Nanobody Technology

Team Nick

Core team:

  • Nick Devoogdt, Molecular Biologist, PhD
  • Jens De Vos, Bioengineer, PhD
  • Sam Massa, doctoral student supported by ‘Research Foundation Flanders', FWO

Description nanobody technology:
We specialize in the generation of 'nanobodies' and their genetic formatting as optimal probes for molecular imaging. But what are nanobodies exactly and what makes them so special ? Nanobodies are derived from a particular type of antibodies that exist in the blood of camelids (llama, alpaca, camel, dromedary, etc). In contrast to conventional antibodies that exist in all mammals (including human beings), the camelid 'heavy-chain antibodies' (HCAbs) lack a light chain (the green domains in the figure). So, while conventional antibodies bind to foreign structures (the 'antigen') through the assembly of the variable domain of the heavy chain (VH) and that of the light chain (VL), the camelid HCAbs bind to antigen with a single domain called the VHH. This VHH (also called 'nanobody') can be easily produced in bacteria or yeast in large quantities, is very stable and binds to the antigen with high affinities and specificities. Also, nanobodies are encoded by small DNA fragments and can be manipulated by genetic engineering in multiple formats and fusions.
Since they are very small, nanobodies efficiently penetrate into dense tissues. When they encounter antigen somewhere, nanobodies get trapped, while 'free' nanobody is rapidly removed from the body through filtration by the renal system. So, when nanobodies are labeled with radioactive or fluorescent dyes, they generate very high contrast at the targeted site and very early after administration. These radioactive or fluorescent signals can be detected three-dimensionally by specialized cameras in both laboratory animals and in patients. As a whole, this process is called 'molecular imaging'.
Nanobody Technology Team 1

Aims of the team:
We specialize in the generation and application of nanobodies targeting disease-related antigens for imaging purposes.
On the one hand these biomarker-targeting nanobodies are used for fundamental scientific research. The main questions we ask are the following:

  • What are the rules to most optimally adopt nanobodies for molecular imaging: affinity, stability, amino acid composition, buffer composition, nanobody format, labeling strategies, pharmocokinetics and -dynamics, nanobody mass injected, application route, time of imaging etc.

  • Develop multiple selection strategies to generate nanobodies against different types of membrane receptors: single-pass versus multi-pass membrane receptors, glycosylated proteins, multimeric receptors, selection on recombinant proteins versus whole cells, selection of species-crossreactive nanobodies or nanobodies recognizing multiple isoforms, etc

  • What type of imaging strategies can we use the nanobodies for ? We are currently testing SPECT and PET applications, fluorescence tomography, MRI, ultrasound, etc.

  • By labeling nanobodies with high-energy radioactive compounds, can we use them for targeted radiotherapy ? How can we format and apply these nanobodies for low toxicity and high effectiveness ?

  • What does imaging of these biomarkers tell us about the biology of the targeted receptor ? Can the nanobodies be used to quantify receptor levels on the targeted cell ? Can we use nanobodies to stratify individuals that are/will be responsive to therapies ?

On the other hand our goal is to make a translation to the clinic. Are nanobodies toxic and immunogenic ? What nanobody format do we need to use ? How do we optimally produce these nanobodies under cGMP conditions ? What are the doses we need to apply ? What radiolabeling strategy and what imaging modality do we use ?

Research projects:
1. Oncology

Our initial projects were to use nanobodies for targeting and imaging of cancer biomarkers. Examples of successful projects include those targeting HER2, EGFR, CEA and paraprotein for breast, lung, colon cancer and multiple myeloma.

  • EGFR

EGFR is a membrane receptor of the Epithelial Growth Factor family and a biomarker for many cancer types. It is targeted by therapeutic antibodies such as Erbitux and by chemical tyrokinase inhibitors such as erlotinib. In collaboration with the company Ablynx (www.ablynx.com) anti-EGFR nanobodies were tested as tracers for imaging of EGFR-positive xenografted tumors in mice.
In a first hallmark study we showed the potential of anti-EGFR nanobodies to target specifically cancer biomarkers in vivo an demonstrate their applicability for fast non-invasive molecular imaging: 99mTc-labeled nanobodies were cleared very fast out of blood and nontarget organs, and accumulated in EGFR-positive tumors with high tumor-to-organ ratios. This allowed us to visualize tumors with high contrast on SPECT/CT images. Comparing two nanobodies that differ only in a few amino acids, we show that general biodistribution and tumor targeting potential can vary significantly (Gainkam et al, 2008, J Nucl Med). In a follow-up study we showed that the anti-EGFR nanobody was useful to monitor tumor regression when mice were under therapy with erlotinib (Tchouate Gainkam et al, 2011, Mol Imaging Biol).

