**3. Phage display technology for new biomarker binder discovery**

Screening phage display libraries are a powerful tool for identifying specific binders from libraries containing a large diversity of phage surface expressed molecules [20, 21]. Libraries construction are achieved by fusing a repertoire of genes (genotype) encoding the peptides/ proteins to a gene encoding a capsid structural protein. The "displayed" peptides/proteins (phenotypes) are included in the capsid layer on the phage surface. Ideally, these proteins should not be interfered with the phage structure [22].

The amplification of eluted phage is carried out by infecting the exponential growth phase of *Escherichia coli*. To assembly and produce the recombinant phage a helper phage is added [45], whereas T7 phages can be directly released from the host by cell lysis [46]. Successive rounds of biopanning varied by types of library and target antigen used. In practice, the enrichment of phages of interest can be obtained within three to six rounds of biopanning. Further rounds of selection may potentially lead to bias by the enrichment of non-specific

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Phage display is a powerful technology for the generation of antibodies for medical applications. Nowadays, approximately 30 monoclonal antibodies have been approved by FDA for use in clinical practice with many more currently being tested in clinical trials. [49, 50]. The principle of the phage display is represented in **Figure 2**, indicating the workflows of library construction, biopanning and clone screening prior to purification for functional

**Figure 2.** Principle of filamentous bacteriophage M13 phage display using a phagemid vector. Antibody genes encoding for millions of variants of libraries are cloned into a phagemid vector carrying the gene encoding for one of five phage coat proteins (pIII). Large phage libraries can be obtained by transforming *E. coli* with phagemids and rescue of phages with helperphage. Hence, phages displaying the specific antibodies against immobilized targets can be selected and isolated by several rounds of biopanning. These steps involve binding, washing, elution, infection and amplification. The eluted bound phages are subsequently screened by ELISA assay and followed by DNA sequencing prior to their

background phages [47, 48].

protein expression and purification.

assays.

The display technologies have enabled exploration of new antibodies from humans or animals, including shark, camel, llama and lamprey [23–26] that may not otherwise be discovered.

#### **3.1. Antibody phage display library**

Antibody phage display libraries have been extensively used for isolation of specific high affinity binders against unique antigens from different targets [27–31]. Three types of antibody library are typically constructed: naïve, synthetic and immunized libraries [32]. A naïve antibody library refers to the repertoire of antibody genes derived from non-immunized donors. Synthetic antibody libraries are constructed using synthesized mutated CDRs and synthetic frameworks whereas immunized libraries are based on a host immunized with a target antigen of disease [33].

The function of the phagemid vector is akin to that of a plasmid whereby the genes of interest can be cloned directly into the multiple cloning sites upstream of the capsid structural phage protein after digestion by appropriate restriction enzymes. Phage display technology has facilitated the selection of different antibody fragments using genetic engineering approaches [34]. Many antibody fragments created (Fab, scFv and diabody) were used to overcome the limitations of conventional IgG antibodies derived from higher organism [19]. Furthermore, the presentation of single domain antibodies (sdAb) of heavy chains derived from different animals are being widely investigated, including camelids VHH or Nanobodies®, sharks VNAR region of IgNAR [35] and the antibody of variable-like lymphocytes (VLRs) from lamprey fish [36].

#### **3.2. Biopanning of phage display**

The selection of high binding clones from antibody libraries using phage display can be undertaken *in vitro* via a process called biopanning. In this process, the antibody fragments displayed on the surface of phages are incubated with an antigen of interest that is immobilized on a surface [37, 38]. Generally, immunoabsorbent ELISA microplates, uncoated cell culture dishes and immunotubes are commonly used for ligand immobilization [39]. Nonspecific or unbound phages are removed by washing, whereas phage that binds specifically to the target is eluted by changing the binding conditions, depending on types of bacteriophages used in the experiment. For instance, acidic solutions of HCl or glycine buffer are used for M13 bacteriophage [40]. Other methods include use of basic solutions of triethylamine [41], enzymatic cleavage of protease site incorporated in the recombinant coat protein [42], competition with excess antigen [38] and direct bacterial elution [43] have been reported for the elution of M13 bacteriophage. For T7 phage display system, the elution buffer is 1% SDS [44].

