Ulrich Wernery

Camelid immunoglobulins and nanobody technology.

It is well established that all camelids have unique antibodies circulating in their blood. Unlike antibodies from other species, these special antibodies are devoid of light chains and are composed of a heavy-chain homodimer. These so-called heavy-chain antibodies (HCAbs) are expressed after a V-D-J rearrangement and require dedicated constant gamma-genes. An immune response is raised in these so-called heavy-chain antibodies following classical immunization protocols. These HCAbs are easily purified from serum, and the antigen-binding fragment interacts with parts of the target that are less antigenic to conventional antibodies. Since the antigen-binding site of the dromedary HCAb is comprised in one single domain, referred to as variable domain of heavy chain of HCAb (VHH) or nanobody (Nb), we designed a strategy to clone the Nb repertoire of an immunized dromedary and to select the Nbs with specificity for our target antigens. The monoclonal Nbs are well produced in bacteria, are very stable and highly soluble, and bind their cognate antigen with high affinity and specificity. We have successfully developed recombinant Nbs for research purposes, as probe in biosensors, to diagnose infections, and to treat diseases like cancer or trypanosomosis.

Preclinical screening of anti-HER2 nanobodies for molecular imaging of breast cancer

Accurate determination of tumor human epidermal growth factor receptor 2 (HER2)‐status in breast cancer patients is possible via noninvasive imaging, provided adequate tracers are used. In this study, we describe the generation of a panel of 38 nanobodies, small HER2‐binding fragments that are derived from heavy‐chain‐only antibodies raised in an immunized dromedary. In search of a lead compound, a subset of nanobodies was biochemically characterized in depth and preclinically tested for use as tracers for imaging of xenografted tumors. The selected compound, 2Rs15d, was found to be stable and to interact specifically with HER2 recombinant protein and HER2‐expressing cells in ELISA, surface plasmon resonance, flow cytometry, and radioligand binding studies with low nanomolar affinities, and did not compete with anti‐HER2 therapeutic antibodies trastuzumab and pertuzumab. Single‐photon‐emission computed tomography (SPECT) imaging quantification and biodistribution analyses showed that 99mTc‐labeled 2Rs15d has a high tumor uptake in 2 HER2+ tumor models, fast blood clearance, low accumulation in nontarget organs except kidneys, and high concomitant tumor‐to‐blood and tumor‐to‐muscle ratios at 1 h after intravenous injection. These values were dramatically lower for an irrelevant control 99mTc‐nanobody and for 99mTc‐2Rs15d targeting a HER2– tumor.—Vaneycken, I., Devoogdt, N., Van Gassen, N., Vincke, C., Xavier, C., Wernery, U., Muyldermans, S., Lahoutte, T., Caveliers, V. Preclinical screening of anti‐HER2 nanobodies for molecular imaging of breast cancer. FASEB J. 25, 2433–2446 (2011). www.fasebj.org

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.

Nanobodies, a promising tool for species-specific diagnosis of Taenia solium cysticercosis

Taenia solium cysticercosis is a major helminth zoonosis in developing countries. Pigs are the intermediate hosts mediating transmission of infection. Specific assays to diagnose living cysts in pigs are lacking. The monoclonal-based antigen detection ELISA is genus-specific and cross-reactions with Taenia hydatigena hamper the use of this test to screen pigs. We, therefore, aimed to introduce nanobodies, camelid-derived single-domain antibodies specific for T. solium cysticercosis, to develop unambiguous tests. Nanobodies were cloned following immunization of two dromedaries with T. solium antigen and eight T. solium-specific nanobodies were selected after phage display. Their binding characteristics and potential for the diagnosis of porcine cysticercosis were investigated. The nanobodies do not cross-react with T. hydatigena, Taenia saginata, Taenia crassiceps or Trichinella spiralis and were categorized into four epitope-binding groups. The target protein was identified as 14kDa diagnostic glycoprotein (Ts14), but the nanobodies also reacted with other proteins of the same family. Nanobodies were tested in a sandwich ELISA with cyst fluid, and one particular nanobody detected its cognate serum antigens in a species-specific inhibition ELISA. Considering their beneficial production and stability properties, these highly specific nanobodies constitute a promising tool to diagnose cysticercosis after further improvement of the sensitivity and future assay validation.

Generation of Single Domain Antibody Fragments Derived from Camelids and Generation of Manifold Constructs

Immunizing a camelid (camels and llamas) with soluble, properly folded proteins raises an affinity-matured immune response in the unique camelid heavy-chain only antibodies (HCAbs). The peripheral blood lymphocytes of the immunized animal are used to clone the antigen-binding antibody fragment from the HCAbs in a phage display vector. A representative aliquot of the library of these antigen-binding fragments is used to retrieve single domain antigen-specific binders by successive rounds of panning. These single domain antibody fragments are cloned in tandem to generate manifold constructs (bivalent, biparatopic or bispecific constructs) to increase their functional affinity, to increase specificity, or to connect two independent antigen molecules.

Surface display of a single-domain antibody library on Gram-positive bacteria

Combinatorial protein engineering for selection of proteins with novel functions, such as enzymes and affinity reagents, is an important tool in biotechnology, drug discovery, and other biochemical fields. Bacterial display is an emerging technology for isolation of new affinity proteins from such combinatorial libraries. Cells have certain properties that are attractive for directed evolution purposes, in particular the option to use quantitative flow-cytometric cell sorting for selection of binders. Here, an immune library of around 107 camelid single-domain antibody fragments (Nanobodies) was displayed on both the Gram-positive bacterium Staphylococcus carnosus and on phage. As demonstrated for the first time, the antibody repertoire was found to be well expressed on the bacterial surface and flow-cytometric sorting yielded a number of Nanobodies with subnanomolar affinity for the target protein, green fluorescent protein (GFP). Interestingly, the staphylococcal output repertoire and the binders from the phage display selection contained two slightly different sets of clones, containing both unique as well as several similar variants. All of the Nanobodies from the staphylococcal selection were also shown to enhance the fluorescence of GFP upon binding, potentially due to the fluorescence-based sorting principle. Our study highlights the impact of the chosen display technology on the variety of selected binders and thus the value of having alternative methods available, and demonstrates in addition that the staphylococcal system is suitable for generation of high-affinity antibody fragments.

In vitro antiviral activity of single domain antibody fragments against poliovirus.

VHHs or Nanobodies are single-domain antigen-binding fragments derived from heavy chain antibodies found in camelids. It has already been shown that complex protein mixtures and even whole organisms elicit good immune responses in camelids; therefore we hypothesized that VHHs selected from a dromedary immunized with poliovirus type 1 might inhibit the in vitro replication of poliovirus through binding to essential biological sites on the viral capsid. In this study, we aimed to determine whether VHHs inhibit wild-type and vaccine strains of poliovirus type 1. Interestingly, VHHs showed a potent antipolio activity with EC50 values in the low nanomolar range. Moreover, these antibody fragments completely blocked viral multiplication at higher concentrations. Remarkably, no (immune) escape variants against some of these VHHs could be generated. In conclusion, VHHs fulfil several in vitro requirements to be assigned as potential antiviral compounds for further development of an anti-poliovirus drugs.

Imaging and radioimmunotherapy of multiple myeloma with anti-idiotypic Nanobodies

