title: High Throughput Combinatorial Formatting of PcrV Nanobodies for Efficient Potency Improvement.
abstract: Improving potencies through concomitant blockage of multiple epitopes and avid binding by fusing multiple (different) monovalent Nanobody building blocks via linker sequences into one multivalent polypeptide chain is an elegant alternative to affinity maturation. We explored a large and random formatting library of bivalent (combinations of two identical) and biparatopic (combinations of two different) Nanobodies for functional blockade of Pseudomonas aeruginosa PcrV. PcrV is an essential part of the P. aeruginosa type III secretion system (T3SS), and its oligomeric nature allows for multiple complex binding and blocking options. The library screening yielded a large number of promising biparatopic lead candidates, revealing significant (and non-trivial) preferences in terms of Nanobody building block and epitope bin combinations and orientations. Excellent potencies were confirmed upon further characterization in two different P. aeruginosa T3SS-mediated cytotoxicity assays. Three biparatopic Nanobodies were evaluated in a lethal mouse P. aeruginosa challenge pneumonia model, conferring 100% survival upon prophylactic administration and reducing lung P. aeruginosa burden by up to 2 logs. At very low doses, they protected the mice from P. aeruginosa infection-related changes in lung histology, myeloperoxidase production, and lung weight. Importantly, the most potent Nanobody still conferred protection after therapeutic administration up to 24 h post-infection. The concept of screening such formatting libraries for potency improvement is applicable to other targets and biological therapeutic platforms.
authors.affiliation: From Ablynx N.V., Technologiepark 21, 9052 Ghent, Belgium.
authors.forename: Evelyn
authors.initials: E
authors.lastname: De Tavernier
authors.affiliation: From Ablynx N.V., Technologiepark 21, 9052 Ghent, Belgium.
authors.forename: Laurent
authors.initials: L
authors.lastname: Detalle
authors.affiliation: From Ablynx N.V., Technologiepark 21, 9052 Ghent, Belgium.
authors.forename: Erika
authors.initials: E
authors.lastname: Morizzo
authors.affiliation: From Ablynx N.V., Technologiepark 21, 9052 Ghent, Belgium.
authors.forename: Annelies
authors.initials: A
authors.lastname: Roobrouck
authors.affiliation: From Ablynx N.V., Technologiepark 21, 9052 Ghent, Belgium.
authors.forename: Severine
authors.initials: S
authors.lastname: De Taeye
authors.affiliation: From Ablynx N.V., Technologiepark 21, 9052 Ghent, Belgium.
authors.forename: Melanie
authors.initials: M
authors.lastname: Rieger
authors.affiliation: From Ablynx N.V., Technologiepark 21, 9052 Ghent, Belgium.
authors.forename: Tom
authors.initials: T
authors.lastname: Verhaeghe
authors.affiliation: From Ablynx N.V., Technologiepark 21, 9052 Ghent, Belgium.
authors.forename: Andreia
authors.initials: A
authors.lastname: Correia
authors.affiliation: From Ablynx N.V., Technologiepark 21, 9052 Ghent, Belgium.
authors.forename: Rob
authors.initials: R
authors.lastname: Van Hegelsom
authors.affiliation: From Ablynx N.V., Technologiepark 21, 9052 Ghent, Belgium.
authors.forename: Rita
authors.initials: R
authors.lastname: Figueirido
authors.affiliation: From Ablynx N.V., Technologiepark 21, 9052 Ghent, Belgium.
authors.forename: Jeroen
authors.initials: J
authors.lastname: Noens
authors.affiliation: From Ablynx N.V., Technologiepark 21, 9052 Ghent, Belgium.
authors.forename: Søren
authors.initials: S
authors.lastname: Steffensen
authors.affiliation: From Ablynx N.V., Technologiepark 21, 9052 Ghent, Belgium.
authors.forename: Thomas
authors.initials: T
authors.lastname: Stöhr
authors.affiliation: From Ablynx N.V., Technologiepark 21, 9052 Ghent, Belgium.
authors.forename: Willem
authors.initials: W
authors.lastname: Van de Velde
authors.affiliation: From Ablynx N.V., Technologiepark 21, 9052 Ghent, Belgium.
authors.forename: Erik
authors.initials: E
authors.lastname: Depla
authors.affiliation: From Ablynx N.V., Technologiepark 21, 9052 Ghent, Belgium bruno.dombrecht@ablynx.com.
authors.forename: Bruno
authors.initials: B
authors.lastname: Dombrecht
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial / Q000276:immunology*; D001427:Bacterial Toxins / Q000276:immunology*; D016923:Cell Death; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D004195:Disease Models, Animal; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D005260:Female; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008810:Mice, Inbred C57BL; D008958:Models, Molecular; D011014:Pneumonia / Q000276:immunology / Q000382:microbiology / Q000473:pathology; D052899:Pore Forming Cytotoxic Proteins / Q000276:immunology*; D011552:Pseudomonas Infections / Q000276:immunology; D011550:Pseudomonas aeruginosa / Q000276:immunology; D061905:Single-Domain Antibodies / Q000276:immunology*; D064166:Vaccine Potency
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D000939:Epitopes; D052899:Pore Forming Cytotoxic Proteins; D061905:Single-Domain Antibodies; C118546:antigen V, Pseudomonas
keywords: PcrV; Pseudomonas aeruginosa (P. aeruginosa); avidity; biparatopic; bivalent; epitope mapping; formatting; high throughput screening (HTS); single-domain antibody (sdAb, Nanobody); type III secretion system (T3SS)