Nanobodies (Nbs) are the binding domain of heavy-chain-only antibodies; they display advantages as high specificity, affinity, and robustness. They are also potent enzyme inhibitors thanks to their long CDR3 region. In lung diseases, interesting targets to inhibit are for instance antibiotic resistance-conferring enzymes that are secreted, including b-lactamases. Such enzymes were involved in several cases of death or grave lung diseases due to infections by resistant B. cereus strains expressing anthrax-like toxins in non-immunodepressed individuals [1Pena-Gonzalez A., Marston C.K., Rodriguez-R L.M., Kolton C.B., Garcia-Diaz J., Theppote A., e al. Draft Genome Sequence of Bacillus cereus LA2007, a Human-Pathogenic Isolate Harboring Anthrax-Like Plasmids Genome Announc 2017 ;  5 : e00181-e217

Cliccare qui per andare alla sezione Riferimenti, 2Wright A.M., Beres S.B., Consamus E.N., Long S.W., Flores A.R., Barrios R., e al. Rapidly progressive, fatal, inhalation anthrax-like infection in a human: case report, pathogen genome sequencing, pathology, and coordinated response Arch Pathol Lab Med 2011 ;  135 : 1447-1459 [cross-ref]

Cliccare qui per andare alla sezione Riferimenti]. However, the use of Nbs as inhibitors may be limited by their small size and low retention time in lungs. The addition of a polyethylene glycol (PEG) group to cAb-BcII10, a nanobody inhibiting the beta-latamase BCII, was thus considered here to address this issue.

A cysteine was added by genetic engineering at the C-terminal of cAbBcII10 to allow its PEGylation with a PEG-maleimide of 10, 20 or 40kDa. The originality of the procedure is the use of TCEP-coated beads which preferentially reduce the intermolecular disulphide bridges leaving intact the internal SS bridge. The affinity of the PEGylated and unmodified Nbs was compared by competitive ELISA and their inhibitory capacity via a chromogenic substrate degradation assay. The presence of aggregates was highlighted by comparing the absorbance at 280nm before and after centrifugation at 10,000g or by dynamic light scattering. Nebulization was performed with a mesh nebulizer.

The PEGylation method developed in this study led to C-ter site-specific PEGylation with yields over 95%, unlike methods using free reducers. Although a decrease in affinity was observed after PEGylation, this did not translate into a loss of inhibitory capacity in vitro. The cAbBcII10 stability was increased by PEGylation. Indeed, 1h and 4h of agitation, nebulization and 10 freeze/thaw cycles respectively led to the aggregation of 59±11, 97±5, 73±9 and 18±6% of the unmodified cAbBcII10 protein while no significant amount of aggregates was detected after exposure of the PEGylated Nbs to these stresses. Similar results were obtained following the PEGylation of two other Nbs inhibiting enzymes relevant to lung diseases.

To conclude, this work led to the development of a rapid and efficient PEGylation method which can be applied to many Nbs. cAbBcII10 PEGylation using this protocol did not alter its inhibitory activity and increased its stability, including upon nebulization, which is an interesting administration method for lung diseases. This approach could also be used to label Nbs with other molecules such as fluorophores for imaging studies thereby broadening Nbs medical potential.