Grafting of "abbreviated" complementarity-determining regions containing specificity-determining residues essential for ligand contact to engineer a less immunogenic humanized monoclonal antibody.
Murine mAb COL-1 reacts with carcinoembryonic Ag (CEA), expressed on a wide range of human carcinomas. In preclinical studies in animals and clinical trials in patients, murine COL-1 showed excellent tumor localization. To circumvent the problem of immunogenicity of the murine Ab in patients, a humanized COL-1 (HuCOL-1) was generated by grafting the complementarity-determining regions (CDRs) of COL-1 onto the frameworks of the variable light and variable heavy regions of human mAbs. To minimize anti-V region responses, a variant of HuCOL-1 was generated by grafting onto the human frameworks only the "abbreviated" CDRs, the stretches of CDR residues that contain the specificity-determining residues that are essential for the surface complementarity of the Ab and its ligand. In competition RIAs, the recombinant variant completely inhibited the binding of radiolabeled murine and humanized COL-1 to CEA. The HuCOL-1 and its variant showed no difference in their binding ability to the CEA expressed on the surface of a CEA-transduced tumor cell line. Compared with HuCOL-1, the HuCOL-1 variant showed lower reactivity to patients' sera carrying anti-V region Abs to COL-1. The final variant of the HuCOL-1, which retains its Ag-binding reactivity and shows significantly lower serum reactivity than that of the parental Ab, can serve as a prototype for the development of a potentially useful clinical reagent.
De Pascalis
Roberto
R
Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Iwahashi
Makoto
M
Tamura
Midori
M
Padlan
Eduardo A
EA
Gonzales
Noreen R
NR
Santos
Ameurfina D
AD
Giuliano
Mariateresa
M
Schuck
Peter
P
Schlom
Jeffrey
J
Kashmiri
Syed V S
SV
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000097:blood / Q000737:chemistry / Q000235:genetics*; D000918:Antibody Specificity / Q000235:genetics*; D000937:Antigen-Antibody Reactions / Q000235:genetics*; D001483:Base Sequence; D001666:Binding Sites, Antibody / Q000235:genetics; D002272:Carcinoembryonic Antigen / Q000097:blood / Q000235:genetics / Q000276:immunology; D002478:Cells, Cultured; D022801:Complementarity Determining Regions / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000097:blood / Q000235:genetics / Q000276:immunology; D005434:Flow Cytometry; D014644:Genetic Variation / Q000276:immunology; D006801:Humans; D007106:Immune Sera / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000302:isolation & purification; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000302:isolation & purification; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000302:isolation & purification; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000097:blood / Q000138:chemical synthesis / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000097:blood; D018411:Spodoptera / Q000235:genetics
D000911:Antibodies, Monoclonal; D002272:Carcinoembryonic Antigen; D022801:Complementarity Determining Regions; D000939:Epitopes; D007106:Immune Sera; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D008024:Ligands; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins