title: Construction, affinity maturation, and biological characterization of an anti-tumor-associated glycoprotein-72 humanized antibody.
abstract: The tumor-associated glycoprotein (TAG)-72 is expressed in the majority of human adenocarcinomas but is rarely expressed in most normal tissues, which makes it a potential target for the diagnosis and therapy of a variety of human cancers. Here we describe the construction, affinity maturation, and biological characterization of an anti-TAG-72 humanized antibody with minimum potential immunogenicity. The humanized antibody was constructed by grafting only the specificity-determining residues (SDRs) within the complementarity-determining regions (CDRs) onto homologous human immunoglobulin germ line segments while retaining two mouse heavy chain framework residues that support the conformation of the CDRs. The resulting humanized antibody (AKA) showed only about 2-fold lower affinity compared with the original murine monoclonal antibody CC49 and 27-fold lower reactivity to patient serum compared with the humanized antibody HuCC49 that was constructed by CDR grafting. The affinity of AKA was improved by random mutagenesis of the heavy chain CDR3 (HCDR3). The highest affinity variant (3E8) showed 22-fold higher affinity compared with AKA and retained the original epitope specificity. Mutational analysis of the HCDR3 residues revealed that the replacement of Asn(97) by isoleucine or valine was critical for the affinity maturation. The 3E8 labeled with (125)I or (131)I showed efficient tumor targeting or therapeutic effects, respectively, in athymic mice with human colon carcinoma xenografts, suggesting that 3E8 may be beneficial for the diagnosis and therapy of tumors expressing TAG-72.
authors.affiliation: Laboratory of Antibody Engineering, Aprogen Inc., Korea Research Institute of Bioscience and Biotechnology, 52 Eoeun-dong, Yuseong-gu, Daejon 305-333.
authors.forename: Sun Ok
authors.initials: SO
authors.lastname: Yoon
authors.forename: Tae Sup
authors.initials: TS
authors.lastname: Lee
authors.forename: Sang Jick
authors.initials: SJ
authors.lastname: Kim
authors.forename: Myung Hee
authors.initials: MH
authors.lastname: Jang
authors.forename: Young Jun
authors.initials: YJ
authors.lastname: Kang
authors.forename: Jae Hyun
authors.initials: JH
authors.lastname: Park
authors.forename: Keun-Soo
authors.initials: KS
authors.lastname: Kim
authors.forename: Hyun Sil
authors.initials: HS
authors.lastname: Lee
authors.forename: Chun Jeih
authors.initials: CJ
authors.lastname: Ryu
authors.forename: Noreen R
authors.initials: NR
authors.lastname: Gonzales
authors.forename: Syed V S
authors.initials: SV
authors.lastname: Kashmiri
authors.forename: Sang Moo
authors.initials: SM
authors.lastname: Lim
authors.forename: Chang Woon
authors.initials: CW
authors.lastname: Choi
authors.forename: Hyo Jeong
authors.initials: HJ
authors.lastname: Hong
mesh_terms: D000230:Adenocarcinoma / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry; D000951:Antigens, Neoplasm / Q000737:chemistry* / Q000276:immunology*; D001667:Binding, Competitive; D019556:COS Cells; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D022801:Complementarity Determining Regions; D004306:Dose-Response Relationship, Immunologic; D004926:Escherichia coli / Q000378:metabolism; D005260:Female; D014644:Genetic Variation; D006023:Glycoproteins / Q000737:chemistry* / Q000276:immunology*; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009368:Neoplasm Transplantation; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D013997:Time Factors; D014018:Tissue Distribution
chemical_list: D000906:Antibodies; D000951:Antigens, Neoplasm; D022801:Complementarity Determining Regions; D006023:Glycoproteins; D007074:Immunoglobulin G; D020033:Protein Isoforms; D011994:Recombinant Proteins; C047052:tumor-associated antigen 72