Open access peer-reviewed chapter

The Development of Single Domain Antibodies for Diagnostic and Therapeutic Applications

Written By

Chiuan Herng Leow, Qin Cheng, Katja Fischer and James McCarthy

Submitted: October 14th, 2016 Reviewed: December 21st, 2017 Published: February 21st, 2018

DOI: 10.5772/intechopen.73324

Chapter metrics overview

2,024 Chapter Downloads

View Full Metrics

Abstract

Monoclonal antibodies have become increasingly accepted as diagnostics and therapeutics for various human diseases due to their high affinity and specificity. However, several practical drawbacks are apparent for the reagents based on conventional IgG antibodies. With the emergence of antibody engineering, many problems were overcome when the recombinant antibody fragments such as Fabs, scFvs, diabodies and single domain antibodies (sdAbs), are developed. These fragments not only retain the specificity of the whole monoclonal antibodies, but are also easy to express and produce in prokaryotic expression systems. Rather unexpectedly, the natural sdAbs namely VHHs, VNARs and variable lymphocyte receptors (VLRs) that comprise excellent biological activities were recently discovered in camelids, cartilaginous fish and lampreys, respectively. Due to their unique characteristics, including small size, high thermostability, stable folding in the nucleus and cytosol and long CDR3 regions which have access to cavities or clefts on the surface of proteins, these new binders are now investigated extensively as a substitute for conventional antibodies. This review describes the potential of sdAbs selected using in vitro display systems and their use in multiple applications.

Keywords

  • recombinant antibody
  • single domain antibody
  • diagnostic and therapeutic single domain antibody
  • scFv
  • IgNAR
  • VHH
  • VLR
  • VNARs

1. Introduction

In research applications, antibodies are widely used as binders due to their high specificity and high affinity. Antibodies can be classified into three different categories such as polyclonal antibodies, monoclonal antibodies and recombinant antibodies [1]. Polyclonal antibodies (polyclonal Abs) are heterogeneous antibody mixtures that are derived from multiple plasma cell lines. Because polyclonal antibodies comprise a mixture of different antibodies carrying numerous paratopes, they have excellent properties for recognizing antigens [2]. A monoclonal antibody (mAb) is a homogeneous antibody generated from a single B lymphocyte clone. Antibodies produced in mAb format have an extremely high specificity against a single epitope on antigens [3]. Recombinant antibodies (rAbs) are antibodies generated using molecular biology techniques. They are aimed to improve the sensitivity, selectivity, stability and immobilization properties in diagnostic applications, for example, in biosensors [4]. In making decision to use or generate polyclonal, monoclonal or recombinant antibodies, several factors should be considered, including commercial availability, possibility to raise animals, types of applications, time length of a project and costs [1]. Although a vast number of rAbs has been proposed [5, 6, 7, 8], the natural sdAb fragments that were recently discovered from camelids (VHHs), sharks (VNARs) and lampreys (VLRs) have shown to possess extraordinary features that are not found in conventional antibodies, such as a small dimension, an elevated stability and the capability of recognizing cavities and clefts on the surface of proteins that cannot be reached by conventional recombinant antibodies [9, 10, 11]. This chapter will discuss the availability of new binders derived from vertebrates and give an overview of their applications in a biomedical platform by recognizing specified targets from various diseases.

Advertisement

2. Monoclonal antibodies and their limitations

The first description of monoclonal antibody (mAbs) production was published by Nobel prize winners, Kohler and Milstein in 1984 [12]. The fusion technique developed between splenic B cells and myeloma cells is termed the hybridoma technique has revolutionized immunology and medicine. The production of mAbs is not influenced by sources of animal used, making mAbs having better homogeneity in scale-up production [13]. The mAb technology has been widely applied in biomedical research and pharmaceutical industries.

Unlike polyclonal Abs, the monospecificity of a mAb enables targeting of a single epitope. This enables a range of applications, including targeting specific members of a protein family and evaluating changes in molecular conformation and targeting protein-protein interactions. However, the specificity and sensitivity of mAbs can be reduced by small changes in the structure of the antigen determining regions, or even by minor changes in pH or salt concentration. An advantage is that, mAbs can be produced at a greater concentration and much higher purity than polyclonal Abs [13].

The conventional mAb predominantly produced as IgG after an immune response, is represented in Figure 1. As determined by their structural and biological properties, IgG molecules have specific features, namely their large size compared to recombinant antibody fragments, higher synthesis rate and longer half-life. IgGs are the most widely used immunoglobulins for antibody-based diagnostic and therapeutic development. Generally, conventional IgGs are characterized by having a high affinity (Ka) ranging from 10−2 to 101 nM, and excellent specificity for its cognate target epitope [14]. The high degree in variations of antibody specificities is conferred by the variable amino acid sequences in the variable regions of the heavy and light chain (VH and VL). Each variable domain is comprised of three hypervariable (HV) regions, separated by four framework regions (FR). The HV regions are known as complementarity-determining regions (CDRs), and are responsible for the identification of the specific epitope of the cognate antigen. The FR regions are major components of the backbone structure for VH and VL regions in antibodies and can potentially influence the conformation of the antigenic binding loops [15].

Figure 1.

Schematic representation of conventional antibodies and natural single domain antibodies. The conventional IgGs derive from mammals while the natural single domain antibodies derive from camelids, sharks and lampreys, respectively. Single V domains are presented as colored ovals; C domains are shown gray colored. The domains in lamprey variable lymphocyte receptor (VLR) are demonstrated on the right. VLRs consist of an N-terminal cap (LRRNT), the first LRR (LRR1), multiple (usually up to seven) 24-residue variable LRRs (LRRVs), a terminal or end LRRV (LRRVe), a connecting peptide (CP) and a C-terminal cap (LRRCT), followed by an invariant 3′-terminal region.

However, several practical drawbacks are apparent for diagnostic reagents based on conventional IgG antibodies. The complex architecture and large molecular size (~150 kDa) may result in weak binding when small size protein antigens are not easily recognized by the concave surfaces of CDR loops [16]. Initial attempts to generate single domain antibody fragments by separating expression of individual human VH or VL units was reported to result in solubility problems in aqueous solvents, higher cost and more time consuming process and the requirements for sophisticated protein engineering approaches [17]. Moreover, the failure of recognition of selected mAbs on conserved epitopes of specific antigens due to unbound reactivities mediated by the Fc region may hinder their utility for diagnostic applications [18, 19].

With the emergence of DNA engineering, surface display has been widely used to discover new antibody fragments as a means for diagnostic and therapeutic applications. An overview of principles in phage display technology, including antibody library construction, biopanning, types of bacteriophages used and antibody fragments applications are further discussed in the following sessions.

Advertisement

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 display technologies have enabled exploration of new antibodies from humans or animals, including shark, camel, llama and lamprey [23, 24, 25, 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, 28, 29, 30, 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 vitrovia 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]. Non-specific 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].

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 transformingE. coliwith 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.

Table 1.

Comparison of M13 filamentous phage with T7 phage.

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 105 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 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, 63, 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 107–108 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. colistrain 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].

Advertisement

4. Engineered sdAb fragments from vertebrates

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, 70, 71, 72, 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 vitrorecombinant 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].

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 scaled-up 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, 83, 84]. The general features of some natural sdAb fragments are described in the following section.

4.1. VHH heavy-chain domain in camelids

Conventional immunoglobulins comprise two major parts such as the antigen-binding fragment (Fab region) and fragment crystallisable region (Fc region), with a typical molecular weight of 150 kDa. The Fab domain is responsible for antigen binding and therefore its specificity. This domain is divided into heavy (H) and light (L) chains with the molecular weights of 25 kDa each [85]. The stability of the molecular complex of an immunoglobulin is conferred by four inter-domain disulfide bonds in the hinge regions. The heavy chain can be subdivided into one variable (VH) region and three constant (C) regions (CH1, CH2 and CH3) while the light chain contains one variable region (VL) and only one constant region (CL). Lacking direct antigen-binding functions, the main role of the Fc domain is to provide effector functions such as binding to cellular receptors on macrophages and complement activation, and determination of the half-life of an antibody [86].

In addition to conventional heterotetrameric antibodies, the sera of Camelidae were discovered to possess special IgG antibodies known as heavy-chain antibodies (HCAbs). Although HCAbs contain both a constant (Fc) and variable domain, these antibodies are slightly different from conventional IgG by devoid of the L chain polypeptide and the first constant domain (CH1) (Figure 1). Therefore, the isolated variable domain region of camelids HCAbs is known as VHH (variable domain of the heavy chain of HCAbs) or Nanobody® (Nb; Ablynx) [87]. VHH constitutes a binding surface to interact with the target antigen. The molecular weight of VHH is 15 kDa, 10 times lower than that of a conventional antibody. It was thereby considered the smallest possible antibody fragment and has attracted the interests of many scientists [88, 89]. Moreover, the capability of camelid antibodies to retain the reversibility and binding activity after heat denaturation has enabled new applications where transient heating may occur [90].

The major advantage of a VHH antibody is their greater solubility compared to classical VH [17]. This is due to the hydrophilic amino acid substitution present in the framework 2 region. Meanwhile, the single coding exon of less than 450 base pairs facilitates genetic engineering of VHH fragments [91]. In addition, on account of its smaller antigen binding surface area, the 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 as the human anti-mouse antibody (HAMA) response [95].

