**1. Introduction**

Single-domain antibodies, or nanobodies, are derived from heavy-chain only antibodies (HcAbs) found in camelids [1]. Essentially, they can be used for the same therapeutic purposes as monoclonal antibodies (mAbs) and single-chain variable fragments (scFvs) but with some advantages brought about by their inherent properties. For one, the small molecular size of nanobodies (~15 kDa) facilitates penetrance to target sites, as nanobodies are half as large as scFvs and five times smaller than human conventional antibodies [2]. This, in combination with more extended loops of the complementarity determining regions 1 and 3 (CDR1 and CDR3), enables binding to a wider range of epitopes with different shapes at sub-nanomolar affinity, potentially increasing the application of nanobodies as drugs. The lack of a light chain in HcAbs also allows nanobodies to exist as a single domain with less susceptibility to aggregation through hydrophobic interactions, as is the case for scFvs [3–5]. Due to their small size and high similarity to the human immunoglobulin variable domain, they provoke little to no immune response [5] which often makes humanization unnecessary.

Amyloidogenic proteins have previously been targeted with nanobodies to inhibit the course of amyloidogenesis [4]. Nanobodies have been shown to inhibit the formation

**Citation:** Percipalle, M.; Hunashal, Y.; Steyaert, J.; Fogolari, F.; Esposito, G. Structure of Nanobody Nb23. *Molecules* **2021**, *26*, 3567.

Academic Editor: Marilisa Leone

Received: 13 April 2021 Accepted: 21 May 2021 Published: 11 June 2021

https://doi.org/10.3390/ molecules26123567

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

of amyloid β (Aβ) fibrils formed in Alzheimer's disease patients, and also to recognize non-conventional epitopes on Aβ fibrils for diagnostic use [6], although the clinical trials to validate antibody drugs have been unsuccessful so far.

Non-neurodegenerative amyloidoses may prove more amenable for nanobody treatment. A paradigmatic amyloidogenic protein, β2-microglobulin (β2m), which is a component of class I major histocompatibility complex (MHC-1), accumulates as amyloid deposits in the joints of patients undergoing long-term haemodialysis [7]. The deposits contain some 30% of ∆N6β2m, the proteolytic variant of β2m devoid of the N-terminal hexapeptide, that forms fibrils also by mild stirring at neutral pH [8]. This amyloidogenic propensity, much stronger than the parent protein, was also observed with D76Nβ2m, a naturally occurring variant of β2m that causes progressive bowel dysfunction and systemic amyloidosis, i.e., deposits in several vital organs [9].

Several nanobodies were raised against wild-type (WT) β2m and ∆N6β2m by immunization of both a camel and a llama. Nb24, a camel-derived nanobody raised against WT β2m has been shown to inhibit the self-aggregation of the very amyloidogenic ∆N6β2m and D76Nβ2m variants in vitro and, indirectly, also in vivo, and the binding thermodynamics and kinetics along with the epitope mapping of the D76Nβ2m-Nb24 complex were characterized [10,11]. In this case, D76Nβ2m self-aggregation was inhibited despite the fact that Nb24 was raised against the WT β2m. The crystal structure of Nb24 complexes with ∆N6β2m (PDB ID 2X89) and P32Gβ2m (PDB ID 4KDT) are known [11,12] whereas no structure is available for the isolated nanobody. Nb23, which is instead llama-derived and raised against ∆N6β2m, inhibits self-aggregation of its raising antigen, but fails to inhibit D76Nβ2m self-aggregation, despite it being raised against a very amyloidogenic variant of β2m. In order to characterize the interaction of Nb23 with a target other than the original antigen, structural information is crucial. In this study, the solution structure of Nb23 has been determined using nuclear magnetic resonance (NMR) spectroscopy, as a first step of a general project aimed at rationalizing the determinants of nanobody performance with β2m variants. In particular, structure knowledge enables systematic analysis of the conformational, thermodynamic, and kinetic properties of the binding to the β2m variants in order to improve the affinity between nanobody and antigen or attenuate their complex lability through rational design.
