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. Monoclonal antibodies (mAbs)4 are increasingly being used as therapeutic agents for cancer and other diseases. Murine mAbs have limited use as therapeutic agents because of a short half-life, an inability to trigger human effector functions, and the induