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	Sun Ok Yoon, Tae Sup Lee, Sang Jick Kim, Myung Hee Jang, Young Jun Kang, Jae Hyun Park, Keun-Soo Kim, Hyun Sil Lee, Chun Jeih Ryu, Noreen R Gonzales, Syed V S Kashmiri, Sang Moo Lim, Chang Woon Choi, Hyo Jeong Hong
Journal	The Journal of biological chemistry (J Biol Chem) Vol. 281 Issue 11 Pg. 6985-92 (Mar 17 2006) ISSN: 0021-9258 [Print] United States
PMID	16407221 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Chemical References	Antibodies Antigens, Neoplasm Complementarity Determining Regions Glycoproteins Immunoglobulin G Protein Isoforms Recombinant Proteins tumor-associated antigen 72
Topics	Adenocarcinoma (metabolism) Amino Acid Sequence Animals Antibodies (chemistry) Antigens, Neoplasm (chemistry, immunology) Binding, Competitive COS Cells Cell Line, Tumor Chlorocebus aethiops Complementarity Determining Regions Dose-Response Relationship, Immunologic Escherichia coli (metabolism) Female Genetic Variation Glycoproteins (chemistry, immunology) Humans Immunoglobulin G (chemistry) Kinetics Mice Mice, Inbred BALB C Molecular Conformation Molecular Sequence Data Neoplasm Transplantation Polymerase Chain Reaction Protein Binding Protein Isoforms Protein Structure, Tertiary Recombinant Proteins (chemistry) Sequence Homology, Amino Acid Surface Plasmon Resonance Time Factors Tissue Distribution

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