Introduction to Heavy-Chain Only Antibody and Derived Single Domain Antibody (sdAb)
Heavy-Chain Only Antibodies in Camelids
The camelids contain old world camelids, including Camelus dromedarius and Camelus bactrianus, and new world camelids, including Lama glama, Lama pacos, Lama guanicoe, and Lama vicugna. The sera from the family of camelids in addition to the classic antibody, also contain a unique class of heavy-chain only antibodies (HCAbs), which are antigen-binding antibodies lacking light chains and CH1 domains. The HCAbs in camelids contribute to the immune response of these animals that is the outcome of adaptive changes.
Three IgG isotypes with distinct molecular weight fractions are isolated from the camelids serum. Thereinto, IgG1 is the conventional antibodies comprising two heavy and two light chains, and two isotypes are distinguished by the difference between hinge region (19 aa for IgG1a, 12 aa for IgG1b). The IgG2 fraction and IgG3 fraction are the so-called heavy-chain only antibodies, in which the variable domain joined directly to the hinge region. The IgG2 fraction from the dromedary sera contains two isotypes, one encoding a hinge of 35 amino acids (IgG2a) and the other encoding a hinge of 15 amino acids (IgG2c). The third type of hinge with 29 amino acids was identified in llama and attributed to the IgG2b subclass. The IgG3 fraction contains a 12 amino acids hinge. The average proportion of heavy chain antibodies to conventional antibodies for old world camelids is about 50%, but this percentage is somewhat lower (approximately 30%) for new world camelids.
Fig.1 Schematic of the VHH domain of a camelid heavy-chain only antibody. (Wesolowski, 2009)
Single Domain Antibodies (sdAb)
- Sequence Adaptations of VHHs
VHHs are distinct from conventional VHs by the substitution of five amino acids conserved in VH domains of classic antibodies. These VH-VHH hallmark substitutions are Leu12Ser, Val42Phe/Tyr, Gly49Glu, Leu50Arg/Cys, and Trp52Gly. These substitutions of VHH are seen as an adaptation to deal with the absence of the CH1 domain and increase the solubility of the sdAbs in an aqueous environment.
Meanwhile, three distinctive features of sdAbs are to be found in the hypervariable regions. First, the CDR1 of VHHs is extended towards the N-terminal end. Second, VHHs have an average longer CDR3 in which the average CDR3 length in dromedary VHHs is 18 amino acids compared to 14 and 11 amino acids in human and mouse VHs, respectively. Third, in addition to the conserved intradomain disulfide bond, the VHHs also often form an additional disulfide bond. All these adaptations can increase the antigen-interaction surface and offer an additional diversity to their antigen-binding repertoire.
- Structural Adaptations of VHHs
The VHH specific amino acid substitutions resurface the region interacting with the VL in the conventional VH domains in order to exhibit an entirely different architecture. The Gly49Glu and Leu50Arg substitutions can increase the hydrophilicity of the surface of the VHH to expose their most hydrophilic parts to the solvent. Moreover, the substitutions at positions 42 and 52 cause a net shift of the bulky hydrophobic groups, and the CDR3 loop usually folds over these residues and makes them solvent inaccessible.
The largest structural differences between the VHs and VHHs occur at the level of the antigen-binding loops in which VHHs contain a larger number of possible loop structures. This additional structural diversity is attributed to the formation of an interloop cystine. Furthermore, sdAbs can form cavities, grooves, flat surfaces, or large protruding loops to accommodate binding with different antigens depending on the size and type of the target. This feature allows sdAbs to recognize epitopes that are usually not antigenic for classical antibodies, such as the catalytic site of enzymes.
Fig.2 Structure of sdAb. (Kolkman, 2010)
- Diversity of VHH in HCAbs
The VHH and VH share a high degree of amino acid sequence identity and are most similar to the human VH of family 3 with approximately 80% sequence identity, which is the most widespread human VH family. Indeed, VHHs are readily diversified by the introduction of an additional disulfide bridge, the high incidence of nucleotide insertions/deletions, gene replacement, and extensive somatic point mutations.
- Importance of VHH
Advances in antibody engineering make smaller recombinant antibody fragments become top listed as an emerging new class of drugs and allow fine-tuning of the various features of antibodies, such as valency or avidity, stability, intrinsic affinity, and size. VHHs, because of their single domain nature with only 12-15 kDa smallest size, offer several advantages for biotechnological applications. Immunized libraries, naïve VHH library, and a synthetic library can be generated to retain full functional diversity through a straightforward cloning procedure without worrying about the disruption of VH/VL pairing and result in the isolation high-affinity antigen binders. Moreover, sdAbs are conferred a high level of solubility and the feature of reversible chemical denaturation, which mainly attributed to the capabilities of efficient refolding.
Furthermore, sdAbs have high sequential and structural homology with human VH domains and outstanding penetrability with the ability to cross the blood-brain barrier. The short plasma half-life and superior clearance make it ideal as diagnostic tools. The recombinant expression of sdAbs is available in various expression systems such as the prokaryotic system, eukaryotic system (Saccharomyces cerevisiae and Pichia pastoris) and transgenic plants. The potential of sdAb is great in downstream engineering to facilitate subsequent molecular manipulation to engineer multivalent formats, fusion protein, and sdAb humanization.
References
- Wesolowski, J.; et al. Single domain antibodies: promising experimental and therapeutic tools in infection and immunity. Medical microbiology and immunology. 2009, 198(3): 157-174.
- Kolkman, J. A.; Law, D. A. sdAbs-from llamas to therapeutic proteins. Drug discovery today: technologies. 2010, 7(2): e139-e146.
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