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

type 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 expres-

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

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

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

sion or fusion to a carrier protein can be performed [38, 48, 78].

described in the following section.

182 Antibody Engineering

**4.1. VHH heavy-chain domain in camelids**

determination of the half-life of an antibody [86].

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 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 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. 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].

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

The Development of Single Domain Antibodies for Diagnostic and Therapeutic Applications

http://dx.doi.org/10.5772/intechopen.73324

185

activity occurred when the incubation period was prolonged more than 1 h at 70°C [126].

**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.

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 vitro* selection method, novel nanobody clones were isolated that showed specificity to *T. evansi* at

species level, and genus-specific reactivity against various *Trypanosoma* species [128].

tional monoclonal antibodies made from mammals.

**5.1. Applications of camelids VHH domains or nanobodies®**

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 vitro* random 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 conven-

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 10<sup>14</sup> 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].

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 tar-

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

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

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

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

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 10<sup>14</sup> 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

get small target epitopes that may not be accessible to conventional IgG [120].

**4.3. VLRs immunoglobulin-like domains in lamprey**

receptors (VLRs) rather than Ig superfamily (**Figure 1**) [122].

conformation [35].

184 Antibody Engineering

sequence analysis [125].

VLR domain has been so far reported.

of areas.

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 vitro* random 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.
