Development of Nanobodies as Theranostic Agents against CMY-2-Like Class C β-Lactamases

Frédéric Cawez; Paola Sandra Mercuri; Francisco Javier Morales-Yãnez; Rita Maalouf; Marylène Vandevenne; Frederic Kerff; Virginie Guérin; Jacques Mainil; Damien Thiry; Marc Saulmont; Alain Vanderplasschen; Pierre Lafaye; Gabriel Aymé; Pierre Bogaerts; Mireille Dumoulin; Moreno Galleni

doi:10.1128/aac.01499-22

Development of Nanobodies as Theranostic Agents against CMY-2-Like Class C β-Lactamases

Antimicrob Agents Chemother. 2023 Apr 18;67(4):e0149922. doi: 10.1128/aac.01499-22. Epub 2023 Mar 9.

Authors

Frédéric Cawez¹, Paola Sandra Mercuri¹, Francisco Javier Morales-Yãnez^{2

3}, Rita Maalouf², Marylène Vandevenne⁴, Frederic Kerff⁵, Virginie Guérin⁶, Jacques Mainil⁶, Damien Thiry⁶, Marc Saulmont⁷, Alain Vanderplasschen^{3

8}, Pierre Lafaye⁹, Gabriel Aymé⁹, Pierre Bogaerts¹⁰, Mireille Dumoulin^{2

3}, Moreno Galleni¹

Affiliations

¹ InBioS, Center for Protein Engineering, Biological Macromolecules, Department of Life Sciences, University of Liège, Liège, Belgium.
² InBioS, Center for Protein Engineering, NEPTUNS, Department of Life Sciences, University of Liège, Liège, Belgium.
³ ALPANANO, Center for Protein Engineering & FARAH, University of Liège, Liège, Belgium.
⁴ InBios, Center for Protein Engineering, ROBOTEIN, Department of Life Sciences, University of Liège, Liège, Belgium.
⁵ InBioS, Center for Protein Engineering, Department of Life Sciences, University of Liège, Liège, Belgium.
⁶ Bacteriology, FARAH and Faculty of Veterinary Medicine, Department of Infectious and Parasitic Diseases, University of Liège, Liège, Belgium.
⁷ Regional Animal Health and Identification Association (ARSIA), Ciney, Belgium.
⁸ Immunology-Vaccinology, FARAH and Faculty of Veterinary Medicine, Department of Infectious and Parasitic Diseases, University of Liège, Liège, Belgium.
⁹ Institut Pasteur, Université Paris Cité, CNRS UMR 328, Paris, France.
¹⁰ National Reference Center for Antibiotic-Resistant Gram-Negative Bacilli, Department of Clinical Microbiology, CHU UCL Namur, Yvoir, Belgium.

Abstract

Three soluble single-domain fragments derived from the unique variable region of camelid heavy-chain antibodies (VHHs) against the CMY-2 β-lactamase behaved as inhibitors. The structure of the complex VHH cAb_CMY-2(254)/CMY-2 showed that the epitope is close to the active site and that the CDR3 of the VHH protrudes into the catalytic site. The β-lactamase inhibition pattern followed a mixed profile with a predominant noncompetitive component. The three isolated VHHs recognized overlapping epitopes since they behaved as competitive binders. Our study identified a binding site that can be targeted by a new class of β-lactamase inhibitors designed on the sequence of the paratope. Furthermore, the use of mono- or bivalent VHH and rabbit polyclonal anti-CMY-2 antibodies enables the development of the first generation of enzyme-linked immunosorbent assay (ELISA) for the detection of CMY-2 produced by CMY-2-expressing bacteria, irrespective of resistotype.

Keywords: CMY-2 β-lactamase; antibiotic resistance; class C beta-lactamase; nanobodies; new antibiotic scaffold; paratopes; structural biology.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Antibodies
Epitopes
Penicillins
Precision Medicine
Rabbits
Single-Domain Antibodies*
beta-Lactamase Inhibitors
beta-Lactamases / chemistry
beta-Lactamases / genetics

Substances

Single-Domain Antibodies
beta-Lactamases
beta-Lactamase Inhibitors
Penicillins
Antibodies
Epitopes