Discovery of nanobodies: a comprehensive review of their applications and potential over the past five years

Abstract

Nanobodies (Nbs) are antibody fragments derived from heavy-chain-only IgG antibodies found in the Camelidae family as well as cartilaginous fish. Their unique structural and functional properties, such as their small size, the ability to be engineered for high antigen-binding affinity, stability under extreme conditions, and ease of production, have made them promising tools for diagnostics and therapeutics. This potential was realized in 2018 with the approval of caplacizumab, the world's first Nb-based drug. Currently, Nbs are being investigated in clinical trials for a broad range of treatments, including targeted therapies against PDL1 and Epidermal Growth Factor Receptor (EGFR), cardiovascular diseases, inflammatory conditions, and neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. They are also being studied for their potential for detecting and imaging autoimmune conditions and infectious diseases such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A variety of methods are now available to generate target-specific Nbs quickly and efficiently at low costs, increasing their accessibility. This article examines these diverse applications of Nbs and their promising roles. Only the most recent articles published in the last five years have been used to summarize the most advanced developments in the field.

Keywords: CRISPR; Cancer; Cardiovascular; Diagnosis; Display techniques; Infectious-disease; Nanobody; Nanobody-imaging; Neurogenerative; Neurogenerative diseases; Production.

Fig. 1

Key Structural Features of Nanobodies Compared to Conventional Antibodies. Unlike conventional antibodies with both heavy and light chains, HCAbs lack light chains, enabling the isolation of VHH genetic fragments, also known as nanobodies (Nb). B. Conventional mAbs consist of two heavy and two light chains, together forming two identical antigen-binding sites. Each binding site comprises non-covalently linked variable domains, designated VH and VL. Adapted illustration [7]

Fig. 2

Generating Antigen-Specific Nb: A Phage Display Library Approach. A schematic diagram of the different methods of generating Nbs. Camelids are immunized with an antigen to generate an immune library, while a naïve library does not require inoculation. Blood is collected to extract lymphocytes and mRNA, and Nb sequences are refined through PCR and gel electrophoresis. In synthetic libraries, a Nb scaffold is used, and CDRs are randomized. These libraries are transformed into phage vectors and E. coli to display Nbs and produce high-affinity phages via biopanning. Successful phages undergo phage-ELISA, sequencing, affinity testing, and are stored in glycerol for future production. Adapted illustration[3]

Fig. 3

From Glycerol Stock to Lab-Scale Production: A Conventional Nb Production Protocol. Various systems are available for nanobody production, including bacterial, yeast, mammalian, and plant expression systems. Among these, the bacterial expression system, particularly using E. coli, is the most common due to its simplicity, cost-effectiveness, and high yield. In this system, LB medium supplemented with glucose, ampicillin, and MgCl2 is inoculated with glycerol stock and incubated overnight. The culture then seeds TB medium for a few hours before nanobody (Nb) expression is induced with IPTG overnight. Post-expression, cells are centrifuged and lysed to extract Nbs. The extract undergoes IMAC purification, followed by imidazole elution, with an optional size exclusion chromatography. Quality is confirmed through SDS-PAGE, Coomassie staining, and western blotting. Adapted illustration [3]

Fig. 4

Sidebar 1 Comparative Analysis of Antibody Library Construction Methods [22, 55]

Fig. 5

Nb targets in the tumor microenvironment

Fig. 6

Exploring the Versatility of Engineered Nanobodies: Types and Modifications. Schematic representation of the different types of engineered nanobodies showcasing their adaptability for a wide range of applications. Adapted from [161]

Fig. 7

Nanobodies in Disease Diagnosis: Applications and Impact

Fig. 8

Sidebar 2 The use of radiolabeled Nb exemplifies the potential of a variety of Nbs in diagnostic applications [164, 165]

Fig. 9

From Discovery to Development: The Evolution of Nanobodies (original illustration) [, , , , , –280]