Molecular Imaging Using Nanobodies

Molecular imaging using Nanobodies

Tony Lahoutte, MD, PhD

Feb 28th 2014

Wetenschappelijke Prijs prof. Roger Van Geen

1. Introduction

Molecular imaging using Nanobodies started as a post-doctoral research topic in 2007 at the In vivo Cellular and Molecular Imaging (ICMI) laboratory of the Vrije Universiteit Brussel (VUB) directed by Tony Lahoutte. The idea of combining Nanobody technology with radionuclides to form radiotracers for Single Photon Emission Computed Tomography (SPECT) and Positron Emission Tomography (PET) imaging originated during an interdisciplinary meeting between the nuclear medicine team of the VUB University Hospital (UZ Brussel - prof. Axel Bossuyt, Tony Lahoutte and Vicky Caveliers) and the VUB research group Cellular and Molecular Immunology (prof. De Baetselier, prof. Serge Muyldermans). Since then this combined technology has been investigated extensively in vitro and in vivo in preclinical models to refine the methodology and explore potential applications for both basic and clinical research. In 2011, the first clinical translation of radiolabeled Nanobodies was initiated and a Phase I clinical trial in breast cancer patients is currently ongoing at the department of Nuclear Medicine of UZ Brussel. This memorandum gives an overview of the Nanobody imaging technology and a description of the different applications that we investigated in the last 5 years.

2. Molecular imaging & Nanobodies

2.1 Molecular imaging Molecular imaging is aimed at the study of molecular and cellular events in intact living subjects. It has the unique advantage that the biology of health and disease can be studied non-invasively in its natural environment, offering new insights and opportunities for discovery. Nuclear imaging is at present the most sensitive and accurate method for molecular imaging. It requires the administration of a targeting probe labeled with a radioisotope. This radiolabeled probe or ‘tracer’ then distributes throughout the body, interacts with a specific molecular target and remains onsite while the unbound tracer is eliminated from the body. The most widely used tracer is 18F-Fluorodeoxyglucose or 18F-FDG in combination with PET imaging. 18F-FDG is intensely accumulated in sites with increased glucose metabolism and therefore used for the sensitive detection of cancer. In order to image a more specific marker of cancer it is possible to target antigens expressed on the cell membrane. A radiolabeled version of a ligand of the receptor has been extensively investigated in the last few decades. The use of radiolabeled antibodies for ‘immuno-imaging’ of cancer has received a lot attention in recent years and several radiolabeled conventional antibodies are currently used in clinical trials. The main limitation of this method is that it requires several days before a high contrasted image can be obtained due to the prolonged blood retention of intact antibodies. Imaging at several days after

124 injection requires a radiolabeling with isotopes with a long half-life such as I (t 1/2: 100,3h) and

89 Zr (t1/2:78,4h) resulting in a relatively high radiation dose per scan (20-40 mSv). As a result research has been focused on the use of radiolabeled fragments of immunoglobulins that are

more rapidly cleared from the circulation. In our research we investigate Nanobodies that are the smallest possible functional fragments that are derived from antibodies.

2.2. Nanobodies Nanobodies are recombinant proteins that are derived from a particular type of antibodies that exist in the blood of camelids (llama, alpaca, vicuna, guanaco, camel and dromedary). 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 below). 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, Nanobodies get trapped, while unbound Nanobody is rapidly removed from the body through filtration by the renal system. As a result, radiolabeled Nanobodies generate very high specific contrast at the targeted site as early as 1 hour after administration. This allows the use of short-lived isotopes such as 68Ga or 18F and a more favorable dosimetry (2-4 mSv per scan)

Figure 1. Comparison of conventional IgG antibody (left) and a Camelid heavy-chain-only nanobody (middle). The VHH fragment represents a Nanobody.

Related references:

Immuno-imaging using Nanobodies. Vaneycken I, D'huyvetter M, Hernot S, De Vos J, Xavier C, Devoogdt N, Caveliers V, Lahoutte T. Curr Opin Biotechnol. 2011 Dec;22(6):877-81

Molecular imaging using Nanobodies: a case study. Devoogdt N, Xavier C, Hernot S, Vaneycken I, D'Huyvetter M, De Vos J, Massa S, De Baetselier P, Caveliers V, Lahoutte T. Methods Mol Biol. 2012;911:559-67

Camelid single-domain antibody-fragment engineering for (pre)clinical in vivo molecular imaging applications: adjusting the bullet to its target. De Vos J, Devoogdt N, Lahoutte T, Muyldermans S. Expert Opin Biol Ther. 2013 May 16

3. Aim of the research The research focused on Molecular imaging using Nanobodies presented here is aimed at the development of new PET tracers for molecular imaging in oncology, inflammatory and cardiovascular diseases. Our major goal is to translate our lab findings into innovative clinical applications for diagnostic PET imaging. However, we also conduct more basic science investigations at our preclinical imaging facilities in order to identify the ideal conditions and formats for nanobodies as probes for molecular imaging. A recently added objective of our studies is to explore the feasibility of radionuclide therapy using the same Nanobody technology.

This Nanobody research is embedded in a larger research theme that also includes the development of optical imaging tracers and detectors for image guided surgery. These applications are non-radioactive and beyond the scope of the current proposal.

4. Nanobody applications

4.1 Molecular imaging of cancer Our first research projects were aimed to develop nanobody-tracers for imaging of membrane antigens on cancer cells. Examples of successful projects include those targeting HER2, EGFR and CEA for breast, lung and colon cancer, respectively, and will be described hereunder. We are currently expanding our list of oncology tracers in collaboration with strategically-chosen academic and industrial partners. Targeted oncology biomarkers in the pipeline include CD20 for lymphomas, PSMA for prostate cancer and paraproteins for multiple myeloma. The choice to perform extensive preclinical imaging of cancer biomarkers is mostly based on its clinical relevance: all selected targets on the respective cancer cells have either prognostic value, can stratify patients eligible for receptor-targeted therapies and/or are suitable for nanobody- coupled radionuclide therapy.

4.1.1 Imaging Epidermal Growth Factor Receptor expression Epidermal Growth Factor Receptor (EGFR) is a membrane receptor of the Epithelial Growth Factor family and a prognostic biomarker for many cancer types. It is targeted by therapeutic antibodies such as Erbitux and by chemical tyrokinase inhibitors such as erlotinib. Anti-EGFR Nanobodies were evaluated as tracers for imaging of EGFR-positive xenografted tumors in mice. In a first hallmark study we showed the potential of the anti-EGFR nanobody 7C12 to target specifically the EGFR in cancer in vivo and demonstrated its applicability for fast non-invasive molecular imaging: 99mTc-labeled Nanobodies were cleared very fast out of blood and non-target organs, and accumulated in A431 EGFR-positive tumors but not in EGFR-negative R1M tumors with high tumor-to-organ ratios (Figure 2). This allowed us to visualize tumors with high contrast on SPECT/CT images within one hour after injection. Comparing two Nanobodies (7C12 and 7D12) that differ only in a few amino acids we showed that these small modifications can result in measurable differences in the general biodistribution and tumor targeting. In a next study we demonstrated that the anti-EGFR nanobody was useful to monitor tumor regression when mice were under therapy with erlotinib.

Figure 2. Transverse, coronal, and sagittal views of fused pinhole SPECT and micro-CT images of mice bearing A431 and R1M xenografts. A431 (A) and R1M (C) tumors injected with 99mTc- 7C12. A431 (B) and R1M (D) tumors injected with 99mTc-7D12. Images were acquired at 1 h after injection. NIH white color scale is used, and images are equally scaled down to 10% relative to maximum activity in image.

Related references:

Correlation between epidermal growth factor receptor-specific nanobody uptake and tumor burden: a tool for noninvasive monitoring of tumor response to therapy. Gainkam LO, Keyaerts M, Caveliers V, Devoogdt N, Vanhove C, Van Grunsven L, Muyldermans S, Lahoutte T. Mol Imaging Biol. 2011 Oct;13(5):940-8.

