NEPTUNS : Nano-Antibodies to Explore Protein Structure and Functions

Mireille DUMOULIN

Research Associate FRS-FNRS

Bât.B6c, Quartier Agora
allée du six Août 11
4000 Liège 1
Belgium
+32 4 3663546
+32 4 3663364

Research interest

Our research is based on the use of camelid single-domain antibody fragments (also referred to as V_HHs) as probes to investigate the structure and functions of proteins under physiological and pathological conditions. V_HHs are single-domain antibody fragments derived from the heavy-chain antibodies (HcAbs) from Camelidae. Due to their small size (˜140 aa, 15 kDa), V_HHs have a number of remarkable properties that distinguish them from conventional monoclonal antibodies and fragments of thereof. These features include: high stability and solubility, easy production in E. coli, easy modification by genetic engineering to further adapt their properties to the applications of choice and high tissue penetration. Besides that, V_HHs have the ability to target, via their long CDR3 (Complementary Determining Region 3), cryptic epitopes generally inaccessible to conventional antibodies, such as enzymes active site; consequently, they can act as very specific enzyme inhibitors. V_HHs have become powerful tools not only for fundamental research but also for imaging/diagnostic and therapeutic applications.

Our research is highly interdisciplinary and involves expertise in Biochemistry, Molecular Biology, Protein Chemistry, Protein Engineering, Protein misfolding and aggregation, Protein-protein interactions and Phage display. We have access to the equipment of the Protein Factory and Robotein platforms of CIP, for the production and purification of proteins as well as a series of techniques for their biophysical/biochemical characterisation including fluorescence, circular dichroism, SEC-MALS, DLS, DSC, ITC, Bio-Layer Interferometry, X-ray crystallography and transmission electron microscopy. We have access to murine in vivo models through our collaborators.

Research summary

Amyloid fibrils: structure and mechanism of formation

The deposition of misfolded peptides and proteins in the form of amyloid fibrils is the hallmark of nearly fifty medical disorders, including Alzheimer’s disease, Parkinson’s disease, prion diseases and type II diabetes. Neither preventing nor curative treatments are available yet for this family of diseases which constitute therefore a medical challenge of great importance. Our approach to better understand how amyloidogenic proteins convert from their soluble form into amyloid fibrils is based on the use of V_HHs as structural probes to dissect, at the molecular level, the mechanism of aggregation and inhibition of thereof. Our studies are focused on model polyglutamine proteins based on the beta-lactamase BlaP, the amyloidogenic variants of human lysozyme and alpha-synuclein.

Nanomedecine for the treatment of cystic fibrosis

Collaboration with R. Vanbever (LDRI, UCLouvain), T. Leal (LTAP, UCLouvain), S. Gohy (PNEU, UCLouvain) & S. Muyldermans (VUB)

Cystic fibrosis (CF) is a severe genetic disorder associated with the loss of function of the protein called CFTR (Cystic Fibrosis Transmembrane Conduct Regulator). CF primarily affects the respiratory system and this is associated with the great morbidity and mortality of the disease. As consequence of the CFTR defect, the airways of CF patients are obstructed by viscous secretions which are favourable to the development of infectious agents. The chronic respiratory infection results in severe inflammation and the release of a series of enzymes whose actions contribute to the misfunctioning of the lungs. We are involved in three interuniversity projects aiming at developing V_HHs-based nanomedicines inhibiting such enzymes for the treatment of the respiratory pathology in CF.

Design of long-acting and targeted nanomedicines for inhalation

Collaboration with R. Vanbever (LDRI, UCLouvain)

Although inhalation aerosols allow a targeted drug therapy in respiratory diseases, the rapid clearance of protein therapeutics from the lung implies the requirement to deliver them once daily. This imposes a significant time burden on the patients and might reduce patient compliance and the therapeutic outcome of the treatment. We are involved in an interuniversity project, aiming at developing V_HHs-based nanomedicines offering a sustained presence of the protein drug within the lung.

Design of a Nanomedecine for the diagnostic and treatment of mesothelioma

Collaboration with D. Cataldo (GIGA-Cancer) & P. Lassaux (MBPEL, GIGA-Cardiovascular Sciences)

Malignant pleural mesothelioma (MPM) is a cancer that develops from mesothelial cells lining the pleura. MPM is associated with a very poor prognosis, as many patients do not respond to available treatments. The objective of this project is to develop V_HHs-based nanomedicines targeting enzymes involved in tumour progression, tumour migration and chemoresistance in MPM.