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 vivoimaging [104], neurological disorders [105] and oncology [106, 107].

4.2. VNAR heavy-chain domain in sharks

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

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

Similar to the camelid VHH, the VNAR has only a heavy-chain domain. However, the cross-species 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. Yet, in combination with the peculiar feature of a long CDR3 region, these VNAR domains tend to more readily penetrate cleft regions of antigens, thereby increasing the opportunity to target small target epitopes that may not be accessible to conventional IgG [120].

In terms of heat stability, VNAR also possess refolding properties as found in camelids VHH. The ability of retaining fully functional antigen-binding activity after exposure up to 95°C may make VNAR ideally suited to protein array and diagnostic applications where transient heating may occur as part of the protein immobilization process [9, 113]. It is partly due to the presence of cysteine residues in these single domain antibodies, resulting in an extraordinary conformation [35].

VNAR domains are more easily produced as recombinant proteins compared to conventional antibodies. Additionally, due to hydrophilic residues present within VNAR surfaces, high yields of expressed proteins associated with high solubility, are achievable and thus they can be easy produced in prokaryotic systems [112]. Therefore, the potential utility of VNARs as alternative binders for clinical applications is now being investigated in a variety of areas.

4.3. VLRs immunoglobulin-like domains in lamprey

Lamprey and hagfish are the only surviving groups of jawless fish, having appeared since the Cambrian period. The adaptive immune system of jawless vertebrates was recognized as unique due to the rearrangement of antigen receptors which is completely different from that used by jawed vertebrates [121]. The somatic rearrangement of the variable (V) gene segments, diversity (D) segments, joining (J) segments and constant (C) segments is commonly observed in conventional Ig-based Ag receptors. However, the immune system in jawless vertebrates is predominantly regulated by recombination activating gene (RAG)-independent combinatorial assembly to generate leucine-rich repeats (LRR) cassettes for Ag recognition. Owing to these differences, antibodies in jawless fish were termed as variable lymphocyte receptors (VLRs) rather than Ig superfamily (Figure 1) [122].

In comparison to CDR loops used by Ig-based antibodies and T-cell receptors in many animals, the antigen-binding regions of VLRs have evolved into variable β-strands and C-terminal loop structural motifs, resulting in a crescent-shaped protein conformation [123, 124]. Due to the prevalence of this unusual pattern, VLRs tend to be more useful for microbial recognition [36]. Thus far, two VLR genes have been identified in lamprey and hagfish, namely VLRA and VLRB. However, the VLRB gene in lamprey shows more complexity in terms of coding sequence analysis [125].

Sequences analysis has revealed that each VLR consists of a signal peptide (SP), hypervariable LRR regions, consisting of a 27–34 residue N-terminal LRR (LRRNT), the first 24-residue LRR (LRR1), up to nine 24-residue variable LRRs (LRRV), one 24-residue end LRRV (LRRVe), one 16-residue connecting peptide LRR (LRRCP) and a 48–63 residue C-terminal LRR (LRRCT) [126]. The assembly of VLRs entails greater recombination events in LRR modules and can efficiently generate more than 1014 unique repertoires at a level comparable to mammalian Ig. Thus, VLRs may be a source of single-chain domains alternative to conventional Ig-based antibodies [123]. Nevertheless no single domain antibody comprising only one engineered VLR domain has been so far reported.

Having undergone evolution over millions of years, VLRs appear to have been optimized as suitable antigen receptors for humoral protection. Further analysis indicates that VLRs are extremely stable in harsh environments. Their antigen binding capability remained unchanged even after it was eluted from a column at a very basic pH (>11) [11]. In addition, the heat stability of VLRs is similar to shark IgNARs and camelids VHH. For example, eluted VLRs can be stored over 1 year at 4°C, 1 month at room temperature and 36 h at 56°C. However, the degradation of Ag-binding activity occurred when the incubation period was prolonged more than 1 h at 70°C [126].

Although VLRs were discovered less than a decade ago [122], they have provided new insights into the potential of ancestral antibodies in biotechnogical applications. Owing to a greater VLR library diversity as well as associated self-tolerance ability, VLRs can be efficiently used to detect antigens that may not be recognized by mammalian Ig, for example, the sensitivity of VLRB mAb targeting against Bacillus anthracis(BclA) was superior to that of a high affinity conventional murine IgG [11]. Furthermore, the simple modular single polypeptide structures facilitate the production of VLRs antibodies through DNA engineering. VLRs combinatorial libraries of high affinity binders can be constructed through in vitrorandom mutagenesis and loop shuffling using a surface display technology approach, for instance, yeast display system [36]. Thus, VLRs may become alternatives for the developments of new reagents in diagnostic applications to overcome the lack of Ag recognition ability of conventional monoclonal antibodies made from mammals.

Advertisement

5. Use of different recombinant antibodies for specific applications

To date, humans and mice remain the main source of complete antibodies for targeting diseases. However, with the aid of DNA technology, a number of new antibody fragments have been engineered as smaller single domain fragments to improve immunoassays, immunosensors and imaging probes in various applications. As described recently, the discovery of natural single heavy domain antibodies from camelids VHH and shark VNAR, and in addition lamprey VLRs offers some advantages over conventional antibodies. This range of natural antibodies is expected to open various applications: to trace molecule trafficking and to inhibit protein function inside the cell as intrabody, to apply them as therapeuticum and they can be used as detection units in biosensors or immunodiagnostics. In this section, we will review the deployment of different binders in specific diagnostic applications and to what extent these binders are used.

5.1. Applications of camelids VHH domains or nanobodies®

To monitor infections, single domain antibodies naturally derived from camelids (nanobodies) may enable superior species-specific antigen detection than classical monoclonal antibodies in immunodiagnostic tests. Trypanosome infection causes African sleeping sickness and Chagas disease. Both are severe parasitic diseases caused by protozoa of the genus Trypanosoma. Sleeping sickness disease is mainly found in rural Africa. The antigenic variation strategy adopted by this parasite represents a major barrier to the immune system to eliminate it. Consequently, it is difficult for specific mAbs to detect genus-specific antigens [127]. By adoption of an in vitroselection method, novel nanobody clones were isolated that showed specificity to T. evansiat species level, and genus-specific reactivity against various Trypanosomaspecies [128].

Cysticercosis is a serious tissue infection caused by larval cysts of the pork tapeworm, which is prevalent in many low-income countries [129]. Monoclonal antibodies that are currently deployed in sandwich ELISAs are mainly genus-specific against Taeniasp., but poorly specific at a species level to identify Taenia solium, the major Taeniaspecies threatening human health [130, 131]. To circumvent such limitations, an in vitroselection of nanobodies from immunized dromedaries was developed to recognize a specific marker on T. solium. After in vitroselection, the nanobodies showed no cross-reactivity against other livestock Taeniaspecies, while having a very specific response to a specific 14 kDa glycoprotein (Ts14) in T. solium. Therefore, nanobodies showed potential as an alternative to genus-species mAb for developing unambiguous ELISA tests for human cysticercosis [97].

Apart from diagnostic reagents for infectious diseases, nanobodies have been identified as alternative binders to analyze the compositions of substances in food and beverages industries. Due to their excellent thermal stability, nanobodies showed superior behavior to classical mouse mAbs in ELISA to measure caffeine concentration in hot and cold beverages [132].

Camelid sdAbs have recently been applied in ELISA methods to detect a wide range of small molecules, including explosive materials (trinitroluene or TNT) [133], agents of bioterrorism (Botulinum A neurotoxin) [90], toxins (ricin, cholera and staphylococcal enterotoxin B) [134], scorpion toxin [135] and viruses (HIV, rotavirus, Vaccinia and Marburg) [136, 137, 138]. Owing to the combination of several favorable properties, camelid nanobodies have also been employed as molecules to diagnose diseases. In small molecule development, the advanced features of highly stable and unique conformational structure of nanobodies have permitted overcoming many problems faced by traditional whole antibodies and scFv fragments such as cross-reactivity and nanoparticle agglutination. The development of biosensors coupled with nanobodies (nanoconjugates system) has enabled significant improvement in the performance of a device to identify harmful bacteria (Staphylococcus aureus) to a nanometer scale within 10 min [139].

Nevertheless, mAbs remain the common binding agents to identify and trace tumor-associated proteins for noninvasive in vivoimaging. However, limitations, particularly large size (150 kDa) and Fc regions, result in mAbs poorly penetrating into solid tumors [140]. The emergence of nanobodies offers the possibility of resolving such problems, and thereby enables nanobodies to diagnose tumor markers such as EGF receptor [141]. This will enable cancer staging predictions in blood circulation such as prostate-specific antigen [142]. More applications using camelids VHH targeting antigens from various diseases are summarized inTable 2.

Target antigensDiseasesApplicationsReference
VEGF-A165NeoangiogenesisDiagnostic and therapeutic[143]
HER2Breast cancerDiagnostic[144, 145]
HPV-16 L1 proteinCervical cancerDiagnostic and therapeutic[146, 147]
HSP-60Brucellosis (livestock)Diagnostic and vaccine[23, 148]
VCAM1Atherosclerotic lesionsMolecular imaging[149, 150, 151]
VEGFR2AngiogenesisTherapeutic[152]
TNTExplosiveDiagnostic[133, 153]
SEBToxinSensor and diagnostic[134]
RicinToxinSensor and diagnostic[134]
BoNT/AToxinSensor and diagnostic[90, 154, 155]
Scorpion AahIIToxinNeutralizing and therapeutic[135]
EGFRTumorsDetection and imaging
DARCMalaria (by P. vivax)Diagnostic or therapeutic
LMM, ES, CSE, TSB, LLGPs, VF of T. soliumNeurocysticercosisImmunodiagnosis[97]
Heat-killed B. melitensisRiv1 lysatesBrucellosisVaccination, diagnostic, therapeutic[141, 156, 157, 158]
Poliovirus type 1 Sabin strain particlesPoliomyelitisDiagnostic and therapeutic[159, 160]
CEAColon cancerIn vivoimaging[161, 162, 163]
RSV protein FAcute lower respiratory tractTherapeutic[164]
CD105Angiogenesis related tumorsDiagnostic and therapeutic[165, 166]
Ts14 glycoproteinT. soliumcysticercosisDiagnostic[97]
vWFThrombosisTherapeuticwww.ablynx.com
TNFα, IL-6R, IgERheumatoid arthritisTherapeuticwww.ablynx.com
RANKLBone metastasisTherapeuticwww.ablynx.com
RSVBronchiolitis and pneumoniaTherapeuticwww.ablynx.com
DR5Solid tumorsTherapeuticwww.ablynx.com
Not statedAlzheimer’s diseaseTherapeuticwww.ablynx.com

Table 2.

The applications of camelids VHH against specified antigens from various diseases.

5.2. Applications of shark VNAR domains

Evidence that IgNAR is part of the shark adaptive immune response was demonstrated in a work where increasing levels of hen egg lysozyme (HEL) led to the development of specific IgNARs developed in the shark sera after 4–5 months of immunization [25]. The peculiar structure of the shark IgNAR variable domain renders it amenable to create synthetic peptide mimetics to target specific epitopes that are inaccessible to conventional antibodies [118]. Therefore, VNAR may be suitable as new molecular reagents for research, diagnostic and immunotherapeutic applications.

Apical membrane antigen-1 (AMA1) is a highly polymorphic 83 kDa merozoite surface protein that is essential for erythrocyte invasion in malaria parasites [143]. A VNAR isolated from a wobbegong shark showed high-binding affinity to Plasmodium falciparumAMA1 through its unique CDR3 region after undergoing affinity maturation [144]. The binding specificity of a monovalent VNAR clone to P. falciparumAMA1 was comparable with commercially available binding reagents, derived from conventional polyclonal sera, monoclonal antibodies, small fragments (Fab and scFv) and peptides [145]. Foley and co-workers demonstrated the heat stability of purified recombinant VNAR was superior to that of conventional mAbs by targeting immobilized P. falciparumAMA1 in various formats at 45°C, and the refolding property of VNAR was retained when the temperature increased to 80°C. The excellent stability property at extreme pH and resistance to proteolytic cleavage was further evidenced by incubating VNAR with homogenized murine stomach tissues under in vivoconditions [9]. From this point of view, it was purposed that VNAR domains have potential for development as alternate binders for malaria diagnostic platforms.

Human periodontal disease is an advanced gingivitis caused by the bacterial pathogen Porphyromonas gingivalis[146]. Late treatments often lead to dental loss due to the accumulation of lysine gingipain (KgP). KgP is a high molecular weight polyprotease produced by P. gingivalis[147]. This bacterial toxin is responsible for destruction of dental tissue of host by suppressing the secretion of specific lytic enzymes from the immune system [148]. Nuttall and co-workers [149] identified two distinct clones specific to KgP from a naïve wobbegong shark VNAR phage display library with synthetic CDR3 loops. The high stability and binding affinity toward P. gingivalisKgP indicated the potential for VNAR sdAbs as a valuable source of single domain binding reagents [149].

In recent studies, shark VNAR domains have been reported to detect markers from viral diseases at greater sensitivity compared to mAbs and scFvs. Ebola virus hemorrhagic fever (EVHF) is a highly lethal disease caused by Bundibugyo virus (BDBV), Sudan virus (SUDV), Tai Forest virus (TAFV) and Zaire Ebolavirus (ZEBOV) [150, 151, 152]. Shark VNAR and murine scFv phage display libraries have been generated against specified markers on Zaire Ebolavirus. The results indicated that the sensitivity and thermal stability of shark VNAR sdAbs against viral nucleoprotein (NP) of ZEBOV was superior in comparison to murine mAbs and scFvs. [116].

As in the case with camelids nanobodies, highly diversified shark VNAR libraries have also been used to detect different kind of toxins, including staphylococcal enterotoxin B (SEB), ricin and botulinum toxin A (BoNT/A) complex toxoid [153] and cholera toxin (CT) [113]. In addition, VNAR sdAbs have been reported to recognize immunosilent targets in human, for example, the 70 kDa translocase of outer membrane (Tom70) [154]. Owing to the findings of negligible cross-reactivity with other antigens and superior heat stability, shark VNAR domains may be a potent source of sdAbs with thermal stability over conventional antibodies in diagnostic and biotherapeutic applications [155, 156]. The applications of recombinant shark VNAR sdAbs against specified antigens from various diseases are summarized inTable 3.

Target antigensDiseasesApplicationsReference
Kgp protease (P. gingivalis)Periodontal diseaseNeutralization[173]
rhTNFαPro-inflammatory cytokineTherapeutic[114, 181]
AMA1 (P. falciparum)MalariaDiagnostic[168, 169]
Nonfibrillar oligomer formationAlzheimer’s diseaseModeling[182]
Zaire ebolavirus viral nucleoproteinEbolavirus Haemorrhagic FeverDiagnostic[116]
HBeAgHepatitis B virusImmunolocalization and diagnostic[183]
Cholera toxinToxinDiagnostic[113]
SEBToxinSensor and diagnostic[177]
RicinToxinSensor and diagnostic[177]
BoNT/AToxinSensor and diagnostic[177]
Tom70Human immunosilent target processesDetection[178]
GPCR’s ion channelsTherapeuticwww.adalta.com.au
Anti-thrombotic drug targetsCardiovacular diseaseDiagnostic and therapeuticwww.adalta.com.au
www.adalta.com.au
Blood brain barrierTherapeuticwww.ossianix.com
Gastrointestinal tractTherapeuticwww.ossianix.com
MyostatinNeurological diseaseTherapeuticwww.ossianix.com
UveitisTherapeuticwww.elasmogen.com

Table 3.

The applications of shark VNAR against specified antigens from various diseases.

5.3. Applications of lamprey VLRs

The variable lymphocyte receptors (VLRs) discovered from jawless fish had recently attracted interests and is leading to the development of new monoclonal antibodies for biomedical applications [11, 36, 157]. Despite possessing an unusual structure, VLRs have been shown to have excellent binding ability to specified targets (Table 4).

Target antigensDiseasesApplicationsReference
BclA glycoproteinB. anthracisspores (anthrax)Diagnostic[11]
HEL, β-gal, cholera toxin subunit B, R-phycoerythrin, and B-trisaccharideComplex protein antigensAffinity determination[39, 189]
C1q and C3 proteinsCytotoxicity for bacteria and tumor cellsBinding interaction[190]

Table 4.

The applications of lamprey VLRs against specified antigens from various diseases.

Cooper and co-workers demonstrated high specificity of recombinant VLRs for BclA, a major anthrax spore coat which could be produced from an immunized sea lamprey (Petromyzon marinus) using hybridoma technology [11]. Bacillus anthracisis the causative agent for anthrax and the only pathogenic species in the genus Bacillus[158]. Due to their extreme dormancy and durability, anthrax spores have long been considered ideal biological weapons [159, 160, 161]. In this work, the recombinant monoclonal VLRs were shown to be capable of identifying bacteria at a genus level, by differentiating the C-terminal domain of BclA Bacillus anthracisfrom non-coated bacteria of Bacillus cereus[11].

In another study, a large library of recombinant VLRs was constructed to target lysozyme, β-gal, cholera toxin subunit B, R-phycoerythrin and B-trisaccharide antigens using yeast surface display technologies [36]. This high-throughput technology platform offers the potential of rapid identification and isolation of monoclonal VLRs that specifically bind to target antigens with affinities in the micromolar to nanomolar range [36]. Using such display methods, the specificity of selected VLR antibodies can recognize the target antigen with high binding affinity up to 100-fold compared to conventional mouse mAb [36]. These data indicate that the function of VLRs is comparable or perhaps better than that of mammalian IgG antibodies. Therefore, it is speculated that VLRs may be an alternative reagent for the future development of therapeutic and diagnostic applications.

Advertisement

6. Conclusion

The fields of antibody engineering have undergone major advancements in the past few decades. New surface display technologies, in particular phage display and yeast display, are powerful tools that could facilitate the isolation of new antibodies with high specificities for a broad range of targets. Due to their affinity, which often is similar to conventional antibodies and reliable production, recombinant antibodies are becoming increasingly important in the field of diagnosis and therapy for targeting a wide range of diseases such as cancer, inflammatory, autoimmune and viral diseases. In view of natural scaffold design, previous studies showed that the sdAbs repertoires derived from animals such as camelid VHHs, shark VNARs and lamprey VLRs contain several advantages over conventional antibodies. One of the unusual characteristics shared among the sdAbs is that they possess better penetration ability. This feature allows the sdAbs to effectively penetrate into antigen clefts (enzyme active sites, viral capsids and cell surface receptors) which are not easily recognized by the concave surfaces of CDR loops of complex conventional antibodies. To date, due to their ability to target both refractory antigens and immunosilent epitopes, the engineered antibody fragments coupled with latest screening technologies have extensively been used in positron emission tomography and high-sensitivity (nonradioactive, noninvasive) laser technologies for medical imaging. To sum up, it is believed that with rapid progress in antibody engineering technologies, sdAbs will become indispensable as clinical and research reagents in the next decades.

