Site-Specific Labeling of Cysteine-Tagged Camelid Single-Domain Antibody-Fragments for Use in Molecular Imaging

Abstract

Site-specific labeling of molecular imaging probes allows the development of a homogeneous tracer population. The resulting batch-to-batch reproducible pharmacokinetic and pharmacodynamic properties are of great importance for clinical translation. Camelid single-domain antibody-fragments (sdAbs)-the recombinantly produced antigen-binding domains of heavy-chain antibodies, also called Nanobodies-are proficient probes for molecular imaging. To safeguard their intrinsically high binding specificity and affinity and to ensure the tracer's homogeneity, we developed a generic strategy for the site-specific labeling of sdAbs via a thio-ether bond. The unpaired cysteine was introduced at the carboxyl-terminal end of the sdAb to eliminate the risk of antigen binding interference. The spontaneous dimerization and capping of the unpaired cysteine required a reduction step prior to conjugation. This was optimized with the mild reducing agent 2-mercaptoethylamine in order to preserve the domain's stability. As a proof-of-concept the reduced probe was subsequently conjugated to maleimide-DTPA, for labeling with indium-111. A single conjugated tracer was obtained and confirmed via mass spectrometry. The specificity and affinity of the new sdAb-based imaging probe was validated in a mouse xenograft tumor model using a modified clinical lead compound targeting the human epidermal growth factor receptor 2 (HER2) cancer biomarker. These data provide a versatile and standardized strategy for the site-specific labeling of sdAbs. The conjugation to the unpaired cysteine results in the production of a homogeneous group of tracers and is a multimodal alternative to the technetium-99m labeling of sdAbs.

131I-labeled Anti-HER2 Camelid sdAb as a Theranostic Tool in Cancer Treatment

“…in two distinct HER2 + mouse models. The clearance of [ 131 I]-2Rs15d from kidneys was faster than ever observed for this sdAb (18,20,21,36,37). For example, dosimetric calculations revealed that 37 MBq of [ 131 I]-2Rs15d achieved an absorbed dose of only 8 Gy to kidneys.…”

“…2Rs15d was validated preclinically labeled with the diagnostic radionuclides 18 F (36), 68 Ga (18,19) and 111 In (37). Labeled with 68 Ga, 2Rs15d was evaluated in a first clinical trial, where we showed that it was safe and accumulated preferentially in primary tumors and metastases of HER2 + BC patients.…”

In vivo imaging with antibodies and engineered fragments

“…Comparisons of the pharmacokinetics and targeting of intact and antibody fragments (enzymatically-derived and recombinant) have been conducted; recent examples include targeted imaging of prostate-specific membrane antigen (PSMA) using 111 In-radiolabeled F(ab′) 2 and Fab (Fragment antigen binding) fragments, and 89 Zr-labeled minibodies and diabodies with small animal SPECT or PET (Lütje et al, 2014c; Viola-Villegas et al, 2014). Continued interest in smaller antibody fragments and protein scaffolds is evidenced by several imaging studies with nanobodies (Bannas et al, 2014; De Vos et al, 2014; Massa et al, 2014) and affibodies (Kim et al, 2014; Strand et al, 2014; Zielinski et al, 2012). Nanobodies (~15kDa) are single-domain variable heavy chain (V H H) fragments, which are derived from heavy chain-only antibodies found in camelids (Schoonooghe et al, 2012), while affibodies (~7kDa) are based on Staphylococcus aureus protein A (Zielinski et al, 2012).…”

“…In contrast to conjugation at random surface residues, which produces a heterogeneous product and may introduce the label into the antigen-binding site (ABS), site-specific labeling creates a homogenous product in which the label can be intentionally located away from the ABS. A common approach is the addition of a cysteine residue at the carboxyl-terminus, which creates a site for specific labeling via a thio-ether bond (Massa et al, 2014; Tavaré et al, 2014a, 2014b). Alternatively, bio-orthogonally reactive unnatural amino acids, which are not naturally encoded in the genetic code but can still be incorporated artificially into proteins, can be inserted to provide specific sites for conjugation (Axup et al, 2012; Kim et al, 2013, 2012).…”

Structurally Defined αMHC‐II Nanobody–Drug Conjugates: A Therapeutic and Imaging System for B‐Cell Lymphoma

“…[18] Similar conjugation methods are generally transferable to antibody fragments. Their conjugates with radioactive tracers, [19, 20] nanomaterials, [21, 22] gene products, [23] immunomodulators, [24] cytotoxic reagents, [25, 26] or a combination [27] have demonstrated great maneuverability in therapeutic and diagnostic applications.…”