  • CEA

Carcinoembryonic antigen (CEA) is a biomarker for multiple cancers including colon. It is the target of therapeutic antibody Arcitumomab and the clinically-approved imaging tracer CEA-Scan, a 99mTc-labeled Fab fragment that is derived from the murine monoclonal antibody.
Similar as studies with anti-EGFR nanobodies, nanobodies have been succesfully used to image CEA-positive xenografted tumors (Cortez et al, Curr Radiopharmaceuticals, 2008). This model nanobody was further used to show the feasibility of CDR-grafting to generate less immunogenic variants with equal potential for molecular imaging (Vaneycken et al, J Nucl Med, 2010).

  • HER2

HER2 is a biomarker for breast and other types of cancers and its expression usually associates with bad prognosis. It is the antigen for many targeted therapies, including monoclonal antibodies Trastuzumab and Pertuzumab and the tyrosine kinase inhibitor Lapitinib. Noninvasive quantification of HER2 expression in primary tumors and metastases would allow to accurately select patients eligible for targeted therapies and to follow them up during and after therapy.
Anti-HER2 nanobodies have been generated and biochemically evaluated for strength and specificity of antigen recognition, stability, targeted epitope and internalization rate. 99mTc-labeled nanobodies were shown to target HER2-positive but not -negative tumors in laboratory mice via dissection analysis and SPECT/CT imaging studies (Vaneycken & Devoogdt et al, FASEB, 2011).
A lead anti-HER2 nanobody was selected and radiochemical procedures were optimized for radiolabeling with 68Ga (Xavier et al, in preparation). A first-in human clinical trial is ongoing in our hospital to evaluate the safety of this radiotracer for PET-imaging in healthy and breast cancer patients (Vaneycken et al, Curr Opin Biotechnol, 2011).
Chemical procedures have been or are being evaluated to label the lead anti-HER2 nanobody with other radionuclides, including 177Lu (D'Huyvetter et al, Contr Media & Mol Imaging, 2012), 18F, 131I, 211At & 111In and near-infrared fluorescent dyes.
Anti-HER2 nanobodies are also coupled to gold nanoparticles for targeted thermotherapy (Van Den Broek et al, ACS Nano, 2011).
Besides for imaging, nanobodies are also labeled with therapeutic radionuclides such as 177Lu and 211At. Anti-HER2 nanobodies are formatted such that kidney retention is kept to a minimum.
Anti-HER2 nanobodies are being in vitro matured and further engineered into multivalent constructs and the effect of engineering on biochemical parameters and in vivo tumor targeting potential is being investigated.

  • Paraprotein

Paraprotein is (part of) the antibody produced by clonal multiple myeloma cells and therefore represents the ultimate cancer and patient-specific biomarker for targeting this disease. Since multiple myeloma is currently uncurable due to the re-growth of residual disease after conventional radio- and chemotherapy, new specific therapies are urgently needed.
Using stringent selection techniques, anti-idiotypic nanobodies have been generated to specifically bind to paraprotein produced by 5T2 multiple myeloma, a widely used in vivo mouse model for this disease. This project is performed in close collaboration with Prof. Karin Vanderkerken at our university. We are currently evaluating 99mTc-labeled anti-idiotype nanobodies for in vivo imaging. After coupling to high-energy radionuclides or to RNAi molecules, we aim to perform efficiently treat residual disease in mice.

  • Miscellaneous

In collaboration with the group of Claude Libert, we are also evaluating anti-integrin nanobodies for their potential to target tumor vasculature, and as a possible synergistic therapeutic for TNF-therapy.