The amplification of eluted phage is carried out by infecting the exponential growth phase of *Escherichia coli*. To assembly and produce the recombinant phage a helper phage is added [45], whereas T7 phages can be directly released from the host by cell lysis [46]. Successive rounds of biopanning varied by types of library and target antigen used. In practice, the enrichment of phages of interest can be obtained within three to six rounds of biopanning. Further rounds of selection may potentially lead to bias by the enrichment of non-specific background phages [47, 48].

**3. Phage display technology for new biomarker binder discovery**

should not be interfered with the phage structure [22].

**3.1. Antibody phage display library**

178 Antibody Engineering

**3.2. Biopanning of phage display**

Screening phage display libraries are a powerful tool for identifying specific binders from libraries containing a large diversity of phage surface expressed molecules [20, 21]. Libraries construction are achieved by fusing a repertoire of genes (genotype) encoding the peptides/ proteins to a gene encoding a capsid structural protein. The "displayed" peptides/proteins (phenotypes) are included in the capsid layer on the phage surface. Ideally, these proteins

The display technologies have enabled exploration of new antibodies from humans or animals, including shark, camel, llama and lamprey [23–26] that may not otherwise be discovered.

Antibody phage display libraries have been extensively used for isolation of specific high affinity binders against unique antigens from different targets [27–31]. Three types of antibody library are typically constructed: naïve, synthetic and immunized libraries [32]. A naïve antibody library refers to the repertoire of antibody genes derived from non-immunized donors. Synthetic antibody libraries are constructed using synthesized mutated CDRs and synthetic frameworks whereas immunized libraries are based on a host immunized with a target antigen of disease [33]. The function of the phagemid vector is akin to that of a plasmid whereby the genes of interest can be cloned directly into the multiple cloning sites upstream of the capsid structural phage protein after digestion by appropriate restriction enzymes. Phage display technology has facilitated the selection of different antibody fragments using genetic engineering approaches [34]. Many antibody fragments created (Fab, scFv and diabody) were used to overcome the limitations of conventional IgG antibodies derived from higher organism [19]. Furthermore, the presentation of single domain antibodies (sdAb) of heavy chains derived from different animals are being widely investigated, including camelids VHH or Nanobodies®, sharks VNAR region of IgNAR [35] and the antibody of variable-like lymphocytes (VLRs) from lamprey fish [36].

The selection of high binding clones from antibody libraries using phage display can be undertaken *in vitro* via a process called biopanning. In this process, the antibody fragments displayed on the surface of phages are incubated with an antigen of interest that is immobilized on a surface [37, 38]. Generally, immunoabsorbent ELISA microplates, uncoated cell culture dishes and immunotubes are commonly used for ligand immobilization [39]. Nonspecific or unbound phages are removed by washing, whereas phage that binds specifically to the target is eluted by changing the binding conditions, depending on types of bacteriophages used in the experiment. For instance, acidic solutions of HCl or glycine buffer are used for M13 bacteriophage [40]. Other methods include use of basic solutions of triethylamine [41], enzymatic cleavage of protease site incorporated in the recombinant coat protein [42], competition with excess antigen [38] and direct bacterial elution [43] have been reported for the elution of M13 bacteriophage. For T7 phage display system, the elution buffer is 1% SDS [44].

Phage display is a powerful technology for the generation of antibodies for medical applications. Nowadays, approximately 30 monoclonal antibodies have been approved by FDA for use in clinical practice with many more currently being tested in clinical trials. [49, 50]. The principle of the phage display is represented in **Figure 2**, indicating the workflows of library construction, biopanning and clone screening prior to purification for functional assays.

**Figure 2.** Principle of filamentous bacteriophage M13 phage display using a phagemid vector. Antibody genes encoding for millions of variants of libraries are cloned into a phagemid vector carrying the gene encoding for one of five phage coat proteins (pIII). Large phage libraries can be obtained by transforming *E. coli* with phagemids and rescue of phages with helperphage. Hence, phages displaying the specific antibodies against immobilized targets can be selected and isolated by several rounds of biopanning. These steps involve binding, washing, elution, infection and amplification. The eluted bound phages are subsequently screened by ELISA assay and followed by DNA sequencing prior to their protein expression and purification.

