title: Framework shuffling of antibodies to reduce immunogenicity and manipulate functional and biophysical properties.
abstract: We report here the humanization of two mouse monoclonal antibodies (mAb) using framework shuffling of human germline genes. mAbs EA2 and 47 were raised against the human receptor tyrosine kinase EphA2 and EphB4, respectively, which exhibit increased expression levels in many cancer cell lines. One- and two-step strategies were carried out, in which the light and heavy chains of each parental mAb were simultaneously or sequentially humanized. We characterized in detail these newly humanized antibodies in terms of binding affinity to their respective antigen, functional activity, thermostability, electric charge and expression yields. Three previously described framework shuffled, humanized versions of another mouse anti-human EphA2 antibody (mAb B233) were similarly characterized. We show that several of these parameters were either maintained or improved in all humanized molecules when compared with their respective chimaeric counterpart. Therefore, this humanization approach is generally applicable to non-human IgGs and allows for the specific selection of antibodies and antibody fragments exhibiting favorable functional, biochemical and biophysical properties.
authors.affiliation: Department of Antibody Discovery and Protein Engineering, MedImmune, Inc., One Medimmune Way, Gaithersburg, MD 20878, USA.
authors.forename: Melissa M
authors.initials: MM
authors.lastname: Damschroder
authors.forename: Lusiana
authors.initials: L
authors.lastname: Widjaja
authors.forename: Parkash S
authors.initials: PS
authors.lastname: Gill
authors.forename: Valery
authors.initials: V
authors.lastname: Krasnoperov
authors.forename: Weidong
authors.initials: W
authors.lastname: Jiang
authors.forename: William F
authors.initials: WF
authors.lastname: Dall'Acqua
authors.forename: Herren
authors.initials: H
authors.lastname: Wu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology*; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D045744:Cell Line, Tumor; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D036104:Receptor, EphA2 / Q000276:immunology*; D036224:Receptor, EphB4 / Q000276:immunology*; D017421:Sequence Analysis; D013329:Structure-Activity Relationship; D013696:Temperature
chemical_list: D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D036104:Receptor, EphA2; D036224:Receptor, EphB4