Site-Specifically Labeled Immunoconjugates for Molecular Imaging—Part 2: Peptide Tags and Unnatural Amino Acids

Abstract

Molecular imaging using radioisotope- or fluorophore-labeled antibodies is increasingly becoming a critical component of modern precision medicine. Yet despite this promise, the vast majority of these immunoconjugates are synthesized via the random coupling of amine-reactive bifunctional probes to lysines within the antibody, a process that can result in heterogeneous and poorly defined constructs with suboptimal pharmacological properties. In an effort to circumvent these issues, the last 5 years have played witness to a great deal of research focused on the creation of effective strategies for the site-specific attachment of payloads to antibodies. These chemoselective modification methods yield immunoconjugates that are more homogenous and better defined than constructs created using traditional synthetic approaches. Moreover, site-specifically labeled immunoconjugates have also been shown to exhibit superior in vivo behavior compared to their randomly modified cousins. The over-arching goal of this two-part review is to provide a broad yet detailed account of the various site-specific bioconjugation approaches that have been used to create immunoconjugates for positron emission tomography (PET), single photon emission computed tomography (SPECT), and fluorescence imaging. In Part 1, we covered site-specific bioconjugation techniques based on the modification of cysteine residues and the chemoenzymatic manipulation of glycans. In Part 2, we will detail two families of bioconjugation approaches that leverage biochemical tools to achieve site-specificity. First, we will discuss modification methods that employ peptide tags either as sites for enzyme-catalyzed ligations or as radiometal coordination architectures. And second, we will examine bioconjugation strategies predicated on the incorporation of unnatural or non-canonical amino acids into antibodies via genetic engineering. Finally, we will compare the advantages and disadvantages of the modification strategies covered in both parts of the review and offer a brief discussion of the overall direction of the field.

Site-Specific Fluorescent Labeling of Antibodies and Diabodies Using SpyTag/SpyCatcher System for In Vivo Optical Imaging

“…One challenge in constructing MTI probes is conjugating fluorophores to antibodies. Fluorophores are most commonly conjugated to antibodies in a non-specific manner, which can result in decreased antigen-binding affinity and poor pharmacological properties [4][5][6][7]. To overcome this problem, methods have been developed to label antibodies at specific locations.…”

“…To overcome this problem, methods have been developed to label antibodies at specific locations. Site-specific conjugation can be achieved by labeling cysteines or by incorporating peptide tags or modified amino acids [6][7][8][9][10][11][12][13][14]. While promising, each of these methodologies has limitations.…”

“…While promising, each of these methodologies has limitations. For example, labeling of cysteines requires the reduction of natural disulfide bonds or the introduction of cysteine resides via genetic engineering; processes that require significant optimization [6,7]. Peptide tags fused to biologic imaging probes can be used for site-specific modifications; however, they often have low labeling efficiencies, require expensive reagents, or result in large fluorophore-protein probes.…”

“…Peptide tags fused to biologic imaging probes can be used for site-specific modifications; however, they often have low labeling efficiencies, require expensive reagents, or result in large fluorophore-protein probes. Peptide tag-based labeling allows the fluorophore to be attached at a specific location on the imaging probe, with minimal off-site labeling [6,7]. Examples of peptide tag-based labeling methods include SNAP/CLIP (O 6 -alkylguanine-DNA alkyltransferase) [8,9], Halo-tag [10], Sfp phosphopantetheinyl transferase (CoA) [11], Sortase A [12], and Avi biotin ligase recognition peptide [7].…”

“…Peptide tag-based labeling allows the fluorophore to be attached at a specific location on the imaging probe, with minimal off-site labeling [6,7]. Examples of peptide tag-based labeling methods include SNAP/CLIP (O 6 -alkylguanine-DNA alkyltransferase) [8,9], Halo-tag [10], Sfp phosphopantetheinyl transferase (CoA) [11], Sortase A [12], and Avi biotin ligase recognition peptide [7]. The fusion of peptide tags increases the size (18-33 kDa) of the imaging probe and can sterically hinder antigen binding [7].…”