Multiple myeloma (MM) is characterized by the monoclonal expansion of malignant plasma cells in the bone marrow (BM) and the production of monoclonal protein (M-protein). With the implementation of autologous stem cell transplantation and highdose chemotherapy using dexamethasone, bortezomib, thalidomide and lenalidomide, the survival rate has improved but MM patients still relapse, even if they achieve complete remission (CR). Therefore, new therapeutic strategies are needed to target residual malignant cells and eliminate minimal residual disease (MRD) in order to improve patient outcome. Nanobodies are the smallest (15 kDa) antigen-binding fragments derived from camelid heavy-chain-only antibodies. Because of their biochemical characteristics including high stability, solubility and target affinity, they are ideal therapeutic and diagnostic tools. Nanobodies can also recognize epitopes that remain undetected by conventional antibodies. Previous work has already demonstrated that Nanobody conjugates are able to reach, bind and kill cancer cells. Moreover, Nanobodies conjugated with radionuclides have been successfully used with the single-positron emission tomography (SPECT) technology combined with micro-computed tomography (micro-CT) for imaging purposes. Despite the extensive Nanobody-based research, there is so far only little evidence about their potential for diagnostic and/or therapeutic applications in MM. Here, we take advantage of the M-protein present in the murine 5T2MM model to prove the potential use of Nanobodies in MM. The 5TMM models are syngeneic, immunocompetent models that resemble human MM clinically and biologically. The best characterized are the 5T33MM and the 5T2MM models. The former represents an aggressive tumor, which develops in a short period of time (4 weeks), whereas the latter represents a more moderate tumor that develops over a period of 3 months. Both express different idiotypes (5T33MMid and 5T2MMid, respectively) on the cell membrane surface. By immunization of a dromedary with purified 5T2MM M-protein and a simple selection method (see Supplementary Materials and Methods), we were able to select, produce and purify a panel of very specific anti-5T2MM-idiotype Nanobodies (a5T2MMid-Nbs) that recognize nearby epitopes on the idiotype (Supplementary Figure 1). After in vitro characterization of these Nanobodies, R3B23 came up as the better binder (see Supplementary Figures 2, 3 and 4) and was therefore selected for in vivo testing. R3B23 was labeled with radionuclides Technetium (Tc) and Lutetium (Lu) using previous established protocols. Tc (half-life: 6 h) is used in SPECT for nuclear medicine imaging techniques, whereas Lu (half-life: 6.7 days) is mainly used for therapeutic applications due to the emission of low-energy b-minus particles. Several radiotherapeutic monoclonal antibodies (MoAbs) are currently under (pre)clinical evaluation in cancers, including B-cell disorders. In MM MRD, which is characterized by a small tumor load in BM, radiotherapeutic MoAbs might not be ideal as radiotoxicity levels to healthy tissues are high, providing a rationale for the use of highly specific and rapidly cleared radiotherapeutic Nanobodies instead. First, we studied the specificity of R3B23 in vivo. At 1 h post injection (p.i.), anesthetized mice were imaged using pinhole SPECT and micro-CT, as described previously. At 30 min after imaging, the mice were killed, different organs were removed, weighed and the radioactivity was measured. Fused SPECT/microCT images obtained from naive mice with nontargeting control Nanobody Tc-cAbBCII10 showed tracer uptake only in the bladder and kidneys (Figure 1a). Biodistribution experiments (Figure 1d) confirmed a high tracer uptake in both kidneys (4200%IA/g) and only marginal levels of uptake in other organs (ranging from 0.20±0.04%IA/g in muscle tissue to 1.02±0.26%IA/g in lungs) as expected for unbound tracers that are eliminated from the body through renal filtration. Importantly, similar results were observed in naive mice injected with Tc-R3B23 (Figures 1e and h) indicating that R3B23 does not bind to circulating immunoglobulins or other in vivo targets. SPECT/micro-CT scan images and biodistribution studies of terminally diseased 5T33MM mice injected with either Tc-cAbBCII10 (Figures 1b and d) or Tc-R3B23 (Figures 1f and h) and 5T2MM mice injected with Tc-cAbBcII10 (Figures 1c and d) showed analogous patterns with a high tracer uptake in kidneys and bladder and a low uptake in all other organs, demonstrating that MM disease does not influence Nanobody uptake. SPECT/micro-CT scan images of 5T2MM mice injected with Tc-R3B23 (Figure 1g) revealed a systemic tracer uptake, which was confirmed by biodistribution studies (Figure 1h). The up to 100-fold-elevated tracer levels in blood (44.56±2.54%IA/g) can be attributed to binding of the antiidiotypic Nanobody to the high levels of circulating M-protein in this late-stage disease model. The elevated tracer blood-pool activity accounts for the decreased uptake observed in the kidneys (circa 8%IA/g) and is responsible for the elevated uptake in other organs (ranging from 0.91±0.04%IA/g in the muscle to 11.63±2.35%IA/g in the lungs). It is noteworthy that the in vivo increase in circulating M-protein can be monitored over time using SPECT/micro-CT scans with Tc-R3B23 (Supplementary Figure 5). In conclusion, the biodistribution studies demonstrate that R3B23 does not bind to any target in healthy mice or to M-protein with a different idiotype, and it is therefore truly antiidiotypic. Finally, we evaluated the effect of Nanobody R3B23 conjugated with Lutetium (Lu-R3B23) on tumor growth. One week after inoculation of 5T2MM cells into naive mice, we started weekly treatments with intravenously administrated Lu-R3B23 or negative control Lu-cAbBCII10. After 5 weeks of treatment, animals were imaged using SPECT/micro-CT scans with TcR3B23 (Figure 2). On the basis of micro-CT images, an ellipsoid region of interest was drawn around the heart. Tracer uptake in heart, as a measurement of blood-pool activity, is expressed as the counts in the tissue divided by the injected activity/cubic centimeter (%IA/cm). The %IA/cm detected in the heart of mice treated with Lu-R3B23 (5.55±1.42) was significantly lower than the values measured in untreated mice (10.03±0.27; Po0.005) and mice treated with control Nanobody Lu-cAbBcII10 (9.19±0.84; Po0.05). The lower blood value of Tc-R3B23 uptake in mice treated with Lu-R3B23 is not due to in vivo competition with the therapeutic Lu-labeled Nanobody, as the latter was already systemically cleared. Indeed, the 10 mg Tc-R3B23 injection and subsequent SPECT/micro-CT scanning were performed 5 days after injection with 10 mg Lu-Nanobody.