ALPANANO: A platform to generate and select Nanobodies

In collaboration with A. Vanderplasschen (Immunology-Vaccinology, Faculty of Veterinary Medicine, ULiege), and the support of ULiege (Crédit d’Opportunités Stratégiques), we have developed a platform to generate and select V_HHs from large libraries of V_HH genes. Our expertise includes: (i) immunisation of alpaca (i.e., with protein or DNA), (ii) Generation of an immune V_HHs library from the immune blood, (iii) Panning on a recombinant protein (immobilized or in solution), (iv) Screening (ELISA or bio-layer interferometry) on the antigen. After sequencing the positive hits, the V_HHs can be produced, purified, labelled (i.e., biotinylating, fluorescent labelling, etc), engineered (i.e., construction of bivalent/bispecific Nbs, improved stability, etc) and characterized (i.e., affinity, specificity, epitope binning, stability, etc) within CIP platforms (Protein Factory and Robotein).

Lab members

Francisco Javier Morales-Yànez, FRS-FNRS post-doctorat fellow (Chargé de Recherche)
Jonathan Vaneyck, Researcher
Paola Redeghieri, PhD student, Aspirante FNRS, Promotor (Co-promotor: Rita Vanbever, UCLouvain)
Marco Morbidelli, PhD student, FRIA fellowship, Co-promotor (Promotor: Didier Cataldo, GIGA-Cancer; co-promotor: Patricia Lassaux, GIGA-Cardiovascular Sciences)
Giada Disconzi, Researcher (post-graduate Erasmus exchange)

Selected publications

Single-domain antibody fragments with high conformational stability. M. Dumoulin, K. Conrath, A. Van Meirhaeghe, F. Meersman, K. Heremans, L. G. J. Frenken, S. Muyldermans, L. Wyns, and A. Matagne. (2002) Protein Sci. 11, 500-515.
A camelid antibody fragment inhibits the formation of amyloid fibrils by human lysozyme. M. Dumoulin, A. M. Last, A. Desmyter, K. Decanniere, D. Canet, G. Larsson, A. Spencer, D. B. Archer, J. Sasse, S. Muyldermans, L. Wyns, C. Redfield, A. Matagne, C. V. Robinson and C. M. Dobson. (2003) Nature 424, 783-788.
Camelid single-domain antibody fragments: Uses and prospects to investigate protein misfolding and aggregation, and to treat diseases associated with these phenomena. Pain C, Dumont J, Dumoulin M. Biochimie, 2015, 111, 82-106.
Multimodal imaging Gd-nanoparticles functionalized with Pittsburgh compound B or a nanobody for amyloid plaques targeting. J. Pansieri, M. Plissonneau, N. Stransky-Heilkron, M. Dumoulin, L. Heinrich-Balard, P Rivory, J-F Morfin, E. Toth, M. J. Saraiva, E. Allémann, O. Tillement, V. Forge, F. Lux, C. Marquette. Nanomedecine, 2017, Nanomedicine, 12(14):1675-1687. doi: 10.2217/nnm-2017-0079.
Modeling Parkinson's Disease With the Alpha-Synuclein Protein. Gómez-Benito M, Granado N, García-Sanz P, Michel A, Dumoulin M, Moratalla R. Front Pharmacol. 2020 Apr 23;11:356. doi: 10.3389/fphar.2020.00356. eCollection 2020.
Characterisation of the structural, dynamic and aggregation properties of the W64R amyloidogenic variant of human lysozyme. N. Vettore, J. Moray, A. Brans, R. Herman, P. Charlier, J. R.Kumita, F. Kerff, C. M. Dobson and M. Dumoulin. (2021) Biophysical Chemistry, 271:106563. doi: 10.1016/j.bpc.2021.106563.
Protein Engineering Strategies for Improved Pharmacokinetics. Rondon, A., Mahri, S., Morales-Yanez, F., Dumoulin, M., Vanbever, R. Advanced Functional Materials, 2021, https://doi.org/10.1002/adfm.202101633.
A non-internalised CD38-binding radiolabelled single-domain antibody fragment to monitor and treat multiple myeloma; Duray E, Lejeune M, Baron F, Beguin Y, Devoogdt N, Krasniqi A, Lauwers Y, Zhao YJ, D'Huyvetter M, Dumoulin M, Caers J. J Hematol Oncol. 2021 Nov 2;14(1):183. doi: 10.1186/s13045-021-01171-6.
Caers Jo, Duray Elodie., Dumoulin Mireille, D'Huyvetter Mathias and Eriksson Karlström Amelie. Anti-CD38 Single-domain antibodies on disease monitoring and treatment (Patent), WO2022242892A1
Caers Jo, Duray Elodie., Dumoulin Mireille & D'Huyvetter Mathias. Anti-CD38 Single-domain antibodies on disease monitoring and treatment (Patent), WO2021229104A1

Fundings

Logo Dumoulin

updated on 4/11/24