Showing papers by "Serge Muyldermans published in 2016"

Targeted alpha therapy using short-lived alpha-particles and the promise of nanobodies as targeting vehicle

Abstract: Introduction: The combination of a targeted biomolecule that specifically defines the target and a radionuclide that delivers a cytotoxic payload offers a specific way to destroy cancer cells. Targeted radionuclide therapy (TRNT) aims to deliver cytotoxic radiation to cancer cells and causes minimal toxicity to surrounding healthy tissues. Recent advances using α-particle radiation emphasizes their potential to generate radiation in a highly localized and toxic manner because of their high level of ionization and short range in tissue.Areas covered: We review the importance of targeted alpha therapy (TAT) and focus on nanobodies as potential beneficial vehicles. In recent years, nanobodies have been evaluated intensively as unique antigen-specific vehicles for molecular imaging and TRNT.Expert opinion: We expect that the efficient targeting capacity and fast clearance of nanobodies offer a high potential for TAT. More particularly, we argue that the nanobodies’ pharmacokinetic properties match per...

Sortase A-mediated site-specific labeling of camelid single-domain antibody-fragments: a versatile strategy for multiple molecular imaging modalities.

Abstract: A generic site-specific conjugation method that generates a homogeneous product is of utmost importance in tracer development for molecular imaging and therapy. We explored the protein-ligation capacity of the enzyme Sortase A to label camelid single-domain antibody-fragments, also known as nanobodies. The versatility of the approach was demonstrated by conjugating independently three different imaging probes: the chelating agents CHX-A"-DTPA and NOTA for single-photon emission computed tomography (SPECT) with indium-111 and positron emission tomography (PET) with gallium-68, respectively, and the fluorescent dye Cy5 for fluorescence reflectance imaging (FRI). After a straightforward purification process, homogeneous single-conjugated tracer populations were obtained in high yield (30-50%). The enzymatic conjugation did not affect the affinity of the tracers, nor the radiolabeling efficiency or spectral characteristics. In vivo, the tracers enabled the visualization of human epidermal growth factor receptor 2 (HER2) expressing BT474M1-tumors with high contrast and specificity as soon as 1 h post injection in all three imaging modalities. These data demonstrate Sortase A-mediated conjugation as a valuable strategy for the development of site-specifically labeled camelid single-domain antibody-fragments for use in multiple molecular imaging modalities. Copyright © 2016 John Wiley & Sons, Ltd.

Individual aggregates of amyloid beta induce temporary calcium influx through the cell membrane of neuronal cells.

Abstract: Local delivery of amyloid beta oligomers from the tip of a nanopipette, controlled over the cell surface, has been used to deliver physiological picomolar oligomer concentrations to primary astrocytes or neurons. Calcium influx was observed when as few as 2000 oligomers were delivered to the cell surface. When the dosing of oligomers was stopped the intracellular calcium returned to basal levels or below. Calcium influx was prevented by the presence in the pipette of the extracellular chaperone clusterin, which is known to selectively bind oligomers, and by the presence a specific nanobody to amyloid beta. These data are consistent with individual oligomers larger than trimers inducing calcium entry as they cross the cell membrane, a result supported by imaging experiments in bilayers, and suggest that the initial molecular event that leads to neuronal damage does not involve any cellular receptors, in contrast to work performed at much higher oligomer concentrations.

Emerging site-specific bioconjugation strategies for radioimmunotracer development

Abstract: Introduction: Radioimmunotracers are a promising class of companion diagnostics for precision medicine. They are composed of an antibody-based targeting agent and a radiolabeled imaging probe. Together with the tendency towards the use of small antibody-derived fragments, the employed conjugation method is gaining increasing attention. Conventional bioconjugation methods result in heterogeneous tracer populations of which the single elements can differ in immunoreactivity, pharmacokinetic behavior and stability. Site-specific conjugation strategies try to overcome these shortcomings and facilitate radioimmunotracer delivery, characterization and manufacturing.Areas covered: An overview is provided of site-specific conjugation strategies for use in radioimmunotracer development. Currently applied strategies are discussed, together with other emerging site-specific conjugation methods that are applicable to diabodies, single-chain variable fragments (scFvs) and camelid single-domain antibody-fragmen...

An Anti-proteome Nanobody Library Approach Yields a Specific Immunoassay for Trypanosoma congolense Diagnosis Targeting Glycosomal Aldolase

