Nanobodies: Chemical Functionalization Strategies and Intracellular Applications

Abstract

Nanobodies can be seen as next-generation tools for the recognition and modulation of antigens that are inaccessible to conventional antibodies. Due to their compact structure and high stability, nanobodies see frequent usage in basic research, and their chemical functionalization opens the way towards promising diagnostic and therapeutic applications. In this Review, central aspects of nanobody functionalization are presented, together with selected applications. While early conjugation strategies relied on the random modification of natural amino acids, more recent studies have focused on the site-specific attachment of functional moieties. Such techniques include chemoenzymatic approaches, expressed protein ligation, and amber suppression in combination with bioorthogonal modification strategies. Recent applications range from sophisticated imaging and mass spectrometry to the delivery of nanobodies into living cells for the visualization and manipulation of intracellular antigens.

Keywords: antigen-binding proteins; cellular delivery; molecular biology; nanobodies; site-specific functionalization.

Figure 1

Depiction of IgGs, nanobodies, and other engineered recombinant antigen‐binding proteins. a) Comparison of nanobodies and IgGs. Conventional IgG molecules contain two heavy and two light chains. Light chains contain one constant (C_L, orange) and one variable (V_L, light gray) domain. The heavy chains contain three constant domains (C_H1–3, red) and one variable domain (V_H, dark gray). Heavy‐chain antibodies (hcAb) from Camelidae lack the C_H1 and C_L domain of conventional antibodies. They recognize their antigen through a single variable domain, V_HH (dark gray). The X‐Ray structure of a nanobody binding its antigen GFP (green) is shown (PDB ID: 3G9A).17 b) A fibronectin‐based monobody binding the SUMO protein (PDB ID: 3RZW).18 c) An affibody based on Protein A, binding to HER 2 (PDB ID: 3MZW).19 d) A libocalin derived anticalin binding to the Alzheimer's disease (AD)‐relevant amyloid‐β (PDB ID: 4MVI).20 e) A designed ankyrin repeat protein (DARPin) in complex with human interleukin‐4 (PDB ID: 4YDY). Antigens are shown in blue, antigen‐binding proteins in gray.

Figure 2

Comparison of the binding regions and surface structures of nanobodies and human‐derived V_H domains. a) A V_HH (nanobody) from Camelidae with GFP as the antigen (PDB ID: 3G9A).17 b) A human derived variable domain (V_H) with vascular endothelial growth factor as the antigen (PDB ID: 2FJF).23 V_HHs contain a significantly enlarged CDR3 framework (black), thus ensuring high binding affinity. Several hydrophobic amino acids that are highly conserved in conventional V_H domains are mutated within nanobodies, which increases their solubility (orange).24

Figure 3

Schematic depiction of nanobody‐based detection of protein–protein interactions in living cells and nanobody‐mediated protein degradation. a) The fluorescent‐3‐hybrid (F3 H)assay is based on a GFP‐binding nanobody fused to the Lac repressor tightly binding to Lac operator DNA repeats stably integrated in the genome, for example, in baby hamster kidney cells. In this way, a GFP‐labelled protein of interest (Protein 1) is recruited to the LacO region within the nucleus. If a second protein (Protein 2) that is labelled with a different fluorescent protein (RFP) interacts with Protein 1, it will be co‐recruited to the LacO region, thereby resulting in strong correlation of the GFP and RFP fluorescence signal. b) Nanobody‐mediated ubiquitin‐dependent protein degradation. A GFP‐binding nanobody is fused to the F‐box domain of the Drosophila melanogaster derived Slmb protein. Together with the S‐phase kinase associated protein 1 (SKP1), Cullin 1 (CUL1), Ring protein (Rbx) subunits, the F‐box forms the E3 enzyme that is responsible for target‐protein recognition and binding of the E2 enzyme. A GFP‐labelled target protein is recruited to the E3 domain upon nanobody binding. Subsequent ubiquitination catalyzed by an E2 enzyme triggers protein degradation of the GFP‐labelled protein.

Figure 4

The nanobody Nb592 binds to the ATP‐binding cassette (ABC) transporter P‐glycoprotein (P‐gp) and inhibits its ATPase activity. a) Crystal structure of P‐gp in complex with Nb592. The nanobody specifically binds to the nucleotide‐binding domain 1 (NBD1) of P‐gp. TMD=transmembrane domain. P‐gp is shown in blue, Nb592 in gray (PDB ID: 4KSD).51 b) Upon ATP binding and dimerization of the NBDs, restructuring of the P‐gp and its TMD occurs and the xenobiotic is transported across the cellular membrane and into the extracellular environment. Upon ATP hydrolysis and the release of ADP and Pi release, the P‐gps ground state is restored. In contrast, once Nb592 is bound to NBD1, NBD dimerization, ATP complexation, and transporter activity is inhibited.

