title: Humanization of the anti-CD18 antibody 6.7: an unexpected effect of a framework residue in binding to antigen.
abstract: Humanization of monoclonal antibodies by complementary determinant region (CDR)-grafting has become a standard procedure to improve the clinical usage of animal antibodies. However, antibody humanization may result in loss of activity that has been attributed to structural constraints in the framework structure. In this paper, we report the complete humanization of the 6.7 anti-human CD18 monoclonal antibody in a scFv form. We used a germline-based approach to design a humanized VL gene fragment and expressed it together with a previously described humanized VH. The designed humanized VL has only 14 mutations compared to the closest human germline sequence. The resulting humanized scFv maintained the binding capacity and specificity to human CD18 expressed on the cell surface of peripheral blood mononuclear cells (PBMC), and showed the same pattern of staining T-lymphocytes sub-populations, in comparison to the original monoclonal antibody. We observed an unexpected effect of a conserved mouse-human framework position (L37) that hinders the binding of the humanized scFv to antigen. This paper reveals a new framework residue that interferes with paratope and antigen binding and also reinforces the germline approach as a successful strategy to humanize antibodies.
authors.affiliation: Departamento de Biologia Celular, Universidade de Brasi;lia, 70910-900, DF, Brasília, Brazil. brigido@unb.br
authors.forename: Cristina
authors.initials: C
authors.lastname: Caldas
authors.forename: Verônica
authors.initials: V
authors.lastname: Coelho
authors.forename: Jorge
authors.initials: J
authors.lastname: Kalil
authors.forename: Ana Maria
authors.initials: AM
authors.lastname: Moro
authors.forename: Andrea Q
authors.initials: AQ
authors.lastname: Maranhão
authors.forename: Marcelo M
authors.initials: MM
authors.lastname: Brígido
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D000915:Antibody Affinity; D000941:Antigens / Q000276:immunology; D018821:CD18 Antigens / Q000276:immunology*; D006801:Humans; D007133:Immunoglobulin Joining Region / Q000737:chemistry; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D007145:Immunoglobulin kappa-Chains / Q000737:chemistry; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D000941:Antigens; D018821:CD18 Antigens; D007133:Immunoglobulin Joining Region; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains; D011993:Recombinant Fusion Proteins