title: "Superhumanized" antibodies: reduction of immunogenic potential by complementarity-determining region grafting with human germline sequences: application to an anti-CD28.
abstract: Humanized Abs are created by combining, at the genetic level, the complementarity-determining regions of a murine mAb with the framework sequences of a human Ab variable domain. This leads to a functional Ab with reduced immunogenic side effects in human therapy. In this study, we report a new approach to humanizing murine mAbs that may reduce immunogenicity even further. This method is applied to humanize the murine anti-human CD28 Ab, 9.3. The canonical structures of the hypervariable loops of murine 9.3 were matched to human genomic V gene sequences whose hypervariable loops had identical or similar canonical structures. Framework sequences for those human V genes were then used, unmodified, with the 9.3 complementarity-determining regions to construct a humanized version of 9.3. The humanized 9.3 and a chimeric 9.3 control were expressed in Escherichia coli as Fab. The humanized Fab showed a moderate loss in avidity in a direct binding ELISA with immobilized CD28-Ig fusion protein (CD28-Ig). Humanized 9.3 blocked ligation of CD28-Ig to cells expressing the CD28 receptor CD80. Lastly, the humanized 9.3 showed biological activity as an immunosuppressant by inhibiting a MLR.
authors.affiliation: Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
authors.forename: Philip
authors.initials: P
authors.lastname: Tan
authors.forename: David A
authors.initials: DA
authors.lastname: Mitchell
authors.forename: Timothy N
authors.initials: TN
authors.lastname: Buss
authors.forename: Margaret A
authors.initials: MA
authors.lastname: Holmes
authors.forename: Claudio
authors.initials: C
authors.lastname: Anasetti
authors.forename: Jefferson
authors.initials: J
authors.lastname: Foote
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D019138:Antibodies, Blocking / Q000235:genetics / Q000494:pharmacology; D018122:B7-1 Antigen / Q000378:metabolism; D001666:Binding Sites, Antibody / Q000235:genetics; D018106:CD28 Antigens / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D022801:Complementarity Determining Regions / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis / Q000235:genetics; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D007166:Immunosuppressive Agents / Q000494:pharmacology; D007959:Lymphocyte Culture Test, Mixed; D051379:Mice; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000138:chemical synthesis / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
chemical_list: D000906:Antibodies; D019138:Antibodies, Blocking; D018122:B7-1 Antigen; D018106:CD28 Antigens; D022801:Complementarity Determining Regions; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D007166:Immunosuppressive Agents; D011993:Recombinant Fusion Proteins