#### **3.3. Types of bacteriophage utilized in phage display system**

In phage display systems, different bacteriophages have been used to display a range of proteins on surface. M13 filamentous bacteriophage [51, 52] and T7 lytic phage are the most commonly used for displaying and production of antibody fragments [53, 54]. A comparison between M13 bacteriophage and T7 lytic phage are discussed in the following section and summarized in **Table 1**.

appropriately folded proteins containing disulfide bonds. Hence, many functional antibody fragments, enzymes and inhibitors have been displayed and selected using this system [28, 59, 60]. However, it also has the minor limitation of poor display of cytoplasmic proteins on the membrane [61]. Moreover, the removal of stop codons in the DNA library can facilitate correct display of the foreign proteins on the coat protein at the N-terminus of M13 bacteriophage [56].

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The bacteriophage display and cloning system using T7, T4 and λ phage was introduced in 1990s, and has several advantageous features over other phage display systems [62–64]. As a lytic phage, the T7 phage contains a linear double-stranded DNA genome. It has a diameter of 55 nm, with the capsid shaped in an icosahedron structure. The Novagen's T7Select® is the commercially available phage display system that takes advantage of the properties of bacteriophage T7. There are three types of vectors available in this system: for peptide display with up to 50 amino acids in high-copy number (415 per phage); 1200 amino acids display in mid-copy number (5–15 phage) and 1200 amino acids display in low-copy number (0.1–1 per phage) [64]. Fusion proteins are displayed at the C-terminal end of the T7 capsid protein (gene 10); the removal of the stop codon from foreign genes is not necessary, resulting in ease construction of a library. The diversity of T7 phage display is often dependent on the packaging efficiency into the capsid. Nevertheless, a successfully constructed library could encode a library of the

clones [64]. In contrast to bacteriophage M13, the secretion of library proteins

through the periplasmic layer of the host cell does not occur in the T7 phage display system. This may lead to the reduction of physiochemical restriction and less bias in the library peptide diversity [65]. In addition, the T7 phage system has the advantages of being able to display a cytoplasmic protein, a major limitation of the M13 filamentous phage [61, 66].

However, folding of cytoplasmic proteins with disulfide bonds in T7 bacterial phage system do not occur quite well. This problem can be resolved by using the complementing hosts such as BLT5615 or BLT 5403 *E. coli* strain included in the T7Select® kit [65, 67, 68]. In term of general features, T7 phage grows much faster than M13. After infection, clear plaques (lawns) of T7 phages can usually be observed within 2–3 h on an LB plate at 37°C. Furthermore, the purification process of T7 phage for ELISA and DNA sequencing is also simple to perform,

With the advent of recombinant DNA technology, antibody genes can be selected and amplified using phage display, yeast display, bacterial display, ribosome display, mRNA display, DNA display or mammalian cell surface display [69–73] and see chapter in this book: "Display technologies for the selection of monoclonal antibodies for clinical use" by Tsuruta et al. A range of mammalian V-gene libraries have been used to undertake *in vitro* recombinant antibodies screening projects using phage display. These include mouse [74], rabbit [75], sheep [76] and human [77]. Unlike hybridoma technology, the direct link between the geno-

with only PEG/NaCl precipitation required to recover the purified phage [47, 65].

**4. Engineered sdAb fragments from vertebrates**

*3.3.2. T7 bacteriophage system*

size 10<sup>7</sup>

–108

#### *3.3.1. Filamentous bacteriophage M13 system*

The filamentous phage M13 is the most extensively used phage for antibody phage display [55]. Other classes of filamentous phages that have been studied include F1 and Ff phages [56, 57]. In the mature virus particle, filamentous phage M13 have a cylindrical-shaped structure, about 930 nm in length and phage proteins are encoded by a circular single-stranded DNA genome. Foreign peptides are typically displayed on the N-terminal of the minor p3 coat protein or on the major p8 coat protein with the copy numbers from 5 to more than 2000 depending on type of vectors used. However, type 3 is the most widely used display format [56, 58]. Generally, this leads to expression of 1–3 copies of the recombinant fusion protein on the phage surface.

The diversity of M13 phage display libraries typically ranges from 10<sup>5</sup> to 1012, and is greatly dependent on the transformation efficiency of the host *E. coli*. As the proteins are secreted through periplasmic layers, the M13 phage display system represents a suitable tool to display the


**Table 1.** Comparison of M13 filamentous phage with T7 phage.

appropriately folded proteins containing disulfide bonds. Hence, many functional antibody fragments, enzymes and inhibitors have been displayed and selected using this system [28, 59, 60]. However, it also has the minor limitation of poor display of cytoplasmic proteins on the membrane [61]. Moreover, the removal of stop codons in the DNA library can facilitate correct display of the foreign proteins on the coat protein at the N-terminus of M13 bacteriophage [56].

