathogen. Recently, Zuercher et al. (2000) tested the effect of exposure of displaying phage to gastric fluid. This approach was examined, since an essential requirement for oral vaccines is the ability to survive the harsh environment of the stomach in an antigenically intact form. The feasibility of this approach was tested in a simulated gastric fluid using two different mimotopes as well as an anti-idiotypic Fab of the non-anaphylactogenic monoclonal anti-IgE antibody BSW17. All phage clones remained infective after this treatment. However, only epitopes displayed on the pVIII protein (polyvalent display) were still recognized by BSW17 whereas pIII-expressed (monovalent display) epitopes were rapidly inactivated. Surprisingly, when used for oral immunization of mice all phage clones induced anti-IgE antibodies. In contrast, oral immunization with the purified, pVIII protein that displayed the mimotope also induced anti-phage antibodies, but no anti-IgE antibodies were obtained. After feeding a single dose of mimotope-displaying bacteriophage, phage DNA could be detected in mouse feces for 10 days. These results have shown that epitope-displaying bacteriophage can be used to induce an epitope-specific antibody response via the oral route. In conclusion, phage that display epitopes or mimotopes may emerge as useful tools for the development of effective vaccines or may serve as vaccine-delivery vehicles themselves. For the application of phage as vaccines it seems that polyvalent rather than monovalent display are more likely to elicit an efficient immune response, that oral administration is the more effective route for immunization, and that adjuvant is usually not required. Such immunization protocols can be less expensive and more effective than the conventional method of synthesis and coupling peptide ligands to particulate synthetic carriers for immunization. The most common application of bacterial surface display has been in the development of live bacterial vaccine delivery systems. In contrast to phage display, which was a tool for epitope discovery and for vaccine delivery, displaying immunogenic peptides or proteins on
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bacteria for the purpose of vaccine development followed a different scheme. Here, an already identified vaccine candidate (an immunogenic peptide or protein) was displayed on bacteria to be used as carriers for vaccine delivery. Gram-positive cells were earlier considered for vaccine delivery. Nguyen et al. (1993) developed a system for the surface expression of heterologous receptors on the cell surface of S. xylosus. That expression system was constructed by subcloning gene fragments encoding for peptides to be displayed on the cell surface into an E. colistaphylococci shuttle vector. The vector was designed for targeting of recombinant fusion proteins to the outer cell surface of the gram-positive host cell. A specific humoral immune response could be elicited in mice by oral immunization with the recombinant S. xylosus cells, suggesting that this type of gram-positive bacteria might offer potential vehicles for oral vaccination. Liljeqvist et al. (1999) assayed the surface expression in S. carnosus of three different fibronectin binding domains (FNBDs), derived from fibronectin binding proteins of Streptococcus dysgalactiae and S. aureus. All three surface-displayed FNBDs were demonstrated to bind fibronectin in whole-cell enzyme-linked binding assays. Furthermore, for one of the constructs, intranasal immunizations with the recombinant bacteria resulted in improved antibody responses to a model immunogen present within the chimeric surface proteins. To avoid the use of engineered pathogens for vaccine delivery, Fischetti et al. (1996) developed systems that allow the expression of heterologous antigens in commensal gram-positive bacteria. In some cases, both a serum IgG and secretory IgA response were induced to the recombinant protein after vaccination, verifying the validity of the approach. These live recombinant bacteria may be used in the future to introduce a protective immune response to pathogenic microorganisms after mucosal colonization. Many of the described gram-negative display systems have initially been evaluated in E. coli for surface display of various antigenic determinants, and subsequently applied to Salmonella spp. These species are of interest for the live, oral delivery of heterologous antigens for immunization. An early study demonstrated that bacteria that express recombinant proteins (although not at the surface in this particular example) could be used as live oral vaccines. In this study, Poirier et al. (1988) investigated the use of attenuated strains of Salmonella to deliver cloned antiphagocytic virulence determinants of unrelated bacteria. Thus, the aroA strain of S. typhimurium SL3261 was transformed with a low-copy plasmid that contained the cloned gene spm5, encoding streptococcal M protein (the major virulence factor of these organisms). The transformed Salmonella expressed type-5 M protein in the cytoplasmic fraction, and when fed orally to BALB/c mice, evoked both serum and salivary IgA, IgG, and IgM antibodies directed against type-5 M protein. The orally immunized mice were completely protected against both intranasal and intraperitoneal challenge infections with virulent S. typhimurium SL1344 or M5 streptococci. In a second study, mice were immunized with live Salmonella dublin, expressing a flagellum with an inserted cholera toxin, hepatitis B virus or influenza virus haemaglutinin epitopes. The immunized mice produced antigen-specific antibodies and in some cases partial protection from virus challenge (Wu et al., 1989; Newton et al., 1989). It was suggested that epitopes exposed at the bacterial cell surface induce antibody responses in a T-cell-independent manner and that the lipopolysaccharides may serve as adjuvants. These studies provided early evidence that an attenuated strain of Salmonella can be used effectively as a general vaccine vehicle to deliver antiphagocytic virulence determinants of unrelated bacteria.