Figure 5

Nanobodies in imaging. a) Upon binding of its antigen, the GFP‐binding nanobody GBP stabilizes and enhances the fluorescence signal of GFP when imaged in living cells. The nanobody (V_HH) is depicted in gray, GFP in green (PDB ID: 3K1K17). b) FP‐tagged nanobodies (so called chromobodies) avoid the need for FP fusion to the protein of interest and maintain its endogenous expression level. Various chromobodies have been engineered and used as powerful imaging tools. The nanobody (V_HH) is depicted in gray, GFP in green (PDB ID: 3G9A).17 c) Nanobodies have the ability to minimize linkage errors during super‐resolution microscopy experiments. They significantly reduce the spatial distance to the actual specimen compared to classical experimental setups composed of full‐length primary and secondary antibodies. The nanobody is depicted in gray (PDB ID: 3G9A17), the secondary and primary antibodies in gray and orange, respectively (PDB ID: 1IGT58).

Figure 6

FP‐binding nanobodies like the GFP‐binding GBP enable the combinatorial analysis of proteins and their interaction partners through imaging and mass spectrometry‐based proteomics. A protein of interest (Protein 1) expressed as an FP fusion can be imaged using conventional methods. A nanobody that binds the FP is chemically immobilized on a solid support, which facilitates enrichment of the target protein and any interacting protein (Proteins 2 and 3). Subsequent MS analysis enables the identification and assignment of the co‐enriched interacting proteins.

Figure 7

Random labeling of nanobodies. a) NHS‐activated probes/drugs are reacted with the nucleophilic $ϵ$ ‐amine of a solvent‐exposed lysine residue, resulting in heterogeneous nanobody conjugate mixtures with partly reduced binding affinities. b) The C‐terminal fusion of a poly‐lysine stretch to nanobodies is intended to prevent unselective NHS‐based labeling of Lys residues within the CDR loops responsible for antigen binding. The nanobody is depicted in gray, the introduced functionality (e.g., fluorophore, drug, tracer) in red. The crystal structure of a GFP‐binding nanobody is used in (a) and (b) [PDB ID: 3G9A].17

Figure 8

Nanobody functionalization through labelling of unpaired cysteine residues. a) Nanobodies do not possess free cysteine residues. Therefore, incorporating a single cysteine residue into a nanobody (mostly to the C terminus) is an easy way to site‐specifically attach a probe or drug through cysteine‐selective chemistry (shown for a maleimide‐functionalized probe). b) A prostate cancer specific nanobody (antigen PSMA) containing a C‐terminal unpaired cysteine was site‐specifically functionalized with a radiolabel by the use of maleimide chemistry. Single‐photon emission computed tomography (SPECT)/CT images of mice bearing prostate cancer tumors in the left shoulder were taken 3 h (left) and 24 h (right) after injection of the nanobody. Scale from 0 to 0.015 kBq (left) and 0 to 0.005 kBq (right).75a c) A tumor necrosis factor alpha (TNF) nanobody was conjugated to linear and branched polyethylene glycol (PEG) chains through maleimide chemistry. Subsequent pharmacokinetic experiments in mice, rats, and monkeys revealed that the branched PEG conjugates show significantly improved in vivo circulation time compared to linear PEG in all tested species as schematically shown in the concentration–time plot. The nanobody is depicted in gray, the introduced functionality in red. The crystal structure of a GFP‐binding nanobody is used in (a) and (c) [PDB ID: 3G9A].17

Figure 9

Chemoenzymatic functionalization of nanobodies. a) The biotin ligase BirA enables the in vivo attachment of biotin to nanobodies functionalized with the 22 amino acid biotin acceptor domain (BAD, light blue). b) Transglutaminases have been used to attach a functionality to nanobodies by generating an isopeptide bond between the glutamine of a short C‐terminal recognition sequence (dark blue) and an amine‐carrying probe. The nanobody is depicted in gray, the introduced functionality in red. The crystal structure of a GFP‐binding nanobody is used in (a) and (b) [PDB ID: 3G9A].17

Figure 10

Sortase A functionalization of nanobodies. a) The transpeptidase Sortase A catalyzes the reversible formation of an amide bond between threonine of the Sortag LPXTG (purple) and a glycine‐functionalized probe. b) Sortase A functionalization have been combined with maleimide chemistry and strain‐promoted azide–alkyne cycloaddition (SPAAC) to generate fluorescently labelled nanobody dimers binding to class II major histocompatibility complex (MHC) and/or CD11b proteins. The nanobody is depicted in gray or orange, the introduced functionality in red. The crystal structure of a GFP‐binding nanobody is used in (a) and (b) [PDB ID: 3G9A].17

Figure 11

Functionalization of nanobodies by using the lipoic acid ligase (LpIA). a) LpIA recognizes the 13 amino acid LAP tag (green) and ligates an aldehyde‐containing lipoic acid derivative to the side chain of a lysine residue. A subsequent oxime‐forming reaction enables site‐specific functionalization of the nanobody. b) A nanobody‐based activation immunotherapeutic is shown. An anti‐HER2 nanobody was site‐specifically functionalized with a dinitrophenyl (DNP) moiety using LpIA. The DNP acts as an endogenous antibody‐recruiting domain, facilitating a targeted immune response upon HER2 binding of the nanobody. The nanobody is depicted in gray, the introduced functionality in red. The crystal structure of a GFP‐binding nanobody is used in (a) and (b) [PDB ID: 3G9A].17

