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`
`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.
`
`Keywords: phage display,
`antibody, library, valency,
`selection
`
`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
`
`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 specific 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
`
`N1, first N-terminal domain of g3p
`N2, second N-terminal domain of g3p
`CT, C-terminal domain of g3p
`IG, intergenic region
`RT, reverse transcription
`
`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 first
`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 N-
`terminus of g3p. Since then, large scFv,
`
`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
`specific 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
`specific 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
`
`& HENRY STEWART PUBLICATIONS 1473-9550. B R I E F I N G S I N F U N C T I O N A L G E N O M I C S A N D P R O T E O M I C S . VOL 1. NO 2. 189–203. JULY 2002
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`1 8 9
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`Lassen - Exhibit 1032, p. 1
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`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 five copies
`each of g7p and g9p. At the proximal end,
`four to five 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 filamentous phage has been
`shown to be associated with a resistance of
`the cell to infection from filamentous
`
`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
`
`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.5–8 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
`sufficient 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 filamentous 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 difficult with
`immunisation/hybridoma techniques.
`
`M13 phage biology
`M13 is a filamentous bacteriophage. The
`native particle is a thin, cylindrical shape,
`usually 900 nm long and 6–7 nm in
`diameter.11,12 It contains a single-stranded
`DNA genome (6,407 base pairs in length)
`which encodes 11 genes,13 five of which
`
`Carmen and Jermutus
`
`The oxidising periplasm
`of E. coli favours
`disulphide bond
`formation
`
`Filamentous phage
`assemblies in the E. Coli
`periplasm
`
`1 9 0
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`& HENRY STEWART PUBLICATIONS 1473-9550. B R I E F I N G S I N F U N C T I O N A L G E N O M I C S A N D P R O T E O M I C S . VOL 1. NO 2. 189–203. JULY 2002
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`Lassen - Exhibit 1032, p. 2
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`Concepts in antibody phage display
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`X
`
`II
`
`V
`
`IG
`
`ori
`
`6.4 kb
`
`IV
`
`VII
`
`IX
`
`VIII
`
`IG
`
`III
`
`I
`
`VI
`
`XI
`
`Figure 2: The structure of M13 phage particle. The phage particle is of cylindrical shape (900 nm long) and only 6–7 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 five copies each
`
`phage. Its presence in the E. coli
`membrane disrupts the membrane
`integrity, causing a number of effects
`including defective F-pili.17
`The five coat proteins have all been
`
`All coat proteins can be
`used for phage display
`
`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 F-
`pilus and the CT is necessary for termination of phage assembly. The
`glycine-rich linkers, G1 and G2, are thought to confer flexibility 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
`
`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
`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
`
`& HENRY STEWART PUBLICATIONS 1473-9550. B R I E F I N G S I N F U N C T I O N A L G E N O M I C S A N D P R O T E O M I C S . VOL 1. NO 2. 189–203. JULY 2002
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`1 9 1
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`Carmen and Jermutus
`
`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 efficiencies
`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 modified intergenic region
`(IG),28 causing it to be replicated and
`packaged less efficiently than the
`phagemid which carries the wild-type
`M13 IG. This ensures that the genotype
`(phagemid) and phenotype (g3p–scFv
`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
`
`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) ScFv–g3p 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
`
`1 9 2
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`& HENRY STEWART PUBLICATIONS 1473-9550. B R I E F I N G S I N F U N C T I O N A L G E N O M I C S A N D P R O T E O M I C S . VOL 1. NO 2. 189–203. JULY 2002
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`Lassen - Exhibit 1032, p. 4
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`Downloaded from https://academic.oup.com/bfg/article-abstract/1/2/189/207941 by guest on 20 July 2019
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`Concepts in antibody phage display
`
`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 protein–ligand
`interactions. The ligand is coupled to
`N1–N2 either by the use of an expression
`vector or, if it is sufficiently 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
`g3p–S–S–scFv 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
`
`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
`
`& HENRY STEWART PUBLICATIONS 1473-9550. B R I E F I N G S I N F U N C T I O N A L G E N O M I C S A N D P R O T E O M I C S . VOL 1. NO 2. 189–203. JULY 2002
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`1 9 3
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`Carmen and Jermutus
`
`Most phage do not
`display a fusion protein
`
`Library sizes have
`continued to increase
`
`Naı¨ve libraries contain
`natural CDRs
`
`Synthetic libraries
`contain artificial CDRs
`
`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 modified
`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 DH5Æ/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 finding 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 filamentous 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
`The genomic information coding for
`antibody variable domains is usually
`derived from B cells of either ‘naı¨ve’
`(non–immunised) or immunised
`donors.36 An antibody repertoire from
`immunisation is generally restricted to
`generating antibodies against the antigen
`of the original immunogenic response,
`
`whereas naı¨ve 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-specific primers to
`amplify the VH and VL gene segments
`from the cDNA template, and restriction-
`based 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 first naı¨ve 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
`affinities.37 About five 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 affinities in the subnanomolar
`range. These results support theoretical
`predictions38 that the size of the library is
`thought to be proportional to the
`affinities 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 purification and choice of strain,
`have been described that allow the
`generation of library sizes of 1010 in a
`single electroporation step.39
`
`Synthetic antibody libraries
`Construction of synthetic libraries
`involves rearranging VH and VL gene
`segments in vitro and introducing artificial
`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 Vº3 (DPL16) light chain
`
`1 9 4
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`& HENRY STEWART PUBLICATIONS 1473-9550. B R I E F I N G S I N F U N C T I O N A L G E N O M I C S A N D P R O T E O M I C S . VOL 1. NO 2. 189–203. JULY 2002
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`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 five 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 non-
`human 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 Specific
`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 affinity,
`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) affinities
`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
`
`Concepts in antibody phage display
`
`avoiding rare codons in E. coli. All six
`CDRs are flanked by restriction sites to
`facilitate affinity 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 flanking
`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.
`
`Antibodies that form
`functional proteins in a
`reducing environment
`are rare
`
`Large library sizes can
`alternatively be created
`in vivo recombination
`
`& HENRY STEWART PUBLICATIONS 1473-9550. B R I E F I N G S I N F U N C T I O N A L G E N O M I C S A N D P R O T E O M I C S . VOL 1. NO 2. 189–203. JULY 2002
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`1 9 5
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`Quality control of antibody
`libraries
`As larger libraries are possible with
`improved technology, the task of assessing
`diversity remains difficult. BstNI
`fingerprinting is a quick way of checking
`for diversity since the scFv sequence can
`be amplified 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 efficient
`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
`affinity (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.45–47 Alternatively,
`clones that display g3p from the helper
`phage, due to absence of
`g3-fusion protein, can be effectively
`removed using a modified helper phage.
`
`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 non-
`infectious 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-specific 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
`
`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 five main
`steps: (i) coating of antigen; (ii) block; (iii)
`incubation of phage with antigen; (iv)
`washing to remove non-specific phage;
`and (v) elution (Figure 6A–D).
`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
`difficult to access by the phage library.
`Antigen can be biotinylated for selections
`in solution. The phage–antibody–antigen
`
`Carmen and Jermutus
`
`Library quality can be
`assessed by its
`performance in
`selections
`
`Co-expression of
`chaperones can
`improve display levels
`
`Small molecules are
`first conjugated to a
`carrier molecule
`
`1 9 6
`
`& HENRY STEWART PUBLICATIONS 1473-9550. B R I E F I N G S I N F U N C T I O N A L G E N O M I C S A N D P R O T E O M I C S . VOL 1. NO 2. 189–203. JULY 2002
`
`Lassen - Exhibit 1032, p. 8
`
`