Pathological studies on camelpox lesions of the respiratory system in the United Arab Emirates (UAE)

Three dromedary camels (Camelus dromedarius), which died from generalized camelpox with lesions in the respiratory system, were investigated. Histopathological lesions in the lung consisted of small, sometimes confluent foci of proliferated bronchial epithelium, necrosis and fibrosis. Orthopoxvirus cameli was demonstrated in all three cases by transmission electron microscopy and the virus was isolated from the lung and trachea on Dubca cells. It was proved by restriction enzyme analysis of the viral DNA that the isolates were identical. Immunohistochemical examination showed numerous poxvirus antigen-positive cells in the bronchial epithelia. Immunolabelled material was found in bronchial epithelial cells with hydropic degeneration and in infiltrating macrophages.

Specific Targeting of Atherosclerotic Plaques in ApoE / Mice Using a New Camelid sdAb Binding the Vulnerable Plaque Marker LOX-1

PurposeMolecular imaging has the potential to provide quantitative information about specific biological aspects of developing atherosclerotic lesions. This requires the generation of reliable, highly specific plaque tracers. This study reports a new camelid single-domain antibody fragment (sdAb) targeting the Lectin-like oxidized low-density lipoprotein receptor (LOX-1), a biomarker for the detection and molecular phenotyping of vulnerable atherosclerotic plaques.ProceduresA camelid sdAb was generated and selected for high affinity binding to LOX-1. Ex vivo biodistribution and in vivo single photon emission computed tomography (SPECT)/computed tomography (CT) imaging studies were performed in wild-type mice and in fat-fed atherosclerotic apolipoprotein E-deficient mice with 99mTc-labeled sdAbs. Gamma-counting and autoradiography analyses were performed on dissected aorta segments with different degrees of plaque burden. The specificity of the LOX-1-targeting sdAb was evaluated by blocking with unlabeled sdAb or by comparison with a nontargeting 99mTc-labeled control sdAb.ResultsWe generated a sdAb binding LOX-1 with a KD of 280xa0pMu2009±u200962xa0pM affinity. After 99mTc-labeling, the tracer had radiochemical purity higher then 99xa0% and retained specificity in in vitro binding studies. Tracer blood clearance was fast with concomitant high kidney retention. At 3xa0h after injection, uptake in tissues other than plaques was low and not different than background, suggesting a restricted expression pattern of LOX-1. Conversely, uptake in aortic segments increased with plaque content and was due to specific LOX-1 binding. In vivo SPECT/CT imaging 160xa0min after injection in atherosclerotic mice confirmed specific targeting of LOX-1-expressing aortic plaques.ConclusionsThe LOX-sdAb specifically targets LOX-1-expressing atherosclerotic plaques within hours after injection. The possibility to image LOX-1 rapidly after administration combined with the favourable biodistribution of a sdAb are beneficial for molecular phenotyping of atherosclerotic plaques and the generation of a future prognostic tracer.