Comparison of the biodistribution and tumor targeting of two 99mTc-labeled anti-EGFR Nanobodies in mice, using pinhole SPECT/micro-CT. Gainkam LO, Huang L, Caveliers V, Keyaerts M, Hernot S, Vaneycken I, Vanhove C, Revets H, De Baetselier P, Lahoutte T. J Nucl Med. 2008 May;49(5):788-95

SPECT imaging with 99mTc-labeled EGFR-specific nanobody for in vivo monitoring of EGFR expression. Huang L, Gainkam LO, Caveliers V, Vanhove C, Keyaerts M, De Baetselier P, Bossuyt A, Revets H, Lahoutte T. Mol Imaging Biol. 2008 May-Jun;10(3):167-75

Localization, mechanism and reduction of renal retention of technetium-99m labeled epidermal growth factor receptor-specific nanobody in mice. Gainkam LO, Caveliers V, Devoogdt N, Vanhove C, Xavier C, Boerman O, Muyldermans S, Bossuyt A, Lahoutte T. Contrast Media Mol Imaging. 2011 Mar-Apr;6(2):85-92

4.1.2 Imaging CEA expression Carcinoembryonic antigen (CEA) is a biomarker for multiple cancer types including colon cancer. It is the target of the 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 successfully used to image CEA-positive xenografted tumors. Although Nanobodies are considered inherently low- immunogenic, a model anti-CEA nanobody was used to show the feasibility of CDR-grafting to generate maximally-humanized nanobodies with equal potential for molecular imaging (Figure 3).

Figure 3. Representative fused pinhole SPECT/micro-CT images 1 h after injection of intravenously injected 99mTc-labeled Nanobodies in xenografted mice show high uptake of both 99mTc-NbCEA5 and 99mTc-humanized CEA5 graft in CEA-positive LS174T tumors. Tumor uptake of 99mTc-humanized scaffold was low.

Related references:

In vitro analysis and in vivo tumor targeting of a humanized, grafted nanobody in mice using pinhole SPECT/micro-CT. Vaneycken I, Govaert J, Vincke C, Caveliers V, Lahoutte T, De Baetselier P, Raes G, Bossuyt A, Muyldermans S, Devoogdt N. J Nucl Med. 2010 Jul;51(7):1099-106

4.1.3 Imaging HER 2 expression 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. All 99mTc- labeled Nanobodies were shown to target HER2-positive (SKOV3) but not HER2-negative (MDA- MB-435D) tumors in xenografted mice via dissection analysis and SPECT/CT imaging studies (Figure 4), and in vitro HER2-binding parameters were compared in function of in vivo tumor targeting potential. As such, a lead nanobody was obtained with nanomolar affinities, noncompetitive binding with Trastuzumab, optimal tumor targeting and low aspecific uptake in other tissues.

Figure 4. 99mTc-labeled anti-HER2 nanobody targeting tumors in mouse xenograft models, 1h p.i. The human cancer cell lines SKOV3 (HER2-positive) and MDA-MB-435D (HER2-negative) were injected subcutaneously in the right hind limb.

A lead anti-HER2 nanobody was selected, and radiochemical procedures were optimized for radiolabeling with 68Ga and a first-in human clinical trial is ongoing in our hospital to evaluate the safety, dosimetry and tumor-targeting potential of the 68Ga-NOTA-anti-HER2 radiotracer for PET-imaging in breast cancer patients (Figure 5).

Figure 5. PET/CT image of a patient with breast cancer. The image shows focal uptake of 68Ga- NOTA-anti-HER2 in the primary breast lesion (HER2 positive) at 90 min post injection.

