title: Germline humanization of a non-human primate antibody that neutralizes the anthrax toxin, by in vitro and in silico engineering.
abstract: Fab 35PA83 is an antibody fragment of non-human primate origin that neutralizes the anthrax lethal toxin. Human antibodies are usually preferred when clinical use is envisioned, even though their framework regions (FR) may carry mutations introduced during affinity maturation. These hypermutations can be immunogenic and therefore FR that are encoded by human germline genes, encountered in IgMs and thus part of the "self" proteins, are preferable. Accordingly, the proportion of FR residues in 35PA83 that were encoded by human V and J germline genes, i.e. the germinality index (GI) of 35PA83, was increased in a multistep cumulative approach. In a first step, the FR1 and FR4 residues of 35PA83 were changed simultaneously into their counterparts coded by 35PA83's closest human germline genes, without prior modelling. The resulting derivative of 35PA83 had the same affinity as its parental Fab. In a second step, the 3D structures of this first 35PA83 derivative, carrying the same type of residue changes but in the FR2 and FR3 regions, were modelled in silico from sequences. Some of the changes in FR2 or FR3 modified the predicted peptide backbone. The changes that did not seem to alter the structure were introduced simultaneously in the Fab by an in vitro method and resulted in a loss of reactivity, which could however be fully restored by a single point mutation. The final 35PA83 derivative had a GI higher than that of a fully human Fab, which had neutralization properties similar to 35PA83 and which was used as a benchmark in this study.
authors.affiliation: Groupe de Biotechnologie des Anticorps, Laboratoire d'Immunobiologie, Centre de Recherches du Service de Santé des Armées, 24 avenue du maquis du Grésivaudan, 38702 La Tronche, France.
authors.forename: Thibaut
authors.initials: T
authors.lastname: Pelat
authors.forename: Hugues
authors.initials: H
authors.lastname: Bedouelle
authors.forename: Anthony R
authors.initials: AR
authors.lastname: Rees
authors.forename: Susan J
authors.initials: SJ
authors.lastname: Crennell
authors.forename: Marie-Paule
authors.initials: MP
authors.lastname: Lefranc
authors.forename: Philippe
authors.initials: P
authors.lastname: Thullier
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000276:immunology*; D000915:Antibody Affinity / Q000276:immunology; D000942:Antigens, Bacterial / Q000276:immunology*; D001427:Bacterial Toxins / Q000276:immunology*; D002460:Cell Line; D019295:Computational Biology; D005854:Germ Cells / Q000276:immunology*; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000737:chemistry; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D017354:Point Mutation / Q000235:genetics; D011323:Primates / Q000276:immunology*; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D016415:Sequence Alignment
chemical_list: D000906:Antibodies; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D007135:Immunoglobulin Variable Region; C030325:anthrax toxin