Advertisement

Acknowledgments

The authors would like to acknowledge support from University of Queensland, QIMR Berghofer Medical Research Institute in Australia and Malaysian Government, including Malaysian Ministry of Higher Education the Higher Institutions Centre of Excellence Program (Grant no.: 311/CIPPM/4401005), RUI Grant no.: RU(1001/CIPPM/811296) and USM Short Term (Grant no.: 304/CIPPM/6313191).

References

  1. 1. Conroy PJ, Hearty S, Leonard P, O’Kennedy RJ. Antibody production, design and use for biosensor-based applications. Semin Cell Dev Biol 2009;20:10-26
  2. 2. Newcombe C, Newcombe AR. Antibody production: polyclonal-derived biotherapeutics. J Chromatogr B Analyt Technol Biomed Life Sci 2007;848:2-7
  3. 3. Milstein C. 12th Sir Hans Krebs Lecture. From antibody diversity to monoclonal antibodies. Eur J Biochem 1981;118:429-436
  4. 4. Torrance L, Ziegler A, Pittman H, Paterson M, Toth R, Eggleston I. Oriented immobilisation of engineered single-chain antibodies to develop biosensors for virus detection. J Virol Methods 2006;134:164-170
  5. 5. Moon SA, Ki MK, Lee S, Hong ML, Kim M, Kim S, Chung J, Rhee SG, Shim H. Antibodies against non-immunizing antigens derived from a large immune scFv library. Mol Cells 2011;31:509-513
  6. 6. Proba K, Wörn A, Honegger A, Plückthun A. Antibody scFv fragments without disulfide bonds made by molecular evolution. J Mol Biol 1998;275:245-253
  7. 7. Dong J, Ihara M, Ueda H. Antibody Fab display system that can perform open-sandwich ELISA. Anal Biochem 2009;386:36-44
  8. 8. Zhang MY, Shu Y, Phogat S, Xiao X, Cham F, Bouma P, Choudhary A, Feng YR, Sanz I, Rybak S, et al. Broadly cross-reactive HIV neutralizing human monoclonal antibody Fab selected by sequential antigen panning of a phage display library. J Immunol Methods 2003;283:17-25
  9. 9. Griffiths K, Dolezal O, Parisi K, Angerosa J, Dogovski, C, Barraclough M, Sanalla A, Casey J, González I, Perugini M, Nuttall S, Foley M. Shark Variable New Antigen Receptor (VNAR) Single Domain Antibody Fragments: Stability and Diagnostic Applications. Antibodies 2013;2:66-81
  10. 10. Muyldermans S, Baral TN, Retamozzo VC, De Baetselier P, De Genst E, Kinne J, Leonhardt H, Magez S, Nguyen VK, Revets H, et al. Camelid immunoglobulins and nanobody technology. Vet Immunol Immunopathol 2009;128:178-183
  11. 11. Herrin BR, Alder MN, Roux KH, Sina C, Ehrhardt GR, Boydston JA, Turnbough CL, Jr., Cooper MD. Structure and specificity of lamprey monoclonal antibodies. Proc Natl Acad Sci U S A 2008;105:2040-2045
  12. 12. Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975;256:495-497
  13. 13. Lipman NS, Jackson LR, Trudel LJ, Weis-Garcia F. Monoclonal versus polyclonal antibodies: distinguishing characteristics, applications, and information resources. ILAR J 2005;46:258-268
  14. 14. Correia IR. Stability of IgG isotypes in serum. MAbs 2010;2:221-232
  15. 15. Janeway CA, Travers JP, WAlport M, Schlomchik M. Immunology. New York: Garland Science; 2001
  16. 16. Wesolowski J, Alzogaray V, Reyelt J, Unger M, Juarez K, Urrutia M, Cauerhff A, Danquah W, Rissiek B, Scheuplein F, et al. Single domain antibodies: promising experimental and therapeutic tools in infection and immunity. Med Microbiol Immunol 2009;198:157-174
  17. 17. Barthelemy PA, Raab H, Appleton BA, Bond CJ, Wu P, Wiesmann C, Sidhu SS. Comprehensive analysis of the factors contributing to the stability and solubility of autonomous human VH domains. J Biol Chem 2008;283:3639-3654
  18. 18. Huang L, Reekmans G, Saerens D, Friedt JM, Frederix F, Francis L, Muyldermans S, Campitelli A, Van Hoof C. Prostate-specific antigen immunosensing based on mixed self-assembled monolayers, camel antibodies and colloidal gold enhanced sandwich assays. Biosens Bioelectron 2005;21:483-490
  19. 19. Hudson PJ, Souriau C. Engineered antibodies. Nat Med 2003;9:129-134
  20. 20. Hoogenboom HR. Selecting and screening recombinant antibody libraries. Nat Biotechnol 2005;23:1105-1116
  21. 21. Jostock T, Vanhove M, Brepoels E, Van Gool R, Daukandt M, Wehnert A, Van Hegelsom R, Dransfield D, Sexton D, Devlin M, et al. Rapid generation of functional human IgG antibodies derived from Fab-on-phage display libraries. J Immunol Methods 2004;289:65-80
  22. 22. McCafferty J, Griffiths AD, Winter G, Chiswell DJ. Phage antibodies: filamentous phage displaying antibody variable domains. Nature 1990;348:552-554
  23. 23. Abbady AQ, Al-Mariri A, Zarkawi M, Al-Assad A, Muyldermans S. Evaluation of a nanobody phage display library constructed from aBrucella-immunised camel. Vet Immunol Immunopathol 2011;142:49-56
  24. 24. Dolk E, van der Vaart M, Lutje Hulsik D, Vriend G, de Haard H, Spinelli S, Cambillau C, Frenken L, Verrips T. Isolation of llama antibody fragments for prevention of dandruff by phage display in shampoo. Appl Environ Microbiol 2005;71:442-450
  25. 25. Dooley H, Flajnik MF, Porter AJ. Selection and characterization of naturally occurring single-domain (IgNAR) antibody fragments from immunized sharks by phage display. Mol Immunol 2003;40:25-33
  26. 26. Xu G, Tasumi S, Pancer Z. Yeast surface display of lamprey variable lymphocyte receptors. Methods Mol Biol 2011;748:21-33
  27. 27. Hussack G, Arbabi-Ghahroudi M, Mackenzie CR, Tanha J. Isolation and characterization ofClostridium difficiletoxin-specific single-domain antibodies. Methods Mol Biol 2012;911:211-239
  28. 28. Liu Y, Regula LK, Stewart A, Lai JR. Synthetic Fab fragments that bind the HIV-1 gp41 heptad repeat regions. Biochem Biophys Res Commun 2011;413:611-615
  29. 29. Shui X, Huang J, Li YH, Xie PL, Li GC. Construction and selection of human Fab antibody phage display library of liver cancer. Hybridoma (Larchmt) 2009;28:341-347
  30. 30. Sowa KM, Cavanagh DR, Creasey AM, Raats J, McBride J, Sauerwein R, Roeffen WF, Arnot DE. Isolation of a monoclonal antibody from a malaria patient-derived phage display library recognising the Block 2 region ofPlasmodium falciparummerozoite surface protein-1. Mol Biochem Parasitol 2001;112:143-147
  31. 31. Yang GH, Yoon SO, Jang MH, Hong HJ. Affinity maturation of an anti-hepatitis B virus PreS1 humanized antibody by phage display. J Microbiol 2007;45:528-533
  32. 32. Carmen S, Jermutus L. Concepts in antibody phage display. Brief Funct Genomic Proteomic 2002;1:189-203
  33. 33. Brichta J, Hnilova M, Viskovic T. Generation of hapten-specific recombinant antibodies: antibody phage display technology: a review. Veterinarni Medicina 2005;50:231-252
  34. 34. Hoogenboom HR, de Bruine AP, Hufton SE, Hoet RM, Arends JW, Roovers RC. Antibody phage display technology and its applications. Immunotechnology 1998;4:1-20
  35. 35. Flajnik MF, Deschacht N, Muyldermans S. A case of convergence: why did a simple alternative to canonical antibodies arise in sharks and camels? PLoS Biol 2011;9:e1001120
  36. 36. Tasumi S, Velikovsky CA, Xu G, Gai SA, Wittrup KD, Flajnik MF, Mariuzza RA, Pancer Z. High-affinity lamprey VLRA and VLRB monoclonal antibodies. Proc Natl Acad Sci USA 2009;106:12891-12896
  37. 37. Ehrlich GK, Berthold W, Bailon P. Phage display technology. Affinity selection by biopanning. Methods Mol Biol 2000;147:195-208
  38. 38. Clackson T, Hoogenboom HR, Griffiths AD, Winter G. Making antibody fragments using phage display libraries. Nature 1991;352:624-628
  39. 39. Takakusagi Y, Takakusagi K, Sugawara F, Sakaguchi K. Use of phage display technology for the determination of the targets for small-molecule therapeutics. Expert Opin Drug Discov 2010;5:361-389
  40. 40. Goletz S, Christensen PA, Kristensen P, Blohm D, Tomlinson I, Winter G, Karsten U. Selection of large diversities of antiidiotypic antibody fragments by phage display. J Mol Biol 2002;315:1087-1097
