**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

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

(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 [5–8], 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 [9–11]. 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.

## **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.

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 complementaritydetermining 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

a connecting peptide (CP) and a C-terminal cap (LRRCT), followed by an invariant 3′-terminal region.

**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),

The Development of Single Domain Antibodies for Diagnostic and Therapeutic Applications

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

177

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

binding loops [15].

in the following sessions.

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 (K<sup>a</sup> ) 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 The Development of Single Domain Antibodies for Diagnostic and Therapeutic Applications http://dx.doi.org/10.5772/intechopen.73324 177

(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 [5–8], 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 [9–11]. 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.

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

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

ity for its cognate target epitope [14]. The high degree in variations of antibody specificities is

) ranging from 10−2 to 101 nM, and excellent specific-

**2. Monoclonal antibodies and their limitations**

higher purity than polyclonal Abs [13].

176 Antibody Engineering

characterized by having a high affinity (K<sup>a</sup>

**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.

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 complementaritydetermining 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].

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.
