Site-Specific Labeling of Single Domain Antibody (sdAb)
sdAb has emerged as a new class for medical diagnostics and basic research over the last years. It is well established that sdAb with a small size (~13 kDa) is very stable and resistant to unfolding at elevated temperature, in various solutions, and under stress conditions. Due to the compact structure, high stability and specificity, sdAbs see frequent usage for applications like affinity purification or protein detection and localization. Mostly, its chemical functionalization, such as site-specific labeling, opens the way towards promising diagnostic and therapeutic applications.
Conventional bioconjugation technologies have proven unfavorable for in vivo therapy and diagnostics since unselective functionalization of antigen-binding sdAb leads to variations in the number of probes attached, in combination with alteration of epitope recognition, which can lead to impaired pharmacokinetic properties and stability. While early conjugation strategies relied on the random modification of natural amino acids, and more recent studies have focused on the alternative and more reliable site-specific attachment of functional moieties with improved biophysical properties to overcome the limitations of random conjugation of sdAb.
Site-Specific Labeling of sdAb
A generic site-specific conjugation method that generates a homogeneous product is of utmost importance in tracer development for molecular imaging and therapy. In recent years, a number of versatile and standardized strategies for the site-specific functionalization of protein have been applied to homogeneously modify sdAb, such as (1) protein engineering with additional cysteine residues; (2) protein modification at the C or N terminus, especially through enzymatic ligation strategies; and (3) genetic incorporation of bioorthogonal unnatural amino acids (UAAs) into sdAb.
The introduction of a tag for site-specific labeling at the C-terminal end of the sdAb is a valid alternative to obtain a better, more controlled probe without antigen-binding interference since the C-terminal end and the CDR loops are located on opposite sides of the domain. Generally, the primary amines of lysines or the sulfhydryls of cysteines serve as conjugation sites. The site-specific coupling method of sdAb is using maleimide chemistry on a cysteine residue that is introduced C-terminally. These versatile techniques manage to couple various molecular reagents, such as biotin, fluorescent dye, bifunctional chelators, etc., to the sdAbs.
Advantages of sdAb Site-Specific Labeling
Site-specific radiolabeling allows for precise chemical modification of sdAb that can be designed to avoid damaging modification to amino acids involved in receptor recognition, thereby preventing antigen-binding interference after conjugation. Using site-specific attachment of tracer to sdAb, a homogeneous tracer population with a well-defined stoichiometry and label position can be obtained. Moreover, the site-specific labeling of targeting probes for diagnostic and therapeutic applications results in a well-defined homogeneous product with uncompromised functionality and straightforward characterization and clinical translation. Finally, the resulting batch-to-batch reproducible pharmacokinetic and pharmacodynamic properties are of great importance for clinical translation.
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