  41. 41. Wang X, Zhong P, Luo PP, Wang KC. Antibody engineering using phage display with a coiled-coil heterodimeric Fv antibody fragment. PLoS One 2011;6:e19023
  42. 42. Ward RL, Clark MA, Lees J, Hawkins NJ. Retrieval of human antibodies from phage-display libraries using enzymatic cleavage. J Immunol Methods 1996;189:73-82
  43. 43. Wind T, Stausbol-Gron B, Kjaer S, Kahns L, Jensen KH, Clark BF. Retrieval of phage displayed scFv fragments using direct bacterial elution. J Immunol Methods 1997;209:75-83
  44. 44. Nowak JE, Chatterjee M, Mohapatra S, Dryden SC, Tainsky MA. Direct production and purification of T7 phage display cloned proteins selected and analyzed on microarrays. Biotechniques 2006;40:220-227
  45. 45. Soltes G, Barker H, Marmai K, Pun E, Yuen A, Wiersma EJ. A new helper phage and phagemid vector system improves viral display of antibody Fab fragments and avoids propagation of insert-less virions. J Immunol Methods 2003;274:233-244
  46. 46. Sharma SC, Memic A, Rupasinghe CN, Duc AC, Spaller MR. T7 phage display as a method of peptide ligand discovery for PDZ domain proteins. Biopolymers 2009;92:183-193
  47. 47. Fukunaga K, Taki M. Practical tips for construction of custom Peptide libraries and affinity selection by using commercially available phage display cloning systems. J Nucleic Acids 2012;2012:295719
  48. 48. Hoogenboom HR. Overview of antibody phage-display technology and its applications. Methods Mol Biol 2002;178:1-37
  49. 49. Nelson AL, Dhimolea E, Reichert JM. Development trends for human monoclonal antibody therapeutics. Nat Rev Drug Discov 2010;9:767-774
  50. 50. Reichert JM. New biopharmaceuticals in the USA: trends in development and marketing approvals 1995-1999. Trends Biotechnol 2000;18:364-369
  51. 51. Tikunova NV, Morozova VV. Phage display on the base of filamentous bacteriophages: application for recombinant antibodies selection. Acta Naturae 2009;1:20-28
  52. 52. Tuckey CD, Noren CJ. Selection for mutants improving expression of an anti-MAP kinase monoclonal antibody by filamentous phage display. J Immunol Methods 2002;270:247-257
  53. 53. Ohtani M, Hikima J, Jung TS, Kondo H, Hirono I, Takeyama H, Aoki T. Variable domain antibodies specific for viral hemorrhagic septicemia virus (VHSV) selected from a randomized IgNAR phage display library. Fish Shellfish Immunol 2013;34:724-728
  54. 54. Sun D, Shi H, Chen J, Shi D, Zhu Q, Zhang H, Liu S, Wang Y, Qiu H, Feng L. Generation of a mouse scFv library specific for porcine aminopeptidase N using the T7 phage display system. J Virol Methods 2012;182:99-103
  55. 55. Smith GP. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 1985;228:1315-1317
  56. 56. Smith GP, Petrenko VA. Phage Display. Chem Rev 1997;97:391-410
  57. 57. Rader C, Barbas CF, 3rd: Phage display of combinatorial antibody libraries. Curr Opin Biotechnol 1997;8:503-508
  58. 58. Sidhu SS. Engineering M13 for phage display. Biomol Eng 2001;18:57-63
  59. 59. Love KR, Swoboda JG, Noren CJ, Walker S. Enabling glycosyltransferase evolution: a facile substrate-attachment strategy for phage-display enzyme evolution. Chembiochem 2006;7:753-756
  60. 60. Zani ML, Moreau T. Phage display as a powerful tool to engineer protease inhibitors. Biochimie 2010;92:1689-1704
  61. 61. Takakusagi Y, Ohta K, Kuramochi K, Morohashi K, Kobayashi S, Sakaguchi K, Sugawara F. Synthesis of a biotinylated camptothecin derivative and determination of the binding sequence by T7 phage display technology. Bioorg Med Chem Lett 2005;15:4846-4849
  62. 62. Castagnoli L, Zucconi A, Quondam M, Rossi M, Vaccaro P, Panni S, Paoluzi S, Santonico E, Dente L, Cesareni G. Alternative bacteriophage display systems. Comb Chem High Throughput Screen 2001;4:121-133
  63. 63. Jespers LS, De Keyser A, Stanssens PE. LambdaZLG6: a phage lambda vector for high-efficiency cloning and surface expression of cDNA libraries on filamentous phage. Gene 1996;173:179-181
  64. 64. Rosenberg A, Griffin K, Stiduer FW, et al. T7 Phage Display System: a powerful new protein display system based on bacteriophage T7.inNovations1998;6:1-6
  65. 65. Krumpe LR, Atkinson AJ, Smythers GW, Kandel A, Schumacher KM, McMahon JB, Makowski L, Mori T. T7 lytic phage-displayed peptide libraries exhibit less sequence bias than M13 filamentous phage-displayed peptide libraries. Proteomics 2006;6:4210-4222
  66. 66. McKenzie KM, Videlock EJ, Splittgerber U, Austin DJ. Simultaneous identification of multiple protein targets by using complementary-DNA phage display and a natural-product-mimetic probe. Angew Chem Int Ed Engl 2004;43:4052-4055
  67. 67. Du XJ, Wu YN, Zhang WW, Dong F, Wang S. Construction and quality examination of murine naive T7 phage display antibody library. Food and Agricultural Immunology 2010;21:81-90
  68. 68. Ohtani M, Hikima J, Jung TS, Kondo H, Hirono I, Aoki T. Construction of an artificially randomized IgNAR phage display library: screening of variable regions that bind to hen egg white lysozyme. Mar Biotechnol (NY) 2013;15:56-62
  69. 69. Colby DW, Kellogg BA, Graff CP, Yeung YA, Swers JS, Wittrup KD. Engineering antibody affinity by yeast surface display. Methods Enzymol 2004;388:348-358
  70. 70. Dantas-Barbosa C, de Macedo Brigido M, Maranhao AQ. Antibody phage display libraries: contributions to oncology. Int J Mol Sci 2012;13:5420-5440
  71. 71. Zhou C, Jacobsen FW, Cai L, Chen Q, Shen WD. Development of a novel mammalian cell surface antibody display platform. MAbs 2010;2:508-518
  72. 72. Bessette PH, Rice JJ, Daugherty PS. Rapid isolation of high-affinity protein binding peptides using bacterial display. Protein Eng Des Sel 2004;17:731-739
  73. 73. Zhou C, Shen WD. Mammalian cell surface display of full length IgG. Methods Mol Biol 2012;907:293-302
  74. 74. Sommavilla R, Lovato V, Villa A, Sgier D, Neri D. Design and construction of a naive mouse antibody phage display library. J Immunol Methods 2010;353:31-43
  75. 75. Rader C. Generation and selection of rabbit antibody libraries by phage display. Methods Mol Biol 2009;525:101-128, xiv
  76. 76. Li Y, Kilpatrick J, Whitelam GC. Sheep monoclonal antibody fragments generated using a phage display system. J Immunol Methods 2000;236:133-146
  77. 77. Solforosi L, Mancini N, Canducci F, Clementi N, Sautto GA, Diotti RA, Clementi M, Burioni R. A phage display vector optimized for the generation of human antibody combinatorial libraries and the molecular cloning of monoclonal antibody fragments. New Microbiol 2012;35:289-294
  78. 78. Johns M. Phage display technology. Methods Mol Med 2000;40:53-62
  79. 79. Harmsen MM, De Haard HJ. Properties, production, and applications of camelid single-domain antibody fragments. Appl Microbiol Biotechnol 2007;77:13-22
  80. 80. Leow CH, Fischer K, Leow CY, Cheng Q, Chuah C, McCarthy J. Single Domain Antibodies as New Biomarker Detectors. Diagnostics (Basel) 2017;7
  81. 81. Boldicke T. Single domain antibodies for the knockdown of cytosolic and nuclear proteins. Protein Sci 2017;26:925-945
  82. 82. Doyle PJ, Arbabi-Ghahroudi M, Gaudette N, Furzer G, Savard ME, Gleddie S, McLean MD, Mackenzie CR, Hall JC. Cloning, expression, and characterization of a single-domain antibody fragment with affinity for 15-acetyl-deoxynivalenol. Mol Immunol 2008;45:3703-3713
  83. 83. Shriver-Lake LC, Goldman ER, Zabetakis D, Anderson GP. Improved production of single domain antibodies with two disulfide bonds by co-expression of chaperone proteins in the Escherichia coli periplasm. J Immunol Methods 2017;443:64-67
