**1. Nanobodies: a concise introduction**

In 1993, Hamers-Casterman discovered the presence of heavy-chain-only antibodies in the sera of Camelidae and assessed that these antibodies are still capable of recognizing an extensive repertoire of antigens despite the absence of the light chain. Single-domain antibodies from camels are called nanobodies. They stated that this discovery could be of inestimable

© 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.

value to the development and engineering of soluble VH domains or new immunological molecules for diagnostic, therapeutic, and biochemical purposes [1]. This discovery gave rise to a whole new research field in which single-domain antibodies are used for a wide range of applications. Some of these will be reviewed in the current chapter.

The structural properties of conventional IgG antibodies are well known. These consist of two heavy-chain polypeptides and two light-chain polypeptides, each of which is folded into four and two domains, respectively. A variable domain is situated at the N-terminus of both chains (VH and VL) and, as the name suggests, its sequence diverges between IgG antibodies. Paired VH-VL domains make up the variable fragment (Fab) and are responsible for the recognition and binding of the target antigen. The sequence of the other domains is well conserved between IgGs, which led to the designation of these domains as constant domains. Heavy-chain-only antibodies differ from conventional IgG antibodies by the lack of a lightchain polypeptide and the first constant domain of the heavy-chain polypeptide (CH1). Consequently, the antigen-binding fragment of heavy-chain antibodies from camels consists of one single domain, termed the VHH domain. This unit forms the functional and structural equivalent of the Fab in conventional IgG antibodies [2]. The smallest antibody fragment that can be produced from conventional IgG antibodies is a short-chain variable fragment (ScFv, ±27 kDa), which consists of a VH and VL domain linked via a polypeptide. In the continuous search for smaller antibody formats, HCAbs were a thrilling novelty, because their discovery allowed researchers to produce an even smaller antibody fragment of only ±15 kDa. This antibody format derived from camels consists of an isolated VHH domain also known as a single-domain antibody or a nanobody (Nb) (**Figure 1**). In addition, human single-domain antibodies VH and VL have been engineered from human conventional antibodies [3–5], and sharks develop heavy-chain-only antibodies (HCAbs) too [6].

The structural features of nanobodies are quite similar to those of the variable domain of conventional IgGs. The core structure of the immunoglobulin domain is formed by four framework regions (FR), whereas antigen binding occurs through three complementaritydetermining regions (CDRs). The latter are located in loops in between β-strands that form the variable immunoglobulin domain. Importantly, FR2 of the VHH domain often contains amino acid substitutions of residues that are involved in hydrophobic interactions between the VH and the VL domains of conventional IgGs (V37 → F/Y, G44 → E/Q, L45 → R, and W47 → G/F/L; Kabat numbering). These substitutions lie at the heart of the single-domain nature of nanobodies because they reduce the hydrophobicity of the former VL interface and improve the nonstickiness of the domain. There are other examples of amino acid substitutions that frequently take place, but these appear to be of less importance [7, 8]. Since nanobodies only consist of one domain, one might wonder whether nanobodies have a diverse antibody repertoire. After all, they lack the VH-VL combinatorial diversity in the antigen-binding site. Nanobodies have counterbalanced the absence of the three hypervariable loops of the VL domain by an *extension* of the hypervariable loops in the VHH domain. These loops show substantial variation in both conformation and length compared with the corresponding loops of the VH domain. This implies that a larger structural repertoire and thus a sufficient diversification in antigen-binding sites can be obtained [9]. More specifically, the introduction of additional Cys residues in the CDRs creates extra disulfide bridges within the VHH domain,

and these are in part responsible for the diversification of the structural repertoire. The disulfide bridges crosslink the antigen-binding loops, resulting not only in the stabilization of the domain but also in the induction of constraints in CDR1 or CDR3. These constraints probably lead to novel loop conformations and thus in an increase in paratope repertoire. Furthermore, VHHs are more prone to insertion and deletion events near or within the paratope compared to VHs. This is translated into an increase of the surface area of the hypervariable regions and

**Figure 1.** Schematic representation of a conventional IgG antibody and a camelid heavy-chain-only antibody (HCAb). For both antibody formats, the smallest producible antibody fragment is depicted: a short-chain variable fragment (ScFv,

Use, Applications and Mechanisms of Intracellular Actions of Camelid VHHs

http://dx.doi.org/10.5772/intechopen.70495

207

**2. The therapeutic potential of extracellular and intracellular nanobodies**

Several monoclonal antibodies (mAbs) have already been approved for clinical use [11], but some limitations are still present despite their success. This includes their large size, relative instability, which imposes restrictions on the administration route and their relative expense of manufacturing. The potential of nanobodies as a therapeutic agent was rapidly recognized as they overcome some of the aforementioned limitations of mAbs. The small size of nanobodies in combination with their extended CDR loops allows them to bind into clefts and cavities, whereas mAbs preferably recognize flat and concave surfaces. Many biological

contributes to the structural variation [10].