`

`Concepts in antibody phage display
`
`Downloaded from https://academic.oup.com/bfg/article-abstract/1/2/189/207941 by guest on 20 July 2019
`
`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 amplified. (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 specificity
`
`Reversibly bound
`antigen may reduce the
`number of specific
`antibody hits
`
`complexes are subsequently captured
`using streptavidin-coated paramagnetic
`beads. Selections on cell surfaces have also
`been successfully demonstrated,54–59 as
`well as on protein bands from
`nitrocellulose membranes following two-
`dimensional (2D) electrophoresis.50,60,61
`Antigen purity is important since
`contaminants presented to a naı¨ve
`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 non-
`specific 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 influence 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
`
`& HENRY STEWART PUBLICATIONS 1473-9550. B R I E F I N G S I N F U N C T I O N A L G E N O M I C S A N D P R O T E O M I C S . VOL 1. NO 2. 189–203. JULY 2002
`
`1 9 7
`
`Lassen - Exhibit 1032, p. 9
`
`

`

`Downloaded from https://academic.oup.com/bfg/article-abstract/1/2/189/207941 by guest on 20 July 2019
`
`specific 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
`specific phage encountering antigen.
`Phage displaying scFv that are not able to
`bind the antigen will either remain in
`solution or may bind non-specifically.
`The washing step is thought to be crucial
`for the removal of non-specific phage.
`