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Camelid Antibodies and VHH domain function

Introduction to single domain VHH property:

        Immunoglobulin (Ig) is a protein that has two distinct structural components, the fragment antigen-binding (Fab) domain and the fragment crystallizable (Fc) area, which can be separated by proteolytic cleavage with papain and pepsin. The binding specificity of the entire Ig molecule, especially the two variable domains on the top—variable heavy chain (VH) and variable light chain—is solely dependent on this domain, whereas the Fc region triggers biological processes upon antigen binding, Fab is responsible for antigen recognition (VL).


        As we know camelid antibodies are different from conventional antibodies as they are made up of two heavy chains only. These antibodies do not contain CH1 region, instead, they have an antigen binding domain known as the VHH region. So, VHH antibodies are also known as single domain antibodies.


        Single-chain variable fragments (scFvs) and camelid heavy-chain variable domains (VHHs), often known as nanobodies, are the most widely used types of designed and recombinant antibody forms. Single-chain variable fragment (scFv), which is made up of VH and VL, was previously thought to be the smallest antibody fragment with the same antigen-binding selectivity as the entire Ig molecule. However, it has been shown that a single V-like domain can maintain the affinity of an entire antibody molecule by the discovery of the camelid VHH and shark variable novel antigen receptor (VNAR) (Asaadi et al., 2021).

Properties of VHH antibodies:


The size of nanobody is about 30kDa weight. Due to their smaller size and the fact that they contain only three antigen-binding loops, VHHs are more easily genetically modified to increase their natural propensity to attach to antigen. The smaller size of VHHs also contributes to their short half-life in blood, which is a result of renal filtration and degradation. This property can be advantageous because it increases tissue permeability, but it can also be disadvantageous since it causes complications in some clinical regimens that call for prolonged antibody circulation because their molecular weight is below the glomerular filtration threshold size (65 kDa).


The low immunogenicity of nanobodies in clinical settings is connected with their high sequence identity with the human VH (VH3 gene family). As a result, the humanization process in VHHs is simpler. Additionally, humanization lowers these fragments' binding affinity, and CDR grafting might represent novel immunogenic epitopes.

Solubility and Stability:

The nonpolar to polar transition makes VHHs more stable from a molecular and thermodynamic standpoint than scFvs. As a result, Nbs are more stable under extreme PH or ionic strength conditions and more resistant to chemical denaturants and protease enzymes. The existence of an additional disulfide link, which reduces the likelihood of heat-induced aggregation and restricts the flexibility of VHH, also contributes to this enhanced conformational stability. They exhibit great refolding efficiency as a result of their better stability, which indicates that changing the sample's temperature has no effect on the Nb conformation, causing it to debind or bind to the target, respectively, without any aggregation or denaturation. Since non-native protein aggregation is a common side effect of antibody treatment, increasing the immune response in extreme cases, this rigidity in structure is a preferred trait in the clinic (Bever et al., 2016).

Applications of VHH single domain antibodies:

  • Nanobodies as crystallization chaperones:

Nbs have a solid track record of helping difficult-to-crystallize proteins. These sturdy, single-domain antibody fragments do in fact frequently lock proteins in a specific conformation. The capacity of Nbs to hide aggregating surfaces from contact with solvents or stabilize inherently flexible regions are crucial properties that enable the crystallization process. The advantageous features of Nbs enabled the identification of unknown conformations and the elucidation of an astounding number of highly dynamic protein structures, shedding light on the antigens' biological functions. Nbs may also develop as a vital method for obtaining crucial structural data on partially disordered or amyloid proteins. As an illustration, conformation-sensitive Nbs maintain amyloid-protofibril stability and inhibit the development of mature amyloid fibrils. (Harmsen and De Haard,2007).

Engineering Camelid antibodies:

By panning on antigens and phage display of cloned VHHs, target-specific VHHs are obtained. These recombinant VHHs, also known as nanobodies, are used for a variety of biochemical and biophysical applications as well as large-scale microbe production.


Recombinant VHHs have great specificity and affinity for antigens after being purified by affinity chromatography, making them soluble in aqueous solutions. As a result, these VHHs are resistant to demanding circumstances as well as thermal or chemical denaturation.


The usage of the nanobodies as markers for intracellular targets to break down certain target proteins or to observe the creation or disruption of protein interactions in real time has proved successful. The nanobodies' resistance to unfolding or proteases is increased by adding an extra intra-domain disulfide bond, making them effective biosensor probes. The nanobodies are sufficiently stable thanks to the disulfide bond for usage in oral formulations.


Studies have shown that because of their great sequence homology to human VH domains and steady function, nanobodies do not result in immunogenicity reactions.


Nanobodies' small size aids in their quick removal from the body through the kidneys as well as the blood-brain barrier. With the removal of extra nanobodies from the blood, they are transported quickly through the tissues to reach the target site, which makes them very useful in non-invasive in vivo imaging (Jain, kamal, Batra, 2007).


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