Radiolabeling Strategies of Nanobodies for Imaging Applications

Abstract

Nanobodies are small recombinant antigen-binding fragments derived from camelid heavy-chain only antibodies. Due to their compact structure, pharmacokinetics of nanobodies are favorable compared to full-size antibodies, allowing rapid accumulation to their targets after intravenous administration, while unbound molecules are quickly cleared from the circulation. In consequence, high signal-to-background ratios can be achieved, rendering radiolabeled nanobodies high-potential candidates for imaging applications in oncology, immunology and specific diseases, for instance in the cardiovascular system. In this review, a comprehensive overview of central aspects of nanobody functionalization and radiolabeling strategies is provided.

Keywords: labeling strategies; molecular imaging; nanobodies; positron emission tomography; radiohalogens; radiometals; single photon emission computed tomography.

Figure 1

Schematic representation of conventional immunoglobulin G’s (IgGs), heavy chain-only antibodies (HCAbs) and nanobodies. V_H, variable heavy; V_L, variable light; V_HH, V_H of HCAbs.

Figure 2

Common bioconjugation reactions for the attachment of radiolabels on cysteine and lysine residues within the nanobody [12]. Asterisk (*) indicates a chiral center with an undefined ratio of the two stereoisomers.

Figure 3

Installation of iodine-125 or iodine-131 into tyrosine residues of proteins and peptides via Iodogen [31].

Figure 4

Radiohalogenated prosthetic groups which have been applied to nanobodies in a single final conjugation reaction.

Figure 5

Pre-derivatization on primary amines of the nanobody with 1, followed by ¹⁸F-introduction via 2 in a strain-promoted azide-alkyne cycloaddition reaction [36].

Figure 6

Reaction mechanism of the sortase A-mediated transpeptidation for labeling nanobodies site-specifically at the C-terminus [44]. LPXTG, sortase A-recognition motif; X, any amino acid except cysteine; G, glycine; SH, thiol function of the active site cysteine; C=O, carbonyl carbon of threonine.

Figure 7

Site-specific insertion of 3 at the nanobody’s C-terminus via sortase A and subsequent inverse-electron demand Diels-Alder (IEDDA) cycloaddition with 4.

Figure 8

Sortase A-mediated C-terminal introduction of 5, followed by ¹⁸F-labeling with 6 in an IEDDA reaction.

Figure 9

¹⁸F-labeling of polyethylene glycol-functionalized or homodimeric nanobodies with 7 through IEDDA cycloaddition [42].

Figure 10

Conjugation of (±)-H₃RESCA-TFP to primary amines of the nanobody, followed by Al¹⁸F-labeling [49,50].

Figure 11

Pre-derivatization on primary amines of the nanobody with 8, followed by ¹⁸F-labeling with 9 via IEDDA reaction [15].

Figure 12

1,4,7-Triazacyclononane-N,N′,N″-triacetic acid (NOTA)-based synthetic chelators applied to nanobodies for radiometal labeling. NOTA substructure is highlighted in blue.

Figure 13

Desferrioxamine B (DFO)-based synthetic chelators used for radiometal labeling of nanobodies. DFO substructure is highlighted in pink.

Figure 14

Diethylenetriaminepentaacetic acid (DTPA)-based synthetic chelators employed for labeling nanobodies with radiometals. DTPA substructure is highlighted in violet.

Figure 15

Complex formation between a C-terminal hexahistidine-tag and ^99mTc-tricarbonyl [110].

Figure 16

Assumed complex formation of technetium-99m coordinated by three ligands [112].