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bioRxiv · 10.1101/2022.07.01.498528

Development of nanobodies as theranostic agents against CMY-2-like class C β-lactamases

Cawez, F.·Mercuri, P. S.·Yanez, F. M.·Maalouf, R.·Vandevenne, M.·Kerff, F.·Guerin, V.·Mainil, J. G.·Thiry, D.·Salmon, M.·Vanderplasschen, A.·Lafaye, P.·Ayme, G.·Bogaerts, P.·Dumoulin, M.·Galleni, M.
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Abstract

Soluble single-domain fragments derived from the unique variable region of camelid heavy-chain antibodies (VHHs) against enzymes may behave as potent inhibitors. The immunization of alpacas with the CMY-2 {beta}-lactamase led to the isolation of three VHHs that specifically recognized and inhibited CMY-2. The structure of the complex VHH cAbCMY-2(254)/CMY-2 was determined by X-ray crystallography. We showed that the epitope is close to the active site and that the CDR3 of the VHH protrudes in the catalytic site. The {beta}-lactamase inhibition was found to follow a mixed profile with a predominant non-competitive component. The three isolated VHHs recognized overlapping epitopes since they behaved as competitive binder. Our study identified a binding site that can be targeted by a new class of {beta}-lactamases inhibitors designed with the help of a peptidomimetic approach. Furthermore, the use of mono or bivalent VHH and rabbit polyclonal anti-CMY-2 antibodies enable the development of the first generation of ELISA test for the detection of CMY-2 produced by resistant bacteria. IMPORTANCEThe still increasing antimicrobial resistance in human clinic or veterinary medicine is a major threat for modern chemotherapy. Beside the major caution in the use of current antibiotics, it is important to develop new classes of antibiotics. This work was focused on {beta}-lactamases that are the enzymes involved in the hydrolysis of the major class of antibiotics, the {beta}-lactam compounds. We selected camelid antibodies that inhibit CMY-2, a class C {beta}-lactamase produced by bacteria isolated from the veterinary and human settings. We characterized the conformational epitope present in CMY-2 in order to create a new family of inhibitors based on the paratope of the antibody. Finally, we designed a primary version of a detection system based on an ELISA using VHH and polyclonal antibodies.

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Cawez, F., Mercuri, P. S., Yanez, F. M., Maalouf, R., Vandevenne, M., Kerff, F., Guerin, V., Mainil, J. G., Thiry, D., Salmon, M., Vanderplasschen, A., Lafaye, P., Ayme, G., Bogaerts, P., Dumoulin, M., Galleni, M.. 2022-07-03. Development of nanobodies as theranostic agents against CMY-2-like class C β-lactamases. https://doi.org/10.1101/2022.07.01.498528

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