Related references:

Preclinical screening of anti-HER2 Nanobodies for molecular imaging of breast cancer. Vaneycken I, Devoogdt N, Van Gassen N, Vincke C, Xavier C, Wernery U, Muyldermans S, Lahoutte T, Caveliers V. FASEB J. 2011 Jul;25(7):2433-46

Synthesis, Preclinical Validation, Dosimetry, and Toxicity of 68Ga-NOTA-Anti-HER2 Nanobodies for iPET Imaging of HER2 Receptor Expression in Cancer. Xavier C, Vaneycken I, D'huyvetter M, Heemskerk J, Keyaerts M, Vincke C, Devoogdt N, Muyldermans S, Lahoutte T, Caveliers V. J Nucl Med. 2013 Mar 13, Epub

4.1.4 Radionuclide therapy using Nanobodies The ideal vector molecule for the development of radiopharmaceuticals for radioimmunotherapy should be stable, show fast and specific targeting and the unbound fraction should be cleared rapidly from the circulation. Nanobodies could very well be a protein-format that meets these requirements. The main limitation for this application is the high kidney retention of radiolabeled Nanobodies. However, we have found that the kidney retention is highly dependent on the nanobody format, the radionuclide and the chemistry used to link the radionuclide to the Nanobody. In our initial experiments we evaluated anti-HER2 Nanobodies coupled to 177Lu. We could demonstrate successful labeling of anti-HER2 nanobody labeled with 177Lu and high and sustained tumor uptake up to 48 h p.i. The first therapy experiments are currently ongoing and the preliminary results show a therapeutic effect with no toxicity in preclinical animal models (unpublished results). A second strategy is to label with radio-iodine. This part of the research is performed in collaboration with the radiochemistry lab of prof. Michael Zalutsky at Duke University and SCK- CEN. Successful labeling could be obtained, but kidney retention remains high with the current chemistry method. We are now investigating alternative radiochemistry methods that could result in less kidney retention. We are also actively investigating the renal mechanisms that are responsible for this retention. In one study we observed that renal retention of an EGFR- targeting, 99mTc-labeled nanobody is reduced in the absence of the megalin-receptor, one of the receptors 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 (Figure 6).

Figure 6. Pinhole SPECT/microCT images of megalin-wildtype (A) and megalin-knockout mice (B) at 1 h p.i. of 99mTc-7C12. NIH color scale is used and images are equally scaled to correct for injected activity. In the megalin-knockout there is a dramatic decrease in kidney retention demonstrating that megalin receptor is a major mechanism of Nanobody renal retention.

Related publications

Targeting breast carcinoma with radioiodinated anti-HER2 Nanobody. Pruszynski M, Koumarianou E, Vaidyanathan G, Revets H, Devoogdt N, Lahoutte T, Zalutsky MR. Nucl Med Biol. 2013 Jan;40(1):52-9

Development of 177Lu-Nanobodies for radioimmunotherapy of HER2-positive breast cancer: evaluation of different bifunctional chelators. D'Huyvetter M, Aerts A, Xavier C, Vaneycken I, Devoogdt N, Gijs M, Impens N, Baatout S, Ponsard B, Muyldermans S, Caveliers V, Lahoutte T. Contrast Media Mol Imaging. 2012 Mar-Apr;7(2):254-64

4.2 Imaging inflammation with Nanobodies

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, they also represent potential in vivo sensors for the status of the immune system. One line of our research aims to validate 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 to image 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.

4.2.1 Imaging myeloid cells In a first proof-of-concept study, Nanobodies were generated to recognize particular M&D subsets (collaboration with Prof De Baetselier, CMIM, VUB). 99mTc-labeled Nanobodies were used in SPECT/CT imaging studies to visualize targeted cells in naive mice, hence revealing their natural distribution in various organs. Using CDR grafting we showed the specificity of targeting by these Nanobodies (Figure 7).

Figure 7. MicroSPECT/CT images of a non-targeting Nanobody (left), an iDC specific nanobody 1.8 (middle, top), a pan-myeloid targeting Nanobody (middle, bottom). The images on the right show the data of the Nanobody scaffold grafted with the CDR loops of Nanobody 1.8 (top) and Nanobody 2.1 (bottom). For Nanobody 1.8 the lung targeting can be transplanted onto the scaffold and for Nanobody 2.1 the liver targeting is transferred due to the CDR grafting that confirms the specificity of the targeting.