  84. 84. Liu JL, Goldman ER, Zabetakis D, Walper SA, Turner KB, Shriver-Lake LC, Anderson GP. Enhanced production of a single domain antibody with an engineered stabilizing extra disulfide bond. Microb Cell Fact 2015;14:158
  85. 85. Mian IS, Bradwell AR, Olson AJ. Structure, function and properties of antibody binding sites. J Mol Biol 1991;217:133-151
  86. 86. O’Kennedy R, Roben P. Antibody engineering: an overview. Essays Biochem 1991;26:59-75
  87. 87. Huang L, Muyldermans S, Saerens D. Nanobodies(R): proficient tools in diagnostics. Expert Rev Mol Diagn 2010;10:777-785
  88. 88. Muyldermans S, Atarhouch T, Saldanha J, Barbosa JA, Hamers R. Sequence and structure of VH domain from naturally occurring camel heavy chain immunoglobulins lacking light chains. Protein Eng 1994;7:1129-1135
  89. 89. Muyldermans S. Single domain camel antibodies: current status. J Biotechnol 2001;74:277-302
  90. 90. Goldman ER, Anderson GP, Conway J, Sherwood LJ, Fech M, Vo B, Liu JL, Hayhurst A. Thermostable llama single domain antibodies for detection of botulinum A neurotoxin complex. Anal Chem 2008;80:8583-8591
  91. 91. Saerens D, Ghassabeh GH, Muyldermans S. Single-domain antibodies as building blocks for novel therapeutics. Curr Opin Pharmacol 2008;8:600-608
  92. 92. De Genst E, Silence K, Decanniere K, Conrath K, Loris R, Kinne J, Muyldermans S, Wyns L. Molecular basis for the preferential cleft recognition by dromedary heavy-chain antibodies. Proc Natl Acad Sci U S A 2006;103:4586-4591
  93. 93. Desmyter A, Transue TR, Ghahroudi MA, Thi MH, Poortmans F, Hamers R, Muyldermans S, Wyns L. Crystal structure of a camel single-domain VH antibody fragment in complex with lysozyme. Nat Struct Biol 1996;3:803-811
  94. 94. Vincke C, Loris R, Saerens D, Martinez-Rodriguez S, Muyldermans S, Conrath K. General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold. J Biol Chem 2009;284:3273-3284
  95. 95. Line BR, Breyer RJ, McElvany KD, Earle DC, Khazaeli MB. Evaluation of human anti-mouse antibody response in normal volunteers following repeated injections of fanolesomab (NeutroSpec), a murine anti-CD15 IgM monoclonal antibody for imaging infection. Nucl Med Commun 2004;25:807-811
  96. 96. Vo-Dinh T, Kasili P, Wabuyele M. Nanoprobes and nanobiosensors for monitoring and imaging individual living cells. Nanomedicine 2006;2:22-30
  97. 97. Deckers N, Saerens D, Kanobana K, Conrath K, Victor B, Wernery U, Vercruysse J, Muyldermans S, Dorny P. Nanobodies, a promising tool for species-specific diagnosis ofTaenia soliumcysticercosis. Int J Parasitol 2009;39:625-633
  98. 98. Roovers RC, Laeremans T, Huang L, De Taeye S, Verkleij AJ, Revets H, de Haard HJ, van Bergen en Henegouwen PM. Efficient inhibition of EGFR signaling and of tumour growth by antagonistic anti-EFGR Nanobodies. Cancer Immunol Immunother 2007;56:303-317
  99. 99. Franco EJ, Sonneson GJ, DeLegge TJ, Hofstetter H, Horn JR, Hofstetter O. Production and characterization of a genetically engineered anti-caffeine camelid antibody and its use in immunoaffinity chromatography. J Chromatogr B Analyt Technol Biomed Life Sci 2010;878:177-186
  100. 100. Moutel S, Bery N, Bernard V, Keller L, Lemesre E, de Marco A, Ligat L, Rain JC, Favre G, Olichon A, Perez F. NaLi-H1: A universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies. Elife 2016;5
  101. 101. Peyvandi F, Callewaert F. Caplacizumab for Acquired Thrombotic Thrombocytopenic Purpura. N Engl J Med 2016;374:2497-2498
  102. 102. Behar G, Siberil S, Groulet A, Chames P, Pugniere M, Boix C, Sautes-Fridman C, Teillaud JL, Baty D. Isolation and characterization of anti-FcgammaRIII (CD16) llama single-domain antibodies that activate natural killer cells. Protein Eng Des Sel 2008;21:1-10
  103. 103. McCoy LE, Rutten L, Frampton D, Anderson I, Granger L, Bashford-Rogers R, Dekkers G, Strokappe NM, Seaman MS, Koh W, et al: Molecular evolution of broadly neutralizing Llama antibodies to the CD4-binding site of HIV-1. PLoS Pathog 2014;10:e1004552
  104. 104. Li T, Vandesquille M, Koukouli F, Dudeffant C, Youssef I, Lenormand P, Ganneau C, Maskos U, Czech C, Grueninger F, et al. Camelid single-domain antibodies: A versatile tool for in vivo imaging of extracellular and intracellular brain targets. J Control Release 2016;243:1-10
  105. 105. Nabuurs RJ, Rutgers KS, Welling MM, Metaxas A, de Backer ME, Rotman M, Bacskai BJ, van Buchem MA, van der Maarel SM, van der Weerd L. In vivo detection of amyloid-beta deposits using heavy chain antibody fragments in a transgenic mouse model for Alzheimer’s disease. PLoS One 2012;7:e38284
  106. 106. Araste F, Ebrahimizadeh W, Rasooli I, Rajabibazl M, Mousavi Gargari SL. A novel VHH nanobody against the active site (the CA domain) of tumor-associated, carbonic anhydrase isoform IX and its usefulness for cancer diagnosis. Biotechnol Lett 2014;36:21-28
  107. 107. Yu Y, Li J, Zhu X, Tang X, Bao Y, Sun X, Huang Y, Tian F, Liu X, Yang L. Humanized CD7 nanobody-based immunotoxins exhibit promising anti-T-cell acute lymphoblastic leukemia potential. Int J Nanomedicine 2017;12:1969-1983
  108. 108. Diaz M, Greenberg AS, Flajnik MF. Somatic hypermutation of the new antigen receptor gene (NAR) in the nurse shark does not generate the repertoire: possible role in antigen-driven reactions in the absence of germinal centers. Proc Natl Acad Sci USA 1998;95:14343-14348
  109. 109. Greenberg AS, Avila D, Hughes M, Hughes A, McKinney EC, Flajnik MF. A new antigen receptor gene family that undergoes rearrangement and extensive somatic diversification in sharks. Nature 1995;374:168-173
  110. 110. Roux KH, Greenberg AS, Greene L, Strelets L, Avila D, McKinney EC, Flajnik MF. Structural analysis of the nurse shark (new) antigen receptor (NAR): molecular convergence of NAR and unusual mammalian immunoglobulins. Proc Natl Acad Sci USA 1998;95:11804-11809
  111. 111. Dooley H, Stanfield RL, Brady RA, Flajnik MF. First molecular and biochemical analysis ofin vivoaffinity maturation in an ectothermic vertebrate. Proc Natl Acad Sci USA 2006;103:1846-1851
  112. 112. Nuttall SD, Krishnan UV, Hattarki M, De Gori R, Irving RA, Hudson PJ. Isolation of the new antigen receptor from wobbegong sharks, and use as a scaffold for the display of protein loop libraries. Mol Immunol 2001;38:313-326
  113. 113. Liu JL, Anderson GP, Delehanty JB, Baumann R, Hayhurst A, Goldman ER. Selection of cholera toxin specific IgNAR single-domain antibodies from a naive shark library. Mol Immunol 2007;44:1775-1783
  114. 114. Camacho-Villegas T, Mata-Gonzalez T, Paniagua-Solis J, Sanchez E, Licea A. Human TNF cytokine neutralization with a vNAR fromHeterodontus franciscishark: a potential therapeutic use. MAbs 2013;5:80-85
  115. 115. Streltsov VA, Varghese JN, Carmichael JA, Irving RA, Hudson PJ, Nuttall SD. Structural evidence for evolution of shark Ig new antigen receptor variable domain antibodies from a cell-surface receptor. Proc Natl Acad Sci USA 2004;101:12444-12449
  116. 116. Goodchild SA, Dooley H, Schoepp RJ, Flajnik M, Lonsdale SG. Isolation and characterisation of Ebolavirus-specific recombinant antibody fragments from murine and shark immune libraries. Mol Immunol 2011;48:2027-2037
  117. 117. Barelle C, Gill DS, Charlton K. Shark novel antigen receptors--the next generation of biologic therapeutics? Adv Exp Med Biol 2009;655:49-62
  118. 118. Stanfield RL, Dooley H, Flajnik MF, Wilson IA. Crystal structure of a shark single-domain antibody V region in complex with lysozyme. Science 2004;305:1770-1773
  119. 119. Muyldermans S, Cambillau C, Wyns L. Recognition of antigens by single-domain antibody fragments: the superfluous luxury of paired domains. Trends Biochem Sci 2001;26:230-235