Figure 12

Tub‐tag labelling for the functionalization of nanobodies and recombinant antigen‐binding proteins. a) Versatile two‐step labelling of nanobodies. TTL‐mediated incorporation of tyrosine derivatives containing chemical reporters enables successive conjugation to a functional probe (red) using a bioorthogonal reaction. b) The TTL (PDB ID: 4IHJ)88 forms an extended cavity during the catalytic cycle. This cavity allows lead to a broad substrate tolerance. c) Fluorescent or biotinylated substrates of TTL enable the efficient one‐step labelling of biomolecules. d) Substrates of TTL include ortho‐and para‐functionalized tyrosine derivatives, fluorescent coumarin amino acids. and large biotinylated tyrosine derivatives. The nanobody is depicted in gray, the introduced functionality in red, and TTL in purple. The crystal structure of a GFP‐binding nanobody is used in (a) and (c) [PDB ID: 3G9A].17

Figure 13

Methods that have been used for the site‐specific labelling of nanobodies (gray). a) Amber suppression was used to install the amino acid AmAzZLys into a nanobody. The benzylic amine of the unnatural amino acid was reacted with an aldehyde‐containing PEG chain and the benzylic azide with a dibenzocyclooctyl fluorophore, resulting in a doubly functionalized nanobody. b) AmAzZLys was incorporated into a nanobody targeting the epidermal growth factor receptor (EGFR, PDB ID: 3P0Y99b). The amino group was used for fluorescent labelling, while the azide was activated by UV irradiation, and photo‐cross‐linking between the nanobody and EGFR was performed. c) Expressed protein ligation (EPL) shown for the C‐terminal functionalization of nanobodies. The respective nanobody is expressed as an intein fusion (intein shown in turquoise, PDB ID: 4GIG99a). Activation with a reducing agent like DTT or mercaptoethanol results in the formation of a highly reactive thioester. Transthioesterification initiated by nucleophilic attack of a cysteine‐containing probe, followed by a S‐to‐N acyl transfer results in the formation of a stable amide bond and the site‐specific functionalization of the nanobody. The nanobody is depicted in gray, the introduced functionality in red, EGFR in blue, and intein in turquoise. The crystal structure of a GFP‐binding nanobody is used in (a), (b), and (c) [PDB ID: 3G9A].17

Figure 14

Depiction of delivery mechanisms that have been used for the cellular uptake of nanobodies. Nanobodies have been delivered into cells through the use of a) charged surface mediated transduction, b) endocytosis, or c) cyclic cell‐penetrating peptides (CPPs). The nanobody is depicted in gray, the introduced functionality in red. d) Thiol‐containing mesoporous silica nanoparticles (MSNs) were conjugated to a maleimide‐functionalized nitrilotriacetic acid (NTA) linker. Activation with a metal ion (shown for Ni) facilitated binding of a His₆‐chromobody. The MSN–chromobody complex showed endosomal entrapment upon cellular incubation, thus necessitating endosomal escape triggers like the peptide INF7 to enable cytosolic distribution of the chromobody. e) A cell delivery system based on the anthrax lethal toxin. Recombinant antigen‐binding proteins (shown for a monobody) are expressed as a fusion with the 30 kDa N‐terminal domain of the toxin enzyme lethal factor (LF_N). The protective‐antigen (PA)‐based pore‐forming transporter (PA oligomers shown in green) is bound to a host‐cell receptor. The binder‐LF_N forms a complex with the transporter (step 1) and endocytosis is initiated (step 2 and 3). Due to the acidic environment in the endosome, the PA oligomers form a transmembrane pore, unfolds the binder‐LF_N fusion protein and initiates its translocation to the cytosol (step 4). The nanobody is depicted in gray, RFP (PDB ID: 1GGX106) in red, and the monobody in blue (PDB ID: 3RZW18). The crystal structure of a GFP‐binding nanobody is used in (a), (b), (c), and (d) [PDB ID: 3G9A].17

Figure 15

Charge‐induced membrane transduction of nanobodies initiated by cell penetrating peptides (CPPs). a–b) Expressed protein ligation (EPL) of nanobodies was used to site‐specifically conjugate CPPs through a) a stable amide bond to give non‐cleavable nanobody–CPP conjugates or b) a cleavable disulfide that gets reduced within the reductive cytosolic environment (cleavable and fluorescent nanobody–CPP). c) Incubation with different cell lines revealed efficient uptake in up to 95 % of the cells when incubated with low μm concentrations. Upon incubation of the Cy5‐labelled cleavable nanobody with cells, the conjugate crosses the cellular membrane into the cytosol, CPP cleavage is initiated, and the fluorescently labelled nanobody binds its nuclear antigen GFP. The nanobody is depicted in gray, GFP in green, and Cy5 in red. The crystal structure of a GFP‐binding nanobody is used in (a), (b), and (c) [PDB ID: 3G9A].17