Personnel involved & collaborators:

  • ICMI: Nick Devoogdt & Sam Massa (nanobody generation and engineering), Gezim Bala & Sophie Hernot (fluorescence imaging), Vicky Caveliers, Ilse Van Eycken, Olive Lea Tchouate Gainkam (until 2008), Matthias d'Huyvetter & Catarina Xavier (radiochemistry and preclinical imaging), Marleen Keyaerts & Tony Lahoutte (clinical translation), Cindy Peleman and Isabel Remory (lab technicians)

  • Cellular and Molecular Immunology (CMIM), VUB: Prof Serge Muyldermans, Dr. Cécile Vincke, Dr. Pieter De Pauw.

  • Dept Hematology and Immunology (HEIM), VUB: Drs. Miguel Lemaire and Prof Karin Vanderkerken

  • Duke University, NC, USA: Prof. Michael Zalutsky & Dr. Marek Pruszynski

  • Belgian Nuclear Research Center/Studiecentrum kernenergie (SCK.CEN): Dr. Nathalie Impens, Dr. An Aerts & Dr. Saarah Baatout

  • UGent, Belgium: Prof Claude Libert, Dr Filip Vanhauwermeiren

  • KTH, Sweden: Dr. Vladimir Tolmachev, Dr. John Lofblom and Dr. Torbjorn Graslund.

  • Interuniversity micro-electronics Center (IMEC), Leuven, Belgium: Dr. Bieke Van Den Broek & Prof. G. Borghs

  • KULeuven: Drs Antoine D'Hollander & Prof. U. Himmelreich

  • Ablynx: Dr. Hilde Revets


2. Atherosclerosis

Cardiovascular diseases now represent the first cause of mortality worldwide and coronary artery disease is responsible for more than half of cardiovascular deaths. The vast majority of coronary events are caused by rupture of vulnerable atherosclerotic plaques and subsequent thrombi formation. A marker that could accurately detect vulnerable plaque prior to rupture and enable preventative therapy to be implemented to avoid a heart attack or stroke would address a major unmet clinical need, and a significant market opportunity. However, despite experimental evaluation of a number of radiolabelled tracers, no non-invasive diagnostic tool is yet available for the early clinical detection of vulnerable plaques prior to plaque rupture. We therefore decided to adopt the nanobody-technology to generate imaging tracers targeting vulnerable plaques.
In a collaborative effort with the Unit INSERM1039 from the university of Grenoble, France, we generated nanobodies binding to both mouse and human VCAM1 with high affinities. VCAM1 (Vascular Cell Adhesion Molecule 1) was chosen since it is a validated marker for plaque vulnerability. Using SPECT/CT imaging with 99mTc-labeled nanobodies, we showed in a mouse model of inflamed atherosclerosis that the selected lead anti-VCAM1 nanobody targeted lesions with high lesion-to-background ratios and was able to detect plaques on the images. Besides plaques in the aortas, the tracer also visualized VCAM1-positive lymphoid tissues (Broisat & Hernot et al, Circ Res, 2012).
In a follow-up study, the lead anti-VCAM1 nanobody was site-specifically conjugated to biotin and coupled to microbubbles. Besides showing functionality in vitro, we showed that nanobody-functionalized microbubbles enable target-visualization via echography (Hernot et al, J Contr Release, 2012).
We are currently generating and evaluating new nanobodies for atherosclerosis imaging, including nanobodies targeting the Oxidized LDL-receptor LOX1 and macrophage markers such as MMR and VSIG4.
Finally, a research project was started in collaboration with Ablynx and Boehringer to evaluate target specificity of a panel of their nanobodies.

Personnel involved & collaborators:

  • ICMI: Nick Devoogdt, Jens De Vos, Sophie Hernot (nanobody generation and engineering), Gezim Bala & Sophie Hernot (fluorescence imaging and echography), Vicky Caveliers & Catarina Xavier (radiochemistry), Tony Lahoutte (clinical translation), Cindy Peleman and Isabel Remory (lab technicians)

  • INSERM1039 at university of Grenoble: Dr. Alexis Broisat, Drs Jacub Toczek, Dr. Laurent Riou, Prof Catherine Ghezzi.

