Part:BBa_K3410001
Nanobody scaffold suitable for grafting.
cAbBCII-10 is a nanobody, originally isolated as an antibody capturing and neutralizing enzymes of the β-lactamase B class [1].
As CDR acceptor (also called "scaffold") we used the nanobody cAbBCII-10 [1] (or also NbBcII10 [2] or 3DWT [3], from here on called "3DWT"). This Nanobody is also available as part of a composite part in the iGEM parts reg (BBa_K2082001). However, the nanobody is not available as basic part, so we also contributed in submitting the basic part of cAbBCII-10 (BBa_K3410001). We used the sequence deposited in the PDB by Vincke et al. [2, 3].
Originally, 3DWT was isolated as an antibody capturing and neutralizing enzymes of the β-lactamase B class [1].
A single-domain antibody, also known as nanobody, is an antibody consisting of a single variable antibody domain. First described in 1993 [6], nanobodies are an important research field for multiple pharmaceutical applications today. Mainly, nanobodies are divided in two types of different structures. The most common nanobodies are the VHH fragments, which consist only of a variable domain of a heavy chain and are mostly found in Camelidae species [8]. Another type of nanobodies are the VNAR fragments based on the IgNAR (“Immunoglobin new antigen receptor”) from cartilaginous fish [9]. Having a molecular mass of ∼12 kDa, the vNAR domain is the smallest antibody-like antigen binding domain known so far in the animals [10]. That is why VNAR domains have only two complementarity determining regions CDR1 and CDR3, in contrast to variable mammalian domains [11].
Accordingly, nanobodies are the smallest known antigen-binding proteins with a length of 4 nm and a diameter of 2.5 nm [12]. However, like a whole antibody, they are still able to recognize and bind specifically their target molecules using the single variable domain. Their compact structure and light molecular weight of only 12-15 kDa (compared to conventional antibodies with 150-160 kDa) make nanobodies the smallest active antigen-binding fragments [13]. Due to their versatile binding properties, high stability, manipulable characteristics as well as improved tissue penetration ability the nanobodies are crucial in the field of biotechnology or medicine today [14], [15].
Antibodies consist of two glycosylated heavy chains (H) with a molecular weight of 50 kDA each and two light chains of 25 kDA each, which are covalently linked via disulfide bridges. [16]. On the one hand, two N-terminal variable domains (V) of the heavy (VH) and light chain (VL) form the antigen-binding domain, also called paratrope. On the other hand, the C-terminal regions form the three constant domains CH1, CH2 and CH3. Compared to common antibodies, the nanobody family consist of conventional heterotetramic antibodies with low-affinity binders and as well as unique functional heavy(H)-chain antibodies (HCAbs) with high affinity binders [13]. The H chain of these homodimeric antibodies consist of one antigen-binding domain, the VHH, and two constant domains. The HCAbs fail to incorporate light(L)-chains owing to the deletion of the first constant domain and a reshaped surface of the VHH side, which normally associates with the L-chain in common antibodies [17]. This structural change leads to this enlargement allowing the nanobodies to bind their antigen with a high affinity Furthermore, the hydrophobic amino acids within the conserved region (frame region; FR), which ensure the interaction of VH and VL, are replaced by hydrophilic amino acids [12], [18].
The field of application of nanobodies is very diverse. Their use in diagnostic test systems and therapeutic options is increasing. In contrast to conventional antibodies, nanobodies merge as a new technical achievement due to their low molecular weight (half the size of single-chain variable elements and ten times lighter than conventional antibodies) and small size (2-3 nm) [17]. Moreover, a high expression rate of nanobodies was shown in E. coli with high functionality and stability even in absence of conserved disulphide bonds [19]. The loss of the L-chain leads to missing disulphide modifications in nanobodies, which makes them suitable for production in different bacteria and yeast [20], [21]. Additionally, their high tolerance against acid conditions and temperature compared to mouse monoclonal antibodies [8], high solubility due to a tetrahedron of highly conserved hydrophilic substitutions [22] and very few cleavage sites for enzymes [13] make the nanobodies very promising and an interesting research subject for new applications in the biotechnology. Furthermore, the recombinant nature and single domain of the nanobodies allows easy generation, production and molecular biological manipulation. These include sequential modifications, the generation of fusion proteins with other substances (e.g. pharmaceuticals), the transfer of antigen specificities as well as the transfer of affinity from one nanobody to another [23].
Due to their low mass, the nanobodies are able to easily cross the blood-brain barrier and make them able to quickly leave the human body's circulation via urine after resolving their function inside the cell. Furthermore, nanobodies show a higher availability to tissue penetration and due to their smaller size, they trigger less immunological reaction, which leads to better pharmacokinetics [24]. All stated points are an important property for therapeutic options (cancer therapies) and diagnostic processes (molecular imaging) [23]. Nanobody Grafting Typically, a camelid immune library is needed for the generation of nanobodies, besides ethical concerns, you would have to keep animals and take care of them. One can also obtain nanobodies from synthetic libraries, but this requires large libraries and sophisticated selection mechanisms. An alternative is the so-called grafting of nanobodies. In this method, the CDRs of any antibody are transferred to an existing nanobody to create a mixed nanobody [4]. However, not only the three CDR regions 1 to 3 are transferred, but also amino acids important for the structure and function are transferred from the donor antibody to the nanobody scaffold [25].
The grafting itself is carried out in silico, therefore the amino acid residues from the VH domain from the donor antibody and from the accepting nanobody have to be numbered according to a numbering scheme like Kabat or AHo [26] and CDR1 to 3 have to be defined accordingly [25]. The correct structure of the graft can be checked with the help of visualization programs such as ChimeraX [27]. Here you can see the results of our grafting. Error Prone PCR For means of affinity maturation we needed a library of our grafted sequence. To introduce the required diversity into the grafted sequence, we chose a random mutagenesis approach by polymerase chain reaction (PCR), first described by Cadwell and Joyce in 1992 [28]. This variant of the PCR-technique is termed error prone PCR (epPCR). In this technique, the natural error rate of the Taq polymerase is increased by unfavorable reaction conditions. This is accomplished by: 1) Increased concentration of Taq DNA polymerase; 2) extended polymerase extension time; 3) increased concentration of MgCl2 ions; 4) unbalanced rate of dNTPs; and 5) addition of MnCl2 ions [29].
References
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[2] C. Vincke, R. Loris, D. Saerens, S. Martinez-Rodriguez, S. Muyldermans, and K. Conrath, “General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold,” The Journal of biological chemistry, vol. 284, no. 5, pp. 3273–3284, 2009, doi: 10.1074/jbc.M806889200.
[3] C. e. a. Vincke, 3DWT: Structure of CabBCII-10 nanobody.
[4] S. W. Fanning and J. R. Horn, “An anti-hapten camelid antibody reveals a cryptic binding site with significant energetic contributions from a nonhypervariable loop,” Protein science : a publication of the Protein Society, vol. 20, no. 7, pp. 1196–1207, 2011, doi: 10.1002/pro.648.
References
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Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |