Jens De Vos

Nanobodies Targeting Mouse/Human VCAM1 for the Nuclear Imaging of Atherosclerotic Lesions

Rationale: A noninvasive tool allowing the detection of vulnerable atherosclerotic plaques is highly needed. By combining nanomolar affinities and fast blood clearance, nanobodies represent potential radiotracers for cardiovascular molecular imaging. Vascular cell adhesion molecule-1 (VCAM1) constitutes a relevant target for molecular imaging of atherosclerotic lesions. Objective: We aimed to generate, radiolabel, and evaluate anti-VCAM1 nanobodies for noninvasive detection of atherosclerotic lesions. Methods and Results: Ten anti-VCAM1 nanobodies were generated, radiolabeled with technetium-99m, and screened in vitro on mouse and human recombinant VCAM1 proteins and endothelial cells and in vivo in apolipoprotein E–deficient (ApoE−/−) mice. A nontargeting control nanobody was used in all experiments to demonstrate specificity. All nanobodies displayed nanomolar affinities for murine VCAM1. Flow cytometry analyses using human human umbilical vein endothelial cells indicated murine and human VCAM1 cross-reactivity for 6 of 10 nanobodies. The lead compound cAbVCAM1-5 was cross-reactive for human VCAM1 and exhibited high lesion-to-control (4.95±0.85), lesion-to-heart (8.30±1.11), and lesion-to-blood ratios (4.32±0.48) (P<0.05 versus control C57Bl/6J mice). Aortic arch atherosclerotic lesions of ApoE−/− mice were successfully identified by single-photon emission computed tomography imaging. 99mTc-cAbVCAM1-5 binding specificity was demonstrated by in vivo competition experiments. Autoradiography and immunohistochemistry further confirmed cAbVCAM1-5 uptake in VCAM1-positive lesions. Conclusions: The 99mTc-labeled, anti-VCAM1 nanobody cAbVCAM1-5 allowed noninvasive detection of VCAM1 expression and displayed mouse and human cross-reactivity. Therefore, this study demonstrates the potential of nanobodies as a new class of radiotracers for cardiovascular applications. The nanobody technology might evolve into an important research tool for targeted imaging of atherosclerotic lesions and has the potential for fast clinical translation.

Immuno-imaging using nanobodies

Immuno-imaging is a developing technology that aims at studying disease in patients using imaging techniques such as positron emission tomography in combination with radiolabeled immunoglobulin derived targeting probes. Nanobodies are the smallest antigen-binding antibody-fragments and show fast and specific targeting in vivo. These probes are currently under investigation as therapeutics but preclinical studies indicate that nanobodies could also become the next generation of magic bullets for immuno-imaging. Initial data show that imaging can be performed as early as 1 hour post-injection enabling the use of short-lived radio-isotopes. These unique properties should enable patient friendly and safe imaging protocols. This review focuses on the current status of radiolabeled nanobodies as targeting probes for immuno-imaging.

Camelid single-domain antibody-fragment engineering for (pre)clinical in vivo molecular imaging applications: adjusting the bullet to its target

Introduction: Molecular imaging is a fast developing field and there is a growing need for specific imaging tracers in the clinic. Camelid single-domain antibody-fragments (sdAbs) recently emerged as a new class of molecular imaging tracers. Areas covered: We review the importance of molecular imaging in the clinic and the use of camelid sdAbs as in vivo molecular imaging tracers. Interest in imaging tracers based on antibody fragments or man-made protein scaffolds expanded over the last years. Camelid sdAbs are small, monomeric binding fragments that are derived from unique heavy-chain-only antibodies. In vivo imaging studies with sdAbs targeting various cell membrane receptors in different disease models have been reported and more sdAb imaging tracers are under development. The first clinical trial with a camelid sdAb as a molecular imaging tracer targeting the breast cancer marker Human Epidermal growth factor Receptor 2 is currently ongoing. Expert opinion: We expect that the development and use of sdAbs as tracers for both preclinical and clinical molecular imaging applications will become widespread.

Site-specific labeling of cysteine-tagged camelid single-domain antibody-fragments for use in molecular imaging.

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 tracers 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 domains 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.

