Computational affinity maturation of camelid single-domain intrabodies against the nonamyloid component of alpha-synuclein

Abstract

Improving the affinity of protein-protein interactions is a challenging problem that is particularly important in the development of antibodies for diagnostic and clinical use. Here, we used structure-based computational methods to optimize the binding affinity of VHNAC1, a single-domain intracellular antibody (intrabody) from the camelid family that was selected for its specific binding to the nonamyloid component (NAC) of human α-synuclein (α-syn), a natively disordered protein, implicated in the pathogenesis of Parkinson’s disease (PD) and related neurological disorders. Specifically, we performed ab initio modeling that revealed several possible modes of VHNAC1 binding to the NAC region of α-syn as well as mutations that potentially enhance the affinity between these interacting proteins. While our initial design strategy did not lead to improved affinity, it ultimately guided us towards a model that aligned more closely with experimental observations, revealing a key residue on the paratope and the participation of H4 loop residues in binding, as well as confirming the importance of electrostatic interactions. The binding activity of the best intrabody mutant, which involved just a single amino acid mutation compared to parental VHNAC1, was significantly enhanced primarily through a large increase in association rate. Our results indicate that structure-based computational design can be used to successfully improve the affinity of antibodies against natively disordered and weakly immunogenic antigens such as α-syn, even in cases such as ours where crystal structures are unavailable.

The good and bad of therapeutic strategies that directly target α‐synuclein

“…50 Following findings supporting that intrabodies could effectively reduce α-syn aggregation, 51,52 Mahajan and coauthors recently performed computational optimization of a single-domain intrabody from the camelid family, which was selected for its specific binding to the non-amyloid component (NAC) of human α-syn, located in the central region of the protein: VHNAC1. 53 Similar results were also obtained in vitro by De Genst and coworkers, which optimized NbSyn2, a single-domain camelid nanobody, binding specifically the four C-terminal residues of monomeric α-syn with nanomolar affinity. 54 NbSyn87, which targets a central region within the C-terminal domain (residues 118-128) of α-syn, has also been found to destabilize the secondary and tertiary structures of the protein, 55,56 and inhibit fibril formation and disrupt toxic oligomer species protein in a cell-free system model of fibrillation.…”

AAV mediated delivery of a novel anti-BACE1 VHH reduces Abeta in an Alzheimer’s disease mouse model

“…However, if required, modifications to the system can easily be made by taking advantage of the relative ease with which VHH and AAV can be engineered. For example, VHH affinities in the low picomolar range can be achieved using standard affinity maturation techniques (Mahajan et al, 2018), or via production of bivalent VHH constructs (Beirnaert et al, 2017) that bind distinct epitopes on a given target. Expression levels of a given VHH from a vector-based system can be improved by use of a self-complementary genome configuration, containing multiple copies of the VHH coding sequence (Verhelle et al, 2017).…”

Recombinant expression of nanobodies and nanobody-derived immunoreagents

“…The first is that the affinity improvement reached the low nanomolar range (K D changed from 278 to 3.2 nM) and the second that such positive effect was associated with an increase of the thermal stability of the mutant with respect to the original nanobody (+7.36°C), whereas the two parameters were inversely correlated in other reports dealing with similar projects [123,125]. In another case of in silico nanobody affinity maturation, Mahajan and al [108]. Identified a key single mutation by using a combination of replica exchange molecular dynamics (REMD), umbrella sampling, and weighted histogram method together with numerical techniques.…”

“…For the selective identification of α-synuclein intrabodies, the ability of the E. coli twinarginine translocation (Tat) system was also exploited to non-covalently secrete protein complexes to the periplasm (FLI-TRAP -functional ligand-binding identification by Tat-based recognition of associating proteins). The resulting nanobodies could be expressed as functional binders in cell cytoplasm [108].…”