  120. 120. Streltsov VA, Carmichael JA, Nuttall SD. Structure of a shark IgNAR antibody variable domain and modeling of an early-developmental isotype. Protein Sci 2005;14:2901-2909
  121. 121. Cooper MD, Alder MN. The evolution of adaptive immune systems. Cell 2006;124:815-822
  122. 122. Pancer Z, Amemiya CT, Ehrhardt GR, Ceitlin J, Gartland GL, Cooper MD. Somatic diversification of variable lymphocyte receptors in the agnathan sea lamprey. Nature 2004;430:174-180
  123. 123. Alder MN, Rogozin IB, Iyer LM, Glazko GV, Cooper MD, Pancer Z. Diversity and function of adaptive immune receptors in a jawless vertebrate. Science 2005;310:1970-1973
  124. 124. Kim HM, Oh SC, Lim KJ, Kasamatsu J, Heo JY, Park BS, Lee H, Yoo OJ, Kasahara M, Lee JO. Structural diversity of the hagfish variable lymphocyte receptors. J Biol Chem 2007;282:6726-6732
  125. 125. Rogozin IB, Iyer LM, Liang L, Glazko GV, Liston VG, Pavlov YI, Aravind L, Pancer Z. Evolution and diversification of lamprey antigen receptors: evidence for involvement of an AID-APOBEC family cytosine deaminase. Nat Immunol 2007;8:647-656
  126. 126. Herrin BR, Cooper MD. Alternative adaptive immunity in jawless vertebrates. J Immunol 2010;185:1367-1374
  127. 127. Pays E, Vanhamme L, Perez-Morga D. Antigenic variation inTrypanosoma brucei: facts, challenges and mysteries. Curr Opin Microbiol 2004;7:369-374
  128. 128. Saerens D, Stijlemans B, Baral TN, Nguyen Thi GT, Wernery U, Magez S, De Baetselier P, Muyldermans S, Conrath K. Parallel selection of multiple anti-infectome Nanobodies without access to purified antigens. J Immunol Methods 2008;329:138-150
  129. 129. Hernandez M, Beltran C, Garcia E, Fragoso G, Gevorkian G, Fleury A, Parkhouse M, Harrison L, Sotelo J, Sciutto E. Cysticercosis: towards the design of a diagnostic kit based on synthetic peptides. Immunol Lett 2000;71:13-17
  130. 130. Dorny P, Brandt J, Zoli A, Geerts S. Immunodiagnostic tools for human and porcine cysticercosis. Acta Trop 2003;87:79-86
  131. 131. Garcia HH, Harrison LJ, Parkhouse RM, Montenegro T, Martinez SM, Tsang VC, Gilman RH. A specific antigen-detection ELISA for the diagnosis of human neurocysticercosis. The Cysticercosis Working Group in Peru. Trans R Soc Trop Med Hyg 1998;92:411-414
  132. 132. Ladenson RC, Crimmins DL, Landt Y, Ladenson JH. Isolation and characterization of a thermally stable recombinant anti-caffeine heavy-chain antibody fragment. Anal Chem 2006;78:4501-4508
  133. 133. Anderson GP, Goldman ER. TNT detection using llama antibodies and a two-step competitive fluid array immunoassay. J Immunol Methods 2008;339:47-54
  134. 134. Goldman ER, Anderson GP, Liu JL, Delehanty JB, Sherwood LJ, Osborn LE, Cummins LB, Hayhurst A. Facile generation of heat-stable antiviral and antitoxin single domain antibodies from a semisynthetic llama library. Anal Chem 2006;78:8245-8255
  135. 135. Hmila I, Abdallah RB, Saerens D, Benlasfar Z, Conrath K, Ayeb ME, Muyldermans S, Bouhaouala-Zahar B. VHH, bivalent domains and chimeric Heavy chain-only antibodies with high neutralizing efficacy for scorpion toxin AahI’. Mol Immunol 2008;45:3847-3856
  136. 136. Strokappe N, Szynol A, Aasa-Chapman M, Gorlani A, Forsman Quigley A, Hulsik DL, Chen L, Weiss R, de Haard H, Verrips T. Llama antibody fragments recognizing various epitopes of the CD4bs neutralize a broad range of HIV-1 subtypes A, B and C. PLoS One 2012;7:e33298
  137. 137. Vanlandschoot P, Stortelers C, Beirnaert E, Ibanez LI, Schepens B, Depla E, Saelens X. Nanobodies(R): new ammunition to battle viruses. Antiviral Res 2011;92:389-407
  138. 138. Pant N, Marcotte H, Hermans P, Bezemer S, Frenken L, Johansen K, Hammarstrom L.Lactobacilliproducing bispecific llama-derived anti-rotavirus proteinsin vivofor rotavirus-induced diarrhea. Future Microbiol 2011;6:583-593
  139. 139. Ryan S, Kell AJ, van Faassen H, Tay LL, Simard B, MacKenzie R, Gilbert M, Tanha J. Single-domain antibody-nanoparticles: promising architectures for increasedStaphylococcus aureusdetection specificity and sensitivity. Bioconjug Chem 2009;20:1966-1974
  140. 140. Kenanova V, Wu AM. Tailoring antibodies for radionuclide delivery. Expert Opin Drug Deliv 2006;3:53-70
  141. 141. Huang L, Gainkam LO, Caveliers V, Vanhove C, Keyaerts M, De Baetselier P, Bossuyt A, Revets H, Lahoutte T. SPECT imaging with 99mTc-labeled EGFR-specific nanobody for in vivo monitoring of EGFR expression. Mol Imaging Biol 2008;10:167-175
  142. 142. Pleschberger M, Saerens D, Weigert S, Sleytr UB, Muyldermans S, Sara M, Egelseer EM. An S-layer heavy chain camel antibody fusion protein for generation of a nanopatterned sensing layer to detect the prostate-specific antigen by surface plasmon resonance technology. Bioconjug Chem 2004;15:664-671
  143. 143. Tillib SV, Ivanova TI, Lyssuk EY, Larin SS, Kibardin AV, Korobko EV, Vikhreva PN, Gnuchev NV, Georgiev GP, Korobko IV. Nanoantibodies for detection and blocking of bioactivity of human vascular endothelial growth factor A(165). Biochemistry (Mosc) 2012;77:659-665
  144. 144. Pruszynski M, Koumarianou E, Vaidyanathan G, Revets H, Devoogdt N, Lahoutte T, Zalutsky MR. Targeting breast carcinoma with radioiodinated anti-HER2 Nanobody. Nucl Med Biol 2013;40:52-59
  145. 145. Vaneycken I, Devoogdt N, Van Gassen N, Vincke C, Xavier C, Wernery U, Muyldermans S, Lahoutte T, Caveliers V. Preclinical screening of anti-HER2 nanobodies for molecular imaging of breast cancer. FASEB J 2011;25:2433-2446
  146. 146. Minaeian S, Rahbarizadeh F, Zarkesh-Esfahani SH, Ahmadvand D, Broom OJ. Neutralization of human papillomavirus by specific nanobodies against major capsid protein L1. J Microbiol Biotechnol 2012;22:721-728
  147. 147. Vosjan MJ, Vercammen J, Kolkman JA, Stigter-van Walsum M, Revets H, van Dongen GA. Nanobodies targeting the hepatocyte growth factor: potential new drugs for molecular cancer therapy. Mol Cancer Ther 2012;11:1017-1025
  148. 148. Abbady AQ, Al-Daoude A, Al-Mariri A, Zarkawi M, Muyldermans S. Chaperonin GroEL aBrucellaimmunodominant antigen identified using Nanobody and MALDI-TOF-MS technologies. Vet Immunol Immunopathol 2012;146:254-263
  149. 149. Leung K: 99mTc(CO)3-Anti-vascular cell adhesion molecule-1 nanobody cAbVCAM1-5. In Molecular Imaging and Contrast Agent Database (MICAD). Bethesda (MD)2004
  150. 150. Leung K. Microbubbles conjugated with anti-vascular cell adhesion molecule-1 nanobody cAbVCAM1-5. In Molecular Imaging and Contrast Agent Database (MICAD). Bethesda (MD)2004
  151. 151. Broisat A, Hernot S, Toczek J, De Vos J, Riou LM, Martin S, Ahmadi M, Thielens N, Wernery U, Caveliers V, et al. Nanobodies targeting mouse/human VCAM1 for the nuclear imaging of atherosclerotic lesions. Circ Res 2012;110:927-937
  152. 152. Behdani M, Zeinali S, Khanahmad H, Karimipour M, Asadzadeh N, Azadmanesh K, Khabiri A, Schoonooghe S, Habibi Anbouhi M, Hassanzadeh-Ghassabeh G, Muyldermans S. Generation and characterization of a functional Nanobody against the vascular endothelial growth factor receptor-2; angiogenesis cell receptor. Mol Immunol 2012;50:35-41
  153. 153. Anderson GP, Moreira SC, Charles PT, Medintz IL, Goldman ER, Zeinali M, Taitt CR. TNT detection using multiplexed liquid array displacement immunoassays. Anal Chem 2006;78:2279-2285
  154. 154. Goldman ER, Anderson GP, Bernstein RD, Swain MD. Amplification of immunoassays using phage-displayed single domain antibodies. J Immunol Methods 2010;352:182-185
  155. 155. Swain MD, Anderson GP, Zabetakis D, Bernstein RD, Liu JL, Sherwood LJ, Hayhurst A, Goldman ER. Llama-derived single-domain antibodies for the detection of botulinum A neurotoxin. Anal Bioanal Chem 2010;398:339-348
  156. 156. Altintas I, Kok RJ, Schiffelers RM. Targeting epidermal growth factor receptor in tumors: from conventional monoclonal antibodies via heavy chain-only antibodies to nanobodies. Eur J Pharm Sci 2012;45:399-407