#### *3.3.2. T7 bacteriophage system*

The bacteriophage display and cloning system using T7, T4 and λ phage was introduced in 1990s, and has several advantageous features over other phage display systems [62–64]. As a lytic phage, the T7 phage contains a linear double-stranded DNA genome. It has a diameter of 55 nm, with the capsid shaped in an icosahedron structure. The Novagen's T7Select® is the commercially available phage display system that takes advantage of the properties of bacteriophage T7. There are three types of vectors available in this system: for peptide display with up to 50 amino acids in high-copy number (415 per phage); 1200 amino acids display in mid-copy number (5–15 phage) and 1200 amino acids display in low-copy number (0.1–1 per phage) [64].

Fusion proteins are displayed at the C-terminal end of the T7 capsid protein (gene 10); the removal of the stop codon from foreign genes is not necessary, resulting in ease construction of a library. The diversity of T7 phage display is often dependent on the packaging efficiency into the capsid. Nevertheless, a successfully constructed library could encode a library of the size 10<sup>7</sup> –108 clones [64]. In contrast to bacteriophage M13, the secretion of library proteins through the periplasmic layer of the host cell does not occur in the T7 phage display system. This may lead to the reduction of physiochemical restriction and less bias in the library peptide diversity [65]. In addition, the T7 phage system has the advantages of being able to display a cytoplasmic protein, a major limitation of the M13 filamentous phage [61, 66].

However, folding of cytoplasmic proteins with disulfide bonds in T7 bacterial phage system do not occur quite well. This problem can be resolved by using the complementing hosts such as BLT5615 or BLT 5403 *E. coli* strain included in the T7Select® kit [65, 67, 68]. In term of general features, T7 phage grows much faster than M13. After infection, clear plaques (lawns) of T7 phages can usually be observed within 2–3 h on an LB plate at 37°C. Furthermore, the purification process of T7 phage for ELISA and DNA sequencing is also simple to perform, with only PEG/NaCl precipitation required to recover the purified phage [47, 65].

## **4. Engineered sdAb fragments from vertebrates**

**Table 1.** Comparison of M13 filamentous phage with T7 phage.

**3.3. Types of bacteriophage utilized in phage display system**

The diversity of M13 phage display libraries typically ranges from 10<sup>5</sup>

summarized in **Table 1**.

180 Antibody Engineering

*3.3.1. Filamentous bacteriophage M13 system*

In phage display systems, different bacteriophages have been used to display a range of proteins on surface. M13 filamentous bacteriophage [51, 52] and T7 lytic phage are the most commonly used for displaying and production of antibody fragments [53, 54]. A comparison between M13 bacteriophage and T7 lytic phage are discussed in the following section and

The filamentous phage M13 is the most extensively used phage for antibody phage display [55]. Other classes of filamentous phages that have been studied include F1 and Ff phages [56, 57]. In the mature virus particle, filamentous phage M13 have a cylindrical-shaped structure, about 930 nm in length and phage proteins are encoded by a circular single-stranded DNA genome. Foreign peptides are typically displayed on the N-terminal of the minor p3 coat protein or on the major p8 coat protein with the copy numbers from 5 to more than 2000 depending on type of vectors used. However, type 3 is the most widely used display format [56, 58]. Generally, this leads to expression of 1–3 copies of the recombinant fusion protein on the phage surface.

dependent on the transformation efficiency of the host *E. coli*. As the proteins are secreted through periplasmic layers, the M13 phage display system represents a suitable tool to display the

to 1012, and is greatly

With the advent of recombinant DNA technology, antibody genes can be selected and amplified using phage display, yeast display, bacterial display, ribosome display, mRNA display, DNA display or mammalian cell surface display [69–73] and see chapter in this book: "Display technologies for the selection of monoclonal antibodies for clinical use" by Tsuruta et al. A range of mammalian V-gene libraries have been used to undertake *in vitro* recombinant antibodies screening projects using phage display. These include mouse [74], rabbit [75], sheep [76] and human [77]. Unlike hybridoma technology, the direct link between the genotype and the phenotype of displayed antibodies during selection (biopanning) can facilitate the identification of binding antibodies and corresponding antibody genes. Further, the gene encoding the desired antibody can be manipulated to improve affinity, specificity and expression or fusion to a carrier protein can be performed [38, 48, 78].