±27 kDa) and a nanobody (Nb, ±15 kDa), respectively.

value to the development and engineering of soluble VH domains or new immunological molecules for diagnostic, therapeutic, and biochemical purposes [1]. This discovery gave rise to a whole new research field in which single-domain antibodies are used for a wide range of

The structural properties of conventional IgG antibodies are well known. These consist of two heavy-chain polypeptides and two light-chain polypeptides, each of which is folded into four and two domains, respectively. A variable domain is situated at the N-terminus of both chains (VH and VL) and, as the name suggests, its sequence diverges between IgG antibodies. Paired VH-VL domains make up the variable fragment (Fab) and are responsible for the recognition and binding of the target antigen. The sequence of the other domains is well conserved between IgGs, which led to the designation of these domains as constant domains. Heavy-chain-only antibodies differ from conventional IgG antibodies by the lack of a lightchain polypeptide and the first constant domain of the heavy-chain polypeptide (CH1). Consequently, the antigen-binding fragment of heavy-chain antibodies from camels consists of one single domain, termed the VHH domain. This unit forms the functional and structural equivalent of the Fab in conventional IgG antibodies [2]. The smallest antibody fragment that can be produced from conventional IgG antibodies is a short-chain variable fragment (ScFv, ±27 kDa), which consists of a VH and VL domain linked via a polypeptide. In the continuous search for smaller antibody formats, HCAbs were a thrilling novelty, because their discovery allowed researchers to produce an even smaller antibody fragment of only ±15 kDa. This antibody format derived from camels consists of an isolated VHH domain also known as a single-domain antibody or a nanobody (Nb) (**Figure 1**). In addition, human single-domain antibodies VH and VL have been engineered from human conventional antibodies [3–5], and

The structural features of nanobodies are quite similar to those of the variable domain of conventional IgGs. The core structure of the immunoglobulin domain is formed by four framework regions (FR), whereas antigen binding occurs through three complementaritydetermining regions (CDRs). The latter are located in loops in between β-strands that form the variable immunoglobulin domain. Importantly, FR2 of the VHH domain often contains amino acid substitutions of residues that are involved in hydrophobic interactions between the VH and the VL domains of conventional IgGs (V37 → F/Y, G44 → E/Q, L45 → R, and W47 → G/F/L; Kabat numbering). These substitutions lie at the heart of the single-domain nature of nanobodies because they reduce the hydrophobicity of the former VL interface and improve the nonstickiness of the domain. There are other examples of amino acid substitutions that frequently take place, but these appear to be of less importance [7, 8]. Since nanobodies only consist of one domain, one might wonder whether nanobodies have a diverse antibody repertoire. After all, they lack the VH-VL combinatorial diversity in the antigen-binding site. Nanobodies have counterbalanced the absence of the three hypervariable loops of the VL domain by an *extension* of the hypervariable loops in the VHH domain. These loops show substantial variation in both conformation and length compared with the corresponding loops of the VH domain. This implies that a larger structural repertoire and thus a sufficient diversification in antigen-binding sites can be obtained [9]. More specifically, the introduction of additional Cys residues in the CDRs creates extra disulfide bridges within the VHH domain,

applications. Some of these will be reviewed in the current chapter.

206 Antibody Engineering

sharks develop heavy-chain-only antibodies (HCAbs) too [6].

**Figure 1.** Schematic representation of a conventional IgG antibody and a camelid heavy-chain-only antibody (HCAb). For both antibody formats, the smallest producible antibody fragment is depicted: a short-chain variable fragment (ScFv, ±27 kDa) and a nanobody (Nb, ±15 kDa), respectively.

and these are in part responsible for the diversification of the structural repertoire. The disulfide bridges crosslink the antigen-binding loops, resulting not only in the stabilization of the domain but also in the induction of constraints in CDR1 or CDR3. These constraints probably lead to novel loop conformations and thus in an increase in paratope repertoire. Furthermore, VHHs are more prone to insertion and deletion events near or within the paratope compared to VHs. This is translated into an increase of the surface area of the hypervariable regions and contributes to the structural variation [10].