  • Cardiology dept VUB: Dr. Bernard Cosyns, Prof Guy Van Camp

  • Cellular and Molecular Immunology (CMIM), Vrije Universiteit Brussel: Prof Serge Muyldermans, Dr. Pieter De Pauw.

  • University of Virginia: Prof David Glover

  • Ablynx: Cedric Ververken

  • Boehringer: Dr. Matthias Schneider


3. Diabetes

Diabetes remains a challenging disease and is expected to grow to almost epidemic proportions. This pathology is caused, at least partially, by loss of pancreatic beta cells. However, there are presently no reliable ways to quantify beta cell mass (BCM) in vivo, which hampers the understanding of the pathogenesis and natural history of diabetes, and the development of novel therapies to preserve BCM. In this project we aim to employ Nanobody-technology to develop tracers for the quantification of BCM via non-invasive molecular imaging. This project started as a participation in the FP7-funded BetaImage consortium (www.betaimage.eu), in which our group was responsible for the generation of new BCM-tracers. We are currently generating and evaluating nanobodies targeting both known BCM-biomarkers (e.g. VMAT2 and GLP1R) and novel ones. The novel ones are discovered by Prof Decio Eisirik's group (ULB, Belgium) and validated by Prof Luc Bouwens' group (VUB, Belgium), both BetaImage partners.

Personnel involved & collaborators:

  • ICMI: Nick Devoogdt & Sam Massa (nanobody generation and engineering), Vicky Caveliers & Catarina Xavier (radiochemistry), Tony Lahoutte (clinical translation), Cindy Peleman (lab technician)

  • Diabetes dept VUB: Drs. Iris Mathijs, Prof. Luc Bouwens

  • Cellular and Molecular Immunology (CMIM), Vrije Universiteit Brussel: Prof Serge Muyldermans, Dr. Pieter De Pauw.

  • Diabetes dept ULB Belgium: Prof Decio Eisirik, Dr Guy Bottu, Dr. Thomas Bouckenooghe

  • Turku University, Finland: Prof Pirjo Nuutila

  • Radboud University Nijmegen, the Netherlands: Prof Martin Gotthardt and Drs Maarten Brom

  • Marburg University, Germany: Prof Eberhardt Weihe

  • Lausanne University, Switzerland: Prof Theo Lassers

  • Geneva University, Switzerland: Prof Paolo Meda

  • Paul Scherrer Institute, Switzerland: Dr. Martin Behe


4. Immunology

Macrophages (MΦs) and myeloid dendritic cells (mDCs) play a crucial role in linking innate and adaptive immune responses and in modulating the balance between humoral versus cellular immunity and activation versus suppression of distinct types of immune and inflammatory responses. Moreover, these pleiotropic cells feature a high degree of plasticity and versatility upon activation and/or differentiation in response to various triggers. Therefore, besides playing a critical role in a range of inflammatory diseases as innate effectors, immunomodulators and/or antigen presenting cells (APCs), they also represent potential in vivo sensors for the status of the immune system.
The current project aims at validating MΦs and/or mDC markers (M&D markers) for the purpose of in vivo targeting of myeloid cells (MCs), especially MΦs and mDCs. Specifically, we aim to validate M&D markers as targets for imaging the inflammatory process and its spontaneous evolution and evolution in response to treatment in the living organism on the basis of visualization of (anti )inflammatory MCs, by using labeled nanobodies targeting M&D markers
In a first proof-of-concept study, in collaboration with Prof De Baetselier and Dr Geert Raes (CMIM, VUB), 99mTc-labeled nanobodies against (unknown) myeloid cell markers were used in vivo in naive mice to visualize targeted cells. Using CDR grafting we showed the specificity of targeting by these nanobodies (De Groeve et al, 2010, J Nucl Med).
In a collaborative effort of the flemish consortium 'Inflammatrack', a wide range of nanobodies targeting membrane biomarkers of macrophages in several disease states were/are being generated and validated. These include MMR, Mgl2, VSIG4, PD-L1, PD-L2 and ST-2.
The mouse disease models that we currently investigate to visualize target-expressing macrophages using these nanobodies include arthritis, non-shivering thermogenesis and tumor stroma.
One recent successful study is the use of anti-MMR nanobodies to visualize MMR-positive macrophages in tumor-bearing mice. In collaboration with Prof Jo Van Ginderachter, Dr Geert Raes and Prof Patrick De Baetselier, we observed that MMR-positive macrophages are abundant in several tumors grown in syngeneic mice. Since these macrophages tend to reside in tumor hypoxic zones, visualizing them might give clues to quantify the degree and spatial distribution of hypoxia within these tumors. De Baetselier's group generated nanobodies targeting MMR. Upon 99mTc labeling, anti-MMR nanobodies specifically targeted MMR-positive macrophages residing in the hypoxic tumor zones, as well as macrophages existing in immunological organs. Using MMR-deficient mice we demonstrated the specificity of targeting. Moreover, co-injection of the tracer with an excess of an unlabeled bivalent nanobody construct drastically reduced radioactive signals in extratumoral organs, while maintaining signals in the tumor, allowing us to specifically monitoring hypoxia in the tumors (Movahedi & Schoonooghe & Laoui et al, Cancer Res, 2012).
Our next aim is to proceed a nanobody-based anti-MMR tracer for clinical applications. As a first step, the De Baetselier group is currently generating new nanobodies recognizing both mouse and human MMR in order to select a new lead compound. In collaboration with Prof Guy Bormans (KULeuven) we are currently also optimizing conditions to label the lead nanobody with 18F.