Abstract: Background Infectious diseases pose a severe worldwide threat to human and livestock health. While early diagnosis could enable prompt preventive interventions, the majority of diseases are found in rural settings where basic laboratory facilities are scarce. Under such field conditions, point-of-care immunoassays provide an appropriate solution for rapid and reliable diagnosis. The limiting steps in the development of the assay are the identification of a suitable target antigen and the selection of appropriate high affinity capture and detection antibodies. To meet these challenges, we describe the development of a Nanobody (Nb)-based antigen detection assay generated from a Nb library directed against the soluble proteome of an infectious agent. In this study, Trypanosoma congolense was chosen as a model system. Methodology/Principal Findings An alpaca was vaccinated with whole-parasite soluble proteome to generate a Nb library from which the most potent T. congolense specific Nb sandwich immunoassay (Nb474H-Nb474B) was selected. First, the Nb474-homologous sandwich ELISA (Nb474-ELISA) was shown to detect experimental infections with high Positive Predictive Value (98%), Sensitivity (87%) and Specificity (94%). Second, it was demonstrated under experimental conditions that the assay serves as test-of-cure after Berenil treatment. Finally, this assay allowed target antigen identification. The latter was independently purified through immuno-capturing from (i) T. congolense soluble proteome, (ii) T. congolense secretome preparation and (iii) sera of T. congolense infected mice. Subsequent mass spectrometry analysis identified the target as T. congolense glycosomal aldolase. Conclusions/Significance The results show that glycosomal aldolase is a candidate biomarker for active T. congolense infections. In addition, and by proof-of-principle, the data demonstrate that the Nb strategy devised here offers a unique approach to both diagnostic development and target discovery that could be widely applied to other infectious diseases.

Generation and in vitro characterisation of inhibitory nanobodies towards plasminogen activator inhibitor 1

Abstract: Plasminogen activator inhibitor 1 (PAI-1) is the principal physiological inhibitor of tissue-type plasminogen activator (t-PA) and has been identified as a risk factor in cardiovascular diseases. In order to generate nanobodies against PAI-1 to interfere with its functional properties, we constructed three nanobody libraries upon immunisation of three alpacas with three different PAI-1 variants. Three panels of nanobodies were selected against these PAI-1 variants. Evaluation of the amino acid sequence identity of the complementarity determining region-3 (CDR3) reveals 34 clusters in total. Five nanobodies (VHH-s-a98, VHH-2w-64, VHH-s-a27, VHH-s-a93 and VHH-2g-42) representing five clusters exhibit inhibition towards PAI-1 activity. VHH-s-a98 and VHH-2w-64 inhibit both glycosylated and non-glycosylated PAI-1 variants through a substrate-inducing mechanism, and bind to two different regions close to αhC and the hinge region of αhF; the profibrinolytic effect of both nanobodies was confirmed using an in vitro clot lysis assay. VHH-s-a93 may inhibit PAI-1 activity by preventing the formation of the initial PAI-1t-PA complex formation and binds to the hinge region of the reactive centre loop. Epitopes of VHH-s-a27 and VHH-2g-42 could not be deduced yet. These five nanobodies interfere with PAI-1 activity through different mechanisms and merit further evaluation for the development of future profibrinolytic therapeutics.

Specificity Evaluation and Disease Monitoring in Arthritis Imaging with Complement Receptor of the Ig superfamily targeting Nanobodies

Abstract: Single-photon emission computed tomography combined with micro-CT (SPECT/μCT) imaging using Nanobodies against complement receptor of the Ig superfamily (CRIg), found on tissue macrophages such as synovial macrophages, has promising potential to visualize joint inflammation in experimental arthritis. Here, we further addressed the specificity and assessed the potential for arthritis monitoring. Signals obtained with 99mTc-labelled NbV4m119 Nanobody were compared in joints of wild type (WT) versus CRIg-/- mice with collagen-induced arthritis (CIA) or K/BxN serum transfer-induced arthritis (STIA). In addition, SPECT/μCT imaging was used to investigate arthritis development in STIA and in CIA under dexamethasone treatment. 99mTc-NbV4m119 accumulated in inflamed joints of WT, but not CRIg-/- mice with CIA and STIA. Development and spontaneous recovery of symptoms in STIA was reflected in initially increased and subsequently reduced joint accumulation of 99mTc-NbV4m119. Dexamethasone treatment of CIA mice reduced 99mTc-NbV4m119 accumulation as compared to saline control in most joints except knees. SPECT/μCT imaging with 99mTc-NbV4m119 allows specific assessment of inflammation in different arthritis models and provides complementary information to clinical scoring for quantitatively and non-invasively monitoring the pathological process and the efficacy of arthritis treatment.

Structure and Function of Camelid VHH

Abstract: The humoral immune response of camels and llama's is unique in containing two types of IgG antibodies: classical heterotetrameric and homodimeric heavy chain-only antibodies (HCAbs). Remarkably, these heavy chain antibodies are functional in antigen binding and reach high titers and affinities after immunizing a camelid. Antigen recognition by HCAbs is mediated by one single variable domain, referred to as VHH. In comparison to VHs of classical antibodies, VHHs harbor notable sequence and structural adaptations clarifying their solubility and antigen-binding capacity in absence of a VL. The genome of camelids contains a significant set of dedicated IGHGH genes and IGHVH germ line genes with VHH-sequence adaptations. During B cell maturation, one IGHVH germ line gene recombines with one IGHD and one IGHJ minigene out of a pool, to generate a VHH domain that is eventually rearranged to a dedicated IGHGH gene where the region encoding the first constant domain is removed by splicing, to produce a functional HCAb. Cloning the VHH repertoire of an immunized camelid from which to retrieve antigen-specific VHHs gives access to single-domain, nanometer-sized, antigen-binding fragments, known as nanobodies. These are employed as versatile research tools, in numerous diagnostic tests and as therapeutics.