title: Increasing the potency of neutralizing single-domain antibodies by functionalization with a CD11b/CD18 binding domain.
abstract: Recombinant single domain antibodies (nanobodies) constitute an attractive alternative for the production of neutralizing therapeutic agents. Their small size warrants rapid bioavailability and fast penetration to sites of toxin uptake, but also rapid renal clearance, which negatively affects their performance. In this work, we present a new strategy to drastically improve the neutralizing potency of single domain antibodies based on their fusion to a second nanobody specific for the complement receptor CD11b/CD18 (Mac-1). These bispecific antibodies retain a small size (~30 kDa), but acquire effector functions that promote the elimination of the toxin-immunocomplexes. The principle was demonstrated in a mouse model of lethal toxicity with tetanus toxin. Three anti-tetanus toxin nanobodies were selected and characterized in terms of overlapping epitopes and inhibition of toxin binding to neuron gangliosides. Bispecific constructs of the most promising monodomain antibodies were built using anti Mac-1, CD45 and MHC II nanobodies. When co-administered with the toxin, all bispecific antibodies showed higher toxin-neutralizing capacity than the monomeric ones, but only their fusion to the anti-endocytic receptor Mac-1 nanobody allowed the mice to survive a 10-fold lethal dose. In a model of delayed neutralization of the toxin, the anti- Mac-1 bispecific antibodies outperformed a sheep anti-toxin polyclonal IgG that had shown similar neutralization potency in the co-administration experiments. This strategy should have widespread application in the development of nanobody-based neutralizing therapeutics, which can be produced economically and more safely than conventional antisera.
authors.affiliation: a Cátedra de Inmunología; DEPBIO; Facultad de Química; Instituto de Higiene; Universidad de la República ; Montevideo , Uruguay.
authors.forename: Martin A
authors.initials: MA
authors.lastname: Rossotti
authors.forename: Andrés
authors.initials: A
authors.lastname: González-Techera
authors.forename: Julio
authors.initials: J
authors.lastname: Guarnaschelli
authors.forename: Lucia
authors.initials: L
authors.lastname: Yim
authors.forename: Ximena
authors.initials: X
authors.lastname: Camacho
authors.forename: Marcelo
authors.initials: M
authors.lastname: Fernández
authors.forename: Pablo
authors.initials: P
authors.lastname: Cabral
authors.forename: Carmen
authors.initials: C
authors.lastname: Leizagoyen
authors.forename: José A
authors.initials: JA
authors.lastname: Chabalgoity
authors.forename: Gualberto
authors.initials: G
authors.lastname: González-Sapienza
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000276:immunology*; D000915:Antibody Affinity / Q000276:immunology; D001666:Binding Sites, Antibody / Q000276:immunology; D039481:CD11b Antigen / Q000276:immunology*; D018821:CD18 Antigens / Q000276:immunology*; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D051379:Mice; D061905:Single-Domain Antibodies / Q000276:immunology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013743:Tetanus Antitoxin / Q000276:immunology*
chemical_list: D057134:Antibodies, Neutralizing; D039481:CD11b Antigen; D018821:CD18 Antigens; D061905:Single-Domain Antibodies; D013743:Tetanus Antitoxin
keywords: ANOVA, analysis of variance; ELISA, enzyme-linked immunosorbent assay; FR2, framework 2; IMGT, International ImMunoGeneTics; LD, lethal dose; MALDI-TOF, matrix assisted laser desorption/ionization-time of flight; Poly-IgG, sheep anti-toxin polyclonal IgG;; VHH; effector functions; immunotherapy; nanobody; neutralization; phage display; therapeutic antibodies