  157. 157. Chopra A: [99mTc]Epidermal growth factor receptor-specific nanobody. In Molecular Imaging and Contrast Agent Database (MICAD). Bethesda (MD)2004
  158. 158. Friedman M, Stahl S. Engineered affinity proteins for tumour-targeting applications. Biotechnol Appl Biochem 2009;53:1-29
  159. 159. Thys B, Saerens D, Schotte L, De Bleeser G, Muyldermans S, Hassanzadeh-Ghassabeh G, Rombaut B. A simple quantitative affinity capturing assay of poliovirus antigens and subviral particles by single-domain antibodies using magnetic beads. J Virol Methods 2011;173:300-305
  160. 160. Thys B, Schotte L, Muyldermans S, Wernery U, Hassanzadeh-Ghassabeh G, Rombaut B.in vitroantiviral activity of single domain antibody fragments against poliovirus. Antiviral Res 2010;87:257-264
  161. 161. Leung K: 99mTc(CO)3-Anti-carcinoembryonic antigen (CEA) humanized CEA5 graft nanobody. In Molecular Imaging and Contrast Agent Database (MICAD). Bethesda (MD)2004
  162. 162. Sukhanova A, Even-Desrumeaux K, Kisserli A, Tabary T, Reveil B, Millot JM, Chames P, Baty D, Artemyev M, Oleinikov V, et al: Oriented conjugates of single-domain antibodies and quantum dots: toward a new generation of ultrasmall diagnostic nanoprobes. Nanomedicine 2012;8:516-525
  163. 163. Vaneycken I, Govaert J, Vincke C, Caveliers V, Lahoutte T, De Baetselier P, Raes G, Bossuyt A, Muyldermans S, Devoogdt N.In vitroanalysis andin vivotumor targeting of a humanized, grafted nanobody in mice using pinhole SPECT/micro-CT. J Nucl Med 2010;51:1099-1106
  164. 164. Hultberg A, Temperton NJ, Rosseels V, Koenders M, Gonzalez-Pajuelo M, Schepens B, Ibanez LI, Vanlandschoot P, Schillemans J, Saunders M, et al. Llama-derived single domain antibodies to build multivalent, superpotent and broadened neutralizing anti-viral molecules. PLoS One 2011;6:e17665
  165. 165. Ahmadvand D, Rasaee MJ, Rahbarizadeh F, Kontermann RE, Sheikholislami F. Cell selection and characterization of a novel human endothelial cell specific nanobody. Mol Immunol 2009;46:1814-1823
  166. 166. Ahmadvand D, Rasaee MJ, Rahbarizadeh F, Mohammadi M. Production and characterization of a high-affinity nanobody against human endoglin. Hybridoma (Larchmt) 2008;27:353-360
  167. 167. Narum DL, Thomas AW. Differential localization of full-length and processed forms of PF83/AMA-1 an apical membrane antigen ofPlasmodium falciparummerozoites. Mol Biochem Parasitol 1994;67:59-68
  168. 168. Nuttall SD, Humberstone KS, Krishnan UV, Carmichael JA, Doughty L, Hattarki M, Coley AM, Casey JL, Anders RF, Foley M, et al. Selection and affinity maturation of IgNAR variable domains targetingPlasmodium falciparumAMA1. Proteins 2004;55:187-197
  169. 169. Henderson KA, Streltsov VA, Coley AM, Dolezal O, Hudson PJ, Batchelor AH, Gupta A, Bai T, Murphy VJ, Anders RF, et al. Structure of an IgNAR-AMA1 complex: targeting a conserved hydrophobic cleft broadens malarial strain recognition. Structure 2007;15:1452-1466
  170. 170. Slots J, Ting M.Actinobacillus actinomycetemcomitansandPorphyromonas gingivalisin human periodontal disease: occurrence and treatment. Periodontol 2000 1999;20:82-121
  171. 171. Kadowaki T, Nakayama K, Okamoto K, Abe N, Baba A, Shi Y, Ratnayake DB, Yamamoto K.Porphyromonas gingivalisproteinases as virulence determinants in progression of periodontal diseases. J Biochem 2000;128:153-159
  172. 172. Aduse-Opoku J, Davies NN, Gallagher A, Hashim A, Evans HE, Rangarajan M, Slaney JM, Curtis MA. Generation of lys-gingipain protease activity inPorphyromonas gingivalisW50 is independent of Arg-gingipain protease activities. Microbiology 2000;146(PT 8):1933-1940
  173. 173. Nuttall SD, Krishnan UV, Doughty L, Nathanielsz A, Ally N, Pike RN, Hudson PJ, Kortt AA, Irving RA. A naturally occurring NAR variable domain binds the Kgp protease from Porphyromonas gingivalis. FEBS Lett 2002;516:80-86
  174. 174. Papaneri AB, Wirblich C, Cooper K, Jahrling PB, Schnell MJ, Blaney JE. Further characterization of the immune response in mice to inactivated and live rabies vaccines expressing Ebola virus glycoprotein. Vaccine 2012;30:6136-6141
  175. 175. Kondratowicz AS, Maury WJ. Ebolavirus: a brief review of novel therapeutic targets. Future Microbiol 2012;7:1-4
  176. 176. Fausther-Bovendo H, Mulangu S, Sullivan NJ. Ebolavirus vaccines for humans and apes. Curr Opin Virol 2012;2:324-329
  177. 177. Liu JL, Anderson GP, Goldman ER. Isolation of anti-toxin single domain antibodies from a semi-synthetic spiny dogfish shark display library. BMC Biotechnol 2007;7:78
  178. 178. Nuttall SD, Krishnan UV, Doughty L, Pearson K, Ryan MT, Hoogenraad NJ, Hattarki M, Carmichael JA, Irving RA, Hudson PJ. Isolation and characterization of an IgNAR variable domain specific for the human mitochondrial translocase receptor Tom70. Eur J Biochem 2003;270:3543-3554
  179. 179. Kovaleva M, Ferguson L, Steven J, Porter A, Barelle C. Shark variable new antigen receptor biologics - a novel technology platform for therapeutic drug development. Expert Opin Biol Ther 2014;14:1527-1539
  180. 180. Zielonka S, Empting M, Grzeschik J, Konning D, Barelle CJ, Kolmar H. Structural insights and biomedical potential of IgNAR scaffolds from sharks. MAbs 2015;7:15-25
  181. 181. Bojalil R, Mata-Gonzalez MT, Sanchez-Munoz F, Yee Y, Argueta I, Bolanos L, Amezcua-Guerra LM, Camacho-Villegas TA, Sanchez-Castrejon E, Garcia-Ubbelohde WJ, et al: Anti-tumor necrosis factor VNAR single domains reduce lethality and regulate underlying inflammatory response in a murine model of endotoxic shock. BMC Immunol 2013;14:17
  182. 182. Streltsov VA, Varghese JN, Masters CL, Nuttall SD. Crystal structure of the amyloid-beta p3 fragment provides a model for oligomer formation in Alzheimer’s disease. J Neurosci 2011;31:1419-1426
  183. 183. Walsh R, Nuttall S, Revill P, Colledge D, Cabuang L, Soppe S, Dolezal O, Griffiths K, Bartholomeusz A, Locarnini S. Targeting the hepatitis B virus precore antigen with a novel IgNAR single variable domain intrabody. Virology 2011;411:132-141
  184. 184. Yu C, Ali S, St-Germain J, Liu Y, Yu X, Jaye DL, Moran MF, Cooper MD, Ehrhardt GR. Purification and identification of cell surface antigens using lamprey monoclonal antibodies. J Immunol Methods 2012;386:43-49
  185. 185. Steichen C, Chen P, Kearney JF, Turnbough CL. Identification of the immunodominant protein and other proteins of theBacillus anthracisexosporium. J Bacteriol 2003;185:1903-1910
  186. 186. Tasota FJ, Henker RA, Hoffman LA. Anthrax as a biological weapon: an old disease that poses a new threat. Crit Care Nurse 2002;22:21-32, 34; quiz 35-26
  187. 187. Witkowski JA, Parish LC. The story of anthrax from antiquity to the present: a biological weapon of nature and humans. Clin Dermatol 2002;20:336-342
  188. 188. Inglesby TV, O’Toole T, Henderson DA, Bartlett JG, Ascher MS, Eitzen E, Friedlander AM, Gerberding J, Hauer J, Hughes J, et al. Anthrax as a biological weapon, 2002: updated recommendations for management. JAMA 2002;287:2236-2252
  189. 189. Velikovsky CA, Deng L, Tasumi S, Iyer LM, Kerzic MC, Aravind L, Pancer Z, Mariuzza RA. Structure of a lamprey variable lymphocyte receptor in complex with a protein antigen. Nat Struct Mol Biol 2009;16:725-730
  190. 190. Wu F, Chen L, Liu X, Wang H, Su P, Han Y, Feng B, Qiao X, Zhao J, Ma N, et al. Lamprey variable lymphocyte receptors mediate complement-dependent cytotoxicity. J Immunol 2013;190:922-930

Written By

Chiuan Herng Leow, Qin Cheng, Katja Fischer and James McCarthy

Submitted: October 14th, 2016 Reviewed: December 21st, 2017 Published: February 21st, 2018