Personnel involved & collaborators:

  • ICMI: Nick Devoogdt (nanobody generation and engineering), Vicky Caveliers & Anneleen Blykers (radiochemistry), Tony Lahoutte (clinical translation), Cindy Peleman (lab technician)

  • CMIM, VUB: Dr Steve Schoonooghe, Prof Geert Raes, Dr Kiavash Movahedi, Drs Damya Laoui, Prof Jo Van Ginderachter, Prof Patrick De Baetselier

  • KULeuven, Immunobiology: Prof Patrick Matthijs, Drs Stephanie Put

  • UGent, Molecular Signal Transduction in Inflammation: Prof Rudy Beyaert, Dr Harald Braun

  • UGent, Molecular Immunology: Prof Johan Grooten, Dr Stefaan De Koker

  • VUB, Immunology: Prof Kris Thielemans, Prof Karine Breckpot

  • KULeuven, radiopharmacy: Prof Guy Bormans


5. Kidney re-uptake and organ biodistribution

Due to their small size, high antigen-specificity and their intrinsic hydrophilic character the uptake of nanobody-based tracers in non-targeted organs is usually low and they are cleared very fast from the body and circulation by renal clearance. However, nanobody-based tracers are frequently retained in the kidneys due to re-uptake in the proximal tubuli, but the extent of this is variable. Also, retention in non-targeted organs such as liver is variable. We are currently investigating the factors influencing this variability in biodistribution. These include nanobody stability, size, format, amino acid composition, pI, buffer composition, application route, mass injected and labeling strategy.
In one study we observed that renal retention of an EGFR-targeting, 99mTc-labeled nanobody is reduced in the absence of the megalin-receptor, on of the receptor involved in re-uptake in the proximal tubuli. In addition, kidney radioactive signals, but not those in the targeted tumor, were dramatically reduced by the co-injection of labeled nanobody with positively-charged amino-acids and the plasma expander gelofusin (Tchouate Gainkam et al, Contr Med Mol Imaging, 2011).

Personnel involved & collaborators:

  • ICMI: Nick Devoogdt & Matthias d'Huyvetter (nanobody generation and engineering), Vicky Caveliers, Ilse Van Eycken, Olive Lea Tchouate Gainkam (until 2008) & Catarina Xavier (radiochemistry and preclinical imaging), Tony Lahoutte (clinical translation), Cindy Peleman (lab technicians)

  • Cellular and Molecular Immunology (CMIM), VUB: Prof Serge Muyldermans, Dr. Cécile Vincke.

  • Duke University, NC, USA: Prof. Michael Zalutsky & Dr. Marek Pruszynski

  • KTH, Sweden: Dr. Vladimir Tolmachev, Dr. John Lofblom and Dr. Torbjorn Graslund.

  • Radboud University Nijmegen, the Netherlands: Prof Otto Boerman