Related publications

Nanobodies as tools for in vivo imaging of specific immune cell types. De Groeve K, Deschacht N, De Koninck C, Caveliers V, Lahoutte T, Devoogdt N, Muyldermans S, De Baetselier P, Raes G. J Nucl Med. 2010 May;51(5):782-9

Novel applications of Nanobodies for in vivo bio-imaging of inflamed tissues in inflammatory diseases and cancer. Schoonooghe S, Laoui D, Van Ginderachter JA, Devoogdt N, Lahoutte T, De Baetselier P, Raes G. Immunobiology. 2012 Dec;217(12):1266-72

4.2.2 Imaging alternatively-activated macrophages in oncology

Alternatively-activated macrophages are of the anti-inflammatory, 'healing' type. Under pathological conditions, they counterbalance inflammatory responses and stimulate tissue regeneration in an attempt to return to homeostasis. A particular subset of such alternatively- activated macrophages express a cell-surface protein called 'Macrophage Mannose Receptor, MMR'. We decided to generate anti-MMR Nanobodies to visualize these cells in imaging studies. In comparative biodistribution studies in naive wild-type and MMR-deficient mice we could first convincingly show that radiolabeled anti-MMR-Nanobodies specifically target resident macrophages in liver, spleen, lymph nodes and bone marrow (Figure 8).

Figure 8. MicroSPECT/CT images of a Wild Type (left coronal and transverse panel) and a MMR KnockOut (KO) mouse (right coronal and transverse panel) injected with 99mTc-labeled anti-MMR Nanobody. The KO mouse shows no tracer accumulation in the liver or lymph nodes confirming the specificity of the tracer uptake in the WT mouse.

In collaboration with professors Van Ginderachter and Raes, we observed that MMR+ macrophages are abundant in several tumors grown in syngeneic mice, where they exert pro- angiogenic and immunosuppressive stimuli. 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. Upon 99mTc labeling, anti-MMR Nanobodies specifically targeted these tumor-infiltrating macrophages, besides the resident macrophages in immunological

organs. Co-injection of the tracer with an excess of an unlabeled bigger and higher-affinity 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 (Figure 9).

Figure 9. MicroSPECT/CT images of a WT mouse injected with 99mTc-labeled anti-MMR Nanobody and unlabeled bivalent anti-MMR nanobody. Intense tumor uptake can be observed while other organs and tissues so no uptake expect for the kidneys (renal elimination). The uptake in normal organs is blocked by the unlabeled bivalent anti-MMR nanobody, whereas tumor accumulation remains unchanged.

Our next goal is to proceed a nanobody-based anti-MMR tracer for clinical applications. We recently generated new Nanobodies recognizing both mouse and human MMR and selected a lead compound. In collaboration with Prof Guy Bormans (KULeuven) we are currently further optimizing conditions to label the lead nanobody with 18F, aiming to generate a novel type of tumor hypoxia PET tracer.

Related publication and patent

Nanobody-based targeting of the macrophage mannose receptor for effective in vivo imaging of tumor-associated macrophages. Movahedi K, Schoonooghe S, Laoui D, Houbracken I, Waelput W, Breckpot K, Bouwens L, Lahoutte T, De Baetselier P, Raes G, Devoogdt N, Van Ginderachter JA. Cancer Res. 2012 Aug 15;72(16):4165-77

Anti-Macrophage Mannose Receptor single variable domains for targeting and in vivo imaging of tumor-associated macrophages”, 2013, no. PCT/EP2013/ 055427. Patent

4.2.2 Imaging alternatively-activated macrophages in arthritis

In collaboration with the group headed by professor Matthijs at the KULeuven we also observed that MMR+ macrophages accumulate in the synovial fluid of joints of arthritic mice in an attempt to soothe the ongoing inflammatory reaction. High-resolution SPECT/CT imaging studies with radiolabeled anti-MMR Nanobodies showed intense uptake of the tracer in arthritic knees, ankles and metatarsal joints (Figure 11).