Site-Specific Labeling of His-Tagged Nanobodies with 99m Tc: A Practical Guide

99mTc-tricarbonyl chemistry provides an elegant technology to site-specifically radiolabel histidine-tagged biomolecules. Considering their unique biochemical properties, this straightforward technology is particularly suited for Nanobodies. This chapter gives a detailed guide to generate highly specific Nanobody-derived radiotracers for both in vitro binding studies and in vivo molecular imaging.

Molecular Imaging Using Nanobodies: A Case Study

Molecular imaging is a noninvasive method to measure specific biological processes in animal models and patients using imaging. In recent years there has been a tremendous evolution in hardware and software for imaging purposes. This progress has created an urgent need for new labeled targeted molecular probes. The unique physicochemical and pharmacokinetic properties of Nanobodies match the requirements of the ideal molecular imaging tracer. Preclinical studies show strong and specific targeting in vivo with rapid clearance of unbound probe resulting in high contrasted images at early time points after intravenous administration. These data suggest that the Nanobody platform might become a generic method for the development of next generation molecular imaging probes.

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

Purpose: Camelid single-domain antibody-fragments (sdAb) have beneficial pharmacokinetic properties, and those targeted to HER2 can be used for imaging of HER2-overexpressing cancer. Labeled with a therapeutic radionuclide, they may be used for HER2-targeted therapy. Here, we describe the generation of a 131I-labeled sdAb as a theranostic drug to treat HER2-overexpressing cancer. Experimental Design: Anti-HER2 sdAb 2Rs15d was labeled with 131I using [131I]SGMIB and evaluated in vitro. Biodistribution was evaluated in two HER2+ murine xenograft models by micro-SPECT/CT imaging and at necropsy, and under challenge with trastuzumab and pertuzumab. The therapeutic potential of [131I]SGMIB-2Rs15d was investigated in two HER2+ tumor mouse models. A single-dose toxicity study was performed in mice using unlabeled [127I]SGMIB-sdAb at 1.4 mg/kg. The structure of the 2Rs15d–HER2 complex was determined by X-ray crystallography. Results: [131I]SGMIB-2Rs15d bound specifically to HER2+ cells (Kd = 4.74 ± 0.39 nmol/L). High and specific tumor uptake was observed in both BT474/M1 and SKOV-3 tumor xenografted mice and surpassed kidney levels by 3 hours. Extremely low uptake values were observed in other normal tissues at all time points. The crystal structure revealed that 2Rs15d recognizes HER2 Domain 1, consistent with the lack of competition with trastuzumab and pertuzumab observed in vivo. [131I]SGMIB-2Rs15d alone, or in combination with trastuzumab, extended median survival significantly. No toxicity was observed after injecting [127I]SGMIB-2Rs15d. Conclusions: These findings demonstrate the theranostic potential of [131I]SGMIB-2Rs15d. An initial scan using low radioactive [*I]SGMIB-2Rs15d allows patient selection and dosimetry calculations for subsequent therapeutic [131I]SGMIB-2Rs15d and could thereby impact therapy outcome on HER2+ breast cancer patients. Clin Cancer Res; 23(21); 6616–28. ©2017 AACR.

A nanobody-based tracer targeting DPP6 for non-invasive imaging of human pancreatic endocrine cells

There are presently no reliable ways to quantify endocrine cell mass (ECM) in vivo, which prevents an accurate understanding of the progressive beta cell loss in diabetes or following islet transplantation. To address this unmet need, we coupled RNA sequencing of human pancreatic islets to a systems biology approach to identify new biomarkers of the endocrine pancreas. Dipeptidyl-Peptidase 6 (DPP6) was identified as a target whose mRNA expression is at least 25-fold higher in human pancreatic islets as compared to surrounding tissues and is not changed by proinflammatory cytokines. At the protein level, DPP6 localizes only in beta and alpha cells within the pancreas. We next generated a high-affinity camelid single-domain antibody (nanobody) targeting human DPP6. The nanobody was radiolabelled and in vivo SPECT/CT imaging and biodistribution studies were performed in immunodeficient mice that were either transplanted with DPP6-expressing Kelly neuroblastoma cells or insulin-producing human EndoC-βH1 cells. The human DPP6-expressing cells were clearly visualized in both models. In conclusion, we have identified a novel beta and alpha cell biomarker and developed a tracer for in vivo imaging of human insulin secreting cells. This provides a useful tool to non-invasively follow up intramuscularly implanted insulin secreting cells.