Site-Specifically Labeled Immunoconjugates for Molecular Imaging—Part 1: Cysteine Residues and Glycans

Abstract

Due to their remarkable selectivity and specificity for cancer biomarkers, immunoconjugates have emerged as extremely promising vectors for the delivery of diagnostic radioisotopes and fluorophores to malignant tissues. Paradoxically, however, these tools for precision medicine are synthesized in a remarkably imprecise way. Indeed, the vast majority of immunoconjugates are created via the random conjugation of bifunctional probes (e.g., DOTA-NCS) to amino acids within the antibody (e.g., lysines). Yet antibodies have multiple copies of these residues throughout their macromolecular structure, making control over the location of the conjugation reaction impossible. This lack of site specificity can lead to the formation of poorly defined, heterogeneous immunoconjugates with suboptimal in vivo behavior. Over the past decade, interest in the synthesis and development of site-specifically labeled immunoconjugates—both antibody-drug conjugates as well as constructs for in vivo imaging—has increased dramatically, and a number of reports have suggested that these better defined, more homogeneous constructs exhibit improved performance in vivo compared to their randomly modified cousins. In this two-part review, we seek to provide an overview of the various methods that have been developed to create site-specifically modified immunoconjugates for positron emission tomography, single photon emission computed tomography, and fluorescence imaging. We will begin with an introduction to the structure of antibodies and antibody fragments. This is followed by the core of the work: sections detailing the four different approaches to site-specific modification strategies based on cysteine residues, glycans, peptide tags, and unnatural amino acids. These discussions will be divided into two installments: cysteine residues and glycans will be detailed in Part 1 of the review, while peptide tags and unnatural amino acids will be addressed in Part 2. Ultimately, we sincerely hope that this review fosters interest and enthusiasm for site-specific immunoconjugates within the nuclear medicine and molecular imaging communities.

Site-specific conjugation allows modulation of click reaction stoichiometry for pretargeted SPECT imaging

“…The benefits of site-specific radiolabeling techniques for directly labeled imaging agents are well documented. 52 A dendrimer scaffold bearing 8 TCO groups was recently reported; however, it is unclear whether improvements in tumor signal relative to the 2-TCO control were due to differences in stoichiometry, PEG length, presence/absence of dendrimer, or a combination thereof. 53 To allow a more controlled analysis of how stoichiometry affects pretargeted imaging, we used THIOMAB™ antibody conjugation 46 to install TCO moieties via maleimide chemistry site-specifically at 2, 4, or 6 engineered cysteine residues.…”

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

“…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].…”

Quantitative analysis of cell surface antigen-antibody interaction using Gaussia princeps luciferase antibody fusion proteins

“…For example, sitespecific labeling of IgGs is possible by targeting their N297-linked oligosaccharide chains. 3 Due to the importance of N297 glycosylation for FcgR-binding, 4 such labeling strategies may affect antibody-FcgR interactions. Site-specific labeling of antibodies has been furthermore achieved by genetic incorporation of unnatural amino acids and subsequent labeling by clickchemistry.…”