unique CDR3 region enables the heavy domain of camelids to penetrate into antigen cleft regions that are not easily recognized by conventional antibodies [92, 93]. From a phylogenetic prospect, since camelids are related to the primate lineage [94] it is possible to produce humanized VHH, a process that may be easier to perform than the complicated manipulation required to "humanize" murine or other more distant species to reduce an alloresponse, such

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Furthermore, due to their high intrinsic domain stability, camelids VHH are now under investigation as probes for diagnostics [18, 96]. The diagnostic potential of camelids VHH as probes in immunodetection systems offers the possibilities of improving the diagnosis of infection [97], cancers [98] and food contaminants [99]. Although VHHs do not originate from humans, the humanizations strategies of VHHs have successfully been undertaken by designing a humanized scaffold region onto the antigen-binding loops (CDRs) of specific VHHs can be grafted [100]. In addition, non-humanized and humanized VHHs with therapeutic potential have been applied in multiple areas, including hematology [101], inflammatory diseases [102], infectious diseases [103], *in vivo* imaging [104], neurological disorders [105]

A class of naturally occurring antibodies comprising a variable domain of a heavy chain (VNAR) without a variable light chain domain was discovered in the serum of elasmobranch cartilaginous fish during early of 1990s [108–110]. These natural functional antibody repertoires were termed as immunoglobulin new antigen receptors (IgNARs). IgNARs are an unconventional and unique class of proteins found in sharks, including nurse sharks (*Ginglymostoma cirratum*) [111], wobbegong sharks (*Orectolobus maculatus*) [112], smooth dogfish (*Mustelus canis*) [113], banded hound sharks (*Triakis scyllium*) [68] and horn shark (*Heterodontus francisci*) [114]. Investigations have revealed that IgNARs function as antibody and immune response mediators in sharks. However, until now it is not clear if the IgNARs as single domain antibodies

Several desirable biological properties of IgNAR V domains have been identified, and their potential as alternative antigen binders explored [112, 113, 116]. The natural habitat of sharks has resulted in evolving extraordinary stable antibodies such that the functionality of antibodies can be retained in a harsh environment [117]. Electron microscopic studies have indicated that the intact IgNAR exists as a disulfide-bonded homodimer that consists of a polyprotein

Similar to the camelid VHH, the VNAR has only a heavy-chain domain. However, the crossspecies conservation of the amino acid sequence with a human VH is extremely low in a VNAR domain (~25%), whereas it is more than 80% homologous to VHH scaffolds in camelids VHH [110, 119]. It is hypothesized that IgNARs lack many residues that exist in conventional antibodies. These are replaced by other hydrophilic residues. The greatly truncated CDR2 region, herein defined as HV2 region, has created a signature hallmark for shark VNAR. Due to this unusual structure, the single variable heavy domain proteins of shark IgNARs are currently the smallest antibody fragments observed in animal kingdoms, having a size of only 12 kDa.

arise from TCR domains/L chains or primitive cell surface molecules [109, 115].

with one variable domain (VNAR) and five constant domains (CNAR) (**Figure 1**) [118].

as the human anti-mouse antibody (HAMA) response [95].

and oncology [106, 107].

**4.2. VNAR heavy-chain domain in sharks**

An advantage of sdAb fragments is their ease of genetic manipulation due to their smaller size, in addition ease of expression in bacterial system, low lot-to-lot variation and easy scaledup production [79, 80] . Moreover, sdAb production is not influenced by species-specific cell fusion partner incompabilities. Nowadays, the desired sdAb repertoire can be developed from shark, camels and humans with an appropriate set of specific primers [81] . However, an additional step of point mutations in framework regions and CDR randomization is required to construct human VH and VL sdAbs [81]. Regardless, the generation of sdAbs by bacterial fermentation is significantly cheaper, simpler and quicker than conventional polyclonal Abs or mAbs production [80, 82–84]. The general features of some natural sdAb fragments are described in the following section.
