Transforming nanobodies into high-precision tools for protein function analysis

Abstract

Single domain antibodies, derived from camelid heavy antibodies (nanobodies®) or shark variable new antigen receptors, have attracted increasing attention in recent years due to their extremely versatile nature and opportunities they offer for downstream modification. Discovered more than three decades ago, these 120 amino acid (~15kDa) antibody fragments are known to bind their target with high specificity and affinity . Key features of nanobodies that make them very attractive include their single domain nature, small size, affordable high level expression in prokaroytes, and their cDNAs are routinely obtained in the process of their isolation. This facilitates and stimulates new experimental approaches. Hence, it allows researchers to formulate new answers to complex biomedical questions. Through elementary PCR-based technologies and chemical modification strategies, their primary structure can be altered almost at leisure whilst retaining their specificity and biological activity, transforming them into highly tailored tools that meet the increasing demands of current day biomedical research. In this review, various aspects of camelid Nanobodies are expounded, including intracellular delivery in recombinant format for manipulation of i.e. cytoplasmic targets, their derivatization to improve nanobody orientation as a capturing device, approaches to reversibly bind their target, their potential as protein silencing devices in cells, the development of strategies to transfer nanobodies through the blood brain barrier and their application in CAR-T experimentation. We also discuss some of their disadvantages and conclude with future prospects.

Generation of photocaged nanobodies forin vivoapplications using genetic code expansion and computationally guided protein engineering

“…Using our approach we were able to generate photo-controllable variants of the anti-GFP enhancer nanobody (eNB), which is used as the basis of a wide range of in vivo tools. [2,11,12,[16][17][18] These variants may thus immediately add the ability to use light for the precise spatiotemporal control of these tools, with the photocaged tyrosine that is most amenable to in vivo systems, NPY.…”

“…In recent years, nanobodies have been developed into indispensable tools for investigating protein function, in part due to the fact that they can be easily expressed in vivo. [1,2] This unique set of properties has been leveraged to develop a variety of tools for labelling and manipulating proteins, to gain insights into biological processes. Fluorescently tagged nanobodies, termed "chromobodies", have been used for in vivo antigen labelling.…”

“…[17,18] Nanobodies have been employed as tools in a wide range of model systems, including mammalian cells, mice, zebrafish, and C. elegans. [2] A limitation of native nanobodies is that their binding cannot be modulated after expression. The ability to induce antigen binding would significantly increase the applicability of nanobody-based tools by adding a level of spatiotemporal control absent when using native nanobodies.…”

A comprehensive comparison between camelid nanobodies and single chain variable fragments

“…Unlike RNAi, Nanobodies, by their smaller size, can bind to the effector domain of desired proteins to defunctionalize that particular domain and enable the fundraising of its role in the process. In contrast, by RNA interference, the function of the whole protein would be disrupted, and the role of each domain will remain unclear [ 220 ].…”

Nanobodies restore stability to cancer-associated mutants of tumor suppressor protein p16INK4a

“…Single-domain antibodies derived from naturally occurring camelid heavy-chain antibodies (nanobodies) have several advantages over conventional antibodies (Gettemans and Dobbelaer, 2021;Hamers-Casterman et al, 1993). Their ability to bind non-conventional epitopes with high affinities (Stijlemans et al, 2004), as well as their high thermodynamic stability (Dumoulin et al, 2002) and their small size, present many benefits for biotechnological and clinical applications (Hoey et al, 2019;Jovčevska and Muyldermans, 2020).…”