Figure 11. In vivo imaging with MMR-specific Nanobodies visualizes MMR expression in arthritic joints. SPECT imaging was performed after micro-CT scans. Representative images of mice obtained at 3 h after intravenous injection of 99mTc-labeled MMR-specific Nanobodies or BCII10 control Nanobodies are shown (n = 30 mice). (A) Representative SPECT/micro-CT images of immunized mice that did not demonstrate clinical symptoms of arthritis (asymptomatic) and were injected with labeled MMR Nanobody or BCII10 control Nanobody. Note presence of specific MMR staining in lymph nodes, liver, spleen (arrows), and tail base (arrowhead). Kidneys and bladder showed aspecific signal in each image due to elimination of Nanobodies via this route. (B) Representative images of mice that did display clinical signs of arthritis in both hind limbs (symptomatic) and were injected with labeled MMR Nanobody or BCII10 control Nanobody. As in asymptomatic mice, a signal was apparent in lymph nodes, liver, spleen, and tail base. Additionally, MMR staining was evident in knees, ankles, and metatarsal joints (arrows).

Related publication SPECT Imaging of Joint Inflammation with Nanobodies Targeting the Macrophage Mannose Receptor in a Mouse Model for Rheumatoid Arthritis. Put S, Schoonooghe S, Devoogdt N, Schurgers E, Avau A, Mitera T, D'Huyvetter M, De Baetselier P, Raes G, Lahoutte T, Matthys P. J Nucl Med. 2013 Feb 27. Epub

4.3 Imaging the vulnerable atherosclerotic plaque 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 a team at 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 (Figure 12) and was able to detect plaques on the images. Besides plaques in the aortas, the tracer also visualized VCAM1-positive lymphoid tissues.

Figure 12. Lesion to control ratio of anti-VCAM1 Nanobody uptake in atherosclerotic plaque compared to the ratio obtained with a control Nanobody. The insert show a histological slice with corresponding autoradiography.

In a follow-up study, the lead anti-VCAM1 Nanobody was site-specifically conjugated to biotin and coupled to microbubbles (Figure 13). Besides showing functionality in vitro, nanobody- functionalized microbubbles enable target-visualization via echography.

Figure 13. Schematic of anti-VCAM1 Nanobody conjugated to biotin and coupled to a microbubble.

We are currently generating and evaluating addtional Nanobodies for atherosclerosis imaging, including Nanobodies targeting the Oxidized LDL-receptor LOX1 and other macrophage markers such as MMR and VSIG4.

Related publications and patents

Nanobodies targeting mouse/human VCAM1 for the nuclear imaging of atherosclerotic lesions. Broisat A, Hernot S, Toczek J, De Vos J, Riou LM, Martin S, Ahmadi M, Thielens N, Wernery U, Caveliers V, Muyldermans S, Lahoutte T, Fagret D, Ghezzi C, Devoogdt N. Circ Res. 2012 Mar 30;110(7):927-37.

Nanobody-coupled microbubbles as novel molecular tracer. Hernot S, Unnikrishnan S, Du Z, Shevchenko T, Cosyns B, Broisat A, Toczek J, Caveliers V, Muyldermans S, Lahoutte T, Klibanov AL, Devoogdt N. J Control Release. 2012 Mar 10;158(2):346-53.

Patent application: "Anti-VCAM1 Nanobodies" submitted August 2012, no WO2013/026878A1.

Patent application:"Anti-macrophage mannose receptor single variable domains for targeting and in vivo imaging of vulnerable atherosclerotic plaques" submitted March 2013.

5. Conclusion and future perspectives Molecular imaging using Nanobodies has moved from an idea during an interdisciplinary brainstorming session, to an intense research activity at the lab bench and now into a Phase I clinical trial in breast cancer patients. The fact that the research moved from bench to bedside is very exciting and the results of the first completed clinical trial are eagerly awaited. A second clinical trial aimed at imaging alternatively activated macrophages and hypoxia is currently in preparation. On the other hand the imaging method is also of interest for basic science investigations in animal models of disease in combination with small animal SPECT or PET.

Over the years we have become confident that the imaging method with Nanobodies is a generic method than can be applied to a large number of molecular imaging applications. With the publication of our research results there is a growing interest from other researchers in EU and USA to use our methods and start collaborative projects with our group. We expect that these interactions will bring new ideas and opportunities for our research. More in particular we plan to explore more in detail the use of Nanobodies coupled to therapeutic radionuclides for radionuclide therapy.