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Schorr et al. (1991) used E. coli OmpA as a carrier for the expression of foreign antigens on the surface of gram-negative bacteria. Immunogenic moieties of P. falciparum blood stage antigens were expressed in an attenuated S. typhimurium strain. Live bacteria expressing the fusion proteins reacted with malarial antigen sera. Mice that were immunized orally with S. typhimurium cells expressing malarial antigens on their surface showed a humoral immune response. Leclerc et al. (1991) targeted two foreign B cell antigenic viral epitopes to different locations in the E. coli cell to examine what effect this had on antibody responses elicited by the recombinant bacteria. The two epitopes were peptides derived from the PreS2 region of hepatitis B virus and the C3 neutralization epitope of poliovirus type 1. They were each expressed in two forms either on the surface, as part of the outer-membrane protein LamB, or soluble in the periplasm, as part of the periplasmic protein MalE. When live bacteria expressing the foreign epitopes at the cell surface were used for immunization of mice, they induced T-cell-independent antibody responses characterized by a rapid induction of IgM and IgG antibodies. In contrast, when the same foreign epitopes were inserted into the MalE protein, the antibody response was only detectable after 3 weeks, belonged only to the IgG class and was strictly T-cell dependent. This study has therefore identified two major pathways by which epitopes expressed by bacterial cells can stimulate specific antibody responses. The first pathway is mediated by direct activation of B cells by bacterial cell-surface antigen and does not require T-cell help. The second pathway is T-cell dependent and concerns antigen that can be released from the bacteria in a soluble form. Stentebjerg-Olesen et al. (1997) assessed the potential of the major structural protein of type 1 fimbriae as a display system for heterologous sequences. As a reporter-epitope, a heterologous sequence mimicking a neutralizing epitope of the cholera toxin B chain was inserted into the fimA gene. The chimeric proteins were exposed on the bacterial surface and the cholera toxin epitope was authentically displayed, i.e. it was recognized on bacteria by specific antiserum. Immunization of rabbits with purified chimeric fimbriae resulted in serum, which specifically recognized cholera toxin B chain, confirming the utility of the employed strategy. It can be concluded that several of the developed bacterial surface-display systems, of both gram-positive and gram-negative bacteria merit additional future consideration for application as live or attenuated vaccines. 9. Phage and cell display come together As described in the previous sections, phage display has been used mostly for affinity screening of combinatorial peptide and protein libraries, to define epitopes for monoclonal antibodies, to select enzyme substrates and to screen cloned antibody repertoires. Phage libraries are enriched for specific binding clones by subjecting the phage to repetitive rounds of affinity selection (biopanning) that include binding, washing and elution steps, reinfection of bacteria and growth to re-express the binding molecule on the phage surface (Fig. 1). Several selection schemes may be applied to isolate antigen-specific phage from a heterogeneous population composed mostly of nonbinders. They include capture with immobilized antigen, capture with soluble ligand followed by isolation of phageligand complexes, selection on antigen columns, selection on living or fixed cells and selection in whole animals (Bradbury et al., 1993; Hoogenboom, 1997; Hoogenboom et al., 1998; Persic
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et al., 1999; Pasqualini and Ruoslahti, 1996; Poul and Marks, 1999; Becerril et al., 1999). With most selection approaches, successive panning results in the diminution of diversity with a few dominant phage clones (usually those having the highest affinity or display efficiency) being recovered. It is therefore desirable to isolate binders following fewer, preferably a single panning cycle. This, in principle, could be achieved by linking antigen recognition with phage infectivity, as has been demonstrated in SIP (selectively infective phage) and similar selection strategies (Malmborg et al., 1997; Krebber et al., 1997). However, even with those powerful selection strategies, enrichment factors rarely exceed 104 per cycle. Recently we developed a novel selection scheme that combines bacterial surface display and phage display of polypeptides for the highly efficient isolation of binders, where antigendisplaying E. coli cells are used to capture antibody-displaying phage. Bradbury et al. (1993) used a similar approach to select phage on living or fixed antigen-displaying F bacteria. In that case, an epitope derived from the oncoprotein p21ras was expressed within the E. coli outer membrane protein, LamB. A library of single-chain Fvs was selected on the displaying bacteria. Bound phage were eluted at a high pH and used to infect F + bacteria for phage expansion. In the reported example, an enrichment factor of 13 000 was obtained in a single cycle. Our recently described system (Benhar et al., 2000) was named DIP (delayed infectivity panning). In this approach, the enrichment factor exceeded 106 per cycle. DIP takes advantage of the versatile Lpp-OmpAH bacterial surface-display system (Georgiou et al., 1997) where protein-coding sequences are fused to an Lpp-OmpAH hybrid sequence. In addition to the successful display of enzymes, antibodies, and cellulose-binding proteins that were described in the earlier sections, we could display receptor domains and a bacterial toxin fragment by this system. DIP combines bacterial display with conventional phage-display libraries (Fig. 3) where the selecting bacterial cells are also being infected by the captured phage. This is made possible by the fact that F + E. coli cells grown at low temperatures do not express the F pilus, which is required for phage infection (Novotny and Fives-Taylor, 1974; Novotny and Lavin, 1971). After phage capture and washing procedures, the bacteria are transferred to 37C so pilus expression is induced and infection ensues. Some antigens (in particular cell-surface receptors) are notoriously difficult to produce as recombinant proteins. In DIP, however, a purified antigen is not required for phage selection and identification. Using two model antibodyantigen pairs, we demonstrated enrichment of over 106-fold in a single DIP cycle for phage, which display a specific antibody, over those displaying an irrelevant antibody. We further showed the successful isolation of anti-toxin and anti-receptor antibodies from an immune phage library, a shuffled library and a large synthetic human library. DIP selection is far more efficient than panning with immobilized antigen or compared to other systems where antigen-displaying cells are used to capture antibody-displaying phage. Several factors may contribute to this observation. First, the selection `matrix' is bacteria that are essentially identical to those used to propagate the phage (except that the latter do not display the antigen). This may be regarded as a `counter-selection' step, where most of the antibody-displaying phage that recognize E. coli cell-surface epitopes adhere to the cells that were used to produce them and are thus eliminated. Second, we may have been fortunate in selecting the Lpp-OmpAH bacterial display format as the platform for DIP,
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Fig. 3. Scheme of the DIP selection method. (A) Map of plasmid pIB-Tx used for bacterial display of antigens. (B) Scheme of DIP selection: (1) Antigen displaying E. coli grown at 16C, when the F pilus is not expressed, are mixed with antibody displaying phage. Antigen-specific phages bind to the cells. (2) Unbound phages are washed out. (3) Cells are shifted to 37C, so F pilus expression is induced, bound phage dissociate from antigen and infect the cell through the F pilus. (4) Infected cells where bacterial display plasmid and phage genome co-reside.
in that the antigen density and accessibility to antibodies may be better than antigens adsorbed to solid surfaces. Third, the antigen concentration used in DIP is relatively low: assuming 104 105 displayed molecules per bacterium (Francisco and Georgiou, 1994; Francisco et al., 1993b; Georgiou et al., 1997; Daugherty et al., 1998), with 1010 bacteria per milliliter we are in the nanomolar concentration range. Thus, our selection is more stringent than using micromolar concentrations of antigen, which is the general practice with immobilized antigens. The effectiveness of DIP makes it suitable for the efficient isolation of rare clones present in large libraries. Since a multitude of phage- and cell-based systems for surface display of polypeptides exists, DIP should be suitable for the isolation and characterisation of various proteinprotein interacting pairs. References
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Phage Display and Biotechnological Applications

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Biotechnology Advances 19 (2001) 1 33

Research review paper

Biotechnological applications of phage and cell display

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I. Benhar / Biotechnology Advances 19 (2001) 133

I. Benhar / Biotechnology Advances 19 (2001) 133

Displayed molecules Peptides

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6 Table 2 Bacterial display systems Bacterium

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A. Gram-negative bacteria E. coli LamB OmpA

Pal Pap PhoE TraT Type 1 fimbriae FliC FimH Lpp-OmpA

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I. Benhar / Biotechnology Advances 19 (2001) 133

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I. Benhar / Biotechnology Advances 19 (2001) 133

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I. Benhar / Biotechnology Advances 19 (2001) 133

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I. Benhar / Biotechnology Advances 19 (2001) 133

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I. Benhar / Biotechnology Advances 19 (2001) 133

I. Benhar / Biotechnology Advances 19 (2001) 133

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