Construction, affinity maturation, and biological characterization of an anti-tumor-associated glycoprotein-72 humanized antibody.
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.
Yoon
Sun Ok
SO
Laboratory of Antibody Engineering, Aprogen Inc., Korea Research Institute of Bioscience and Biotechnology, 52 Eoeun-dong, Yuseong-gu, Daejon 305-333.
Lee
Tae Sup
TS
Kim
Sang Jick
SJ
Jang
Myung Hee
MH
Kang
Young Jun
YJ
Park
Jae Hyun
JH
Kim
Keun-Soo
KS
Lee
Hyun Sil
HS
Ryu
Chun Jeih
CJ
Gonzales
Noreen R
NR
Kashmiri
Syed V S
SV
Lim
Sang Moo
SM
Choi
Chang Woon
CW
Hong
Hyo Jeong
HJ
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
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