CDR1 Composition Can Affect Nanobody Recombinant Expression Yields

Abstract

The isolation of nanobodies from pre-immune libraries by means of biopanning is a straightforward process. Nevertheless, the recovered candidates often require optimization to improve some of their biophysical characteristics. In principle, CDRs are not mutated because they are likely to be part of the antibody paratope, but in this work, we describe a mutagenesis strategy that specifically addresses CDR1. Its sequence was identified as an instability hot spot by the PROSS program, and the available structural information indicated that four CDR1 residues bound directly to the antigen. We therefore modified the loop flexibility with the addition of an extra glycine rather than by mutating single amino acids. This approach significantly increased the nanobody yields but traded-off with moderate affinity loss. Accurate modeling coupled with atomistic molecular dynamics simulations enabled the modifications induced by the glycine insertion and the rationale behind the engineering design to be described in detail.

Novel bispecific nanobody mitigates experimental intestinal inflammation in mice by targeting TNF‐α and IL‐23p19 bioactivities

“…We optimized the linker length and reciprocal position of the two nanobodies at the N‐terminal and C‐terminal to ensure stability and functionality 49,50 . Furthermore, the rational design of the structure holds the potential for further improving the present BsNbs, leveraging their monomeric nature and short sequence for in silico modelling and computational enhancements of their biophysical features, as well as humanization 32,51–53 …”

“…49,50 Furthermore, the rational design of the structure holds the potential for further improving the present BsNbs, leveraging their monomeric nature and short sequence for in silico modelling and computational enhancements of their biophysical features, as well as humanization. 32,[51][52][53] To enhance molecule stability and circulation half-life, we incorporated an Fc domain into the BsNbs. This modification resulted in an increased yield and prolonged in vivo circulation half-time, thereby potentially enhancing the therapeutic effects, as demonstrated in our experimental colitis model in mice (Figure 3).…”

“…Among these options, BsAbs consisting of fusions of Fc and nanobodies offer the advantage of multivalency with reduced mass 30,31 . Nanobodies, which preserve the entire binding capacity in a single, stable domain, provide design flexibility and allow the creation of immunoreagents with structures tailored for specific applications 32–34 . Fusion with Fc domains modifies their clearance kinetics, avidity and receptor‐mediated effector functions, leading to a significant increase in therapeutic response 35 .…”

Native llama Nanobody Library Panning Performed by Phage and Yeast Display Provides Binders Suitable for C-Reactive Protein Detection

“…Nevertheless, they must be considered as hits requiring further improvement to become lead reagents for CRP diagnostics. In silico methods have recently been shown to be highly efficient to improve the biophysical characteristics of nanobodies because their short sequence requires relatively limited computational resources for rational modeling [ 38 , 39 , 40 , 41 ]. We have demonstrated, with some examples, how simple nanobody engineering is in different formats suitable for different applications and expect that such anti-CRP might become a valid alternative to the conventional antibodies used thus far for CRP quantification.…”

Efficient polyethylene terephthalate degradation at moderate temperature: a protein engineering study of LC‐cutinase highlights the key role of residue 243

“…The binding free energy (ΔG bind ) of the protein-ligand interaction was estimated by molecular mechanics energies combined with the generalized Born and surface area continuum solvation using MMPBSA.PY [48] from the AMBER-TOOLS21 package [49]. The ΔG bind values were averaged over the mean values calculated for each simulation replicate by decomposing their contribution with the per-residue effective free-energy decomposition (prEFED) protocol, as done in [25,50]. Residues were defined as a hot spot of interaction with 3PET if their energy contribution was ≤ −1.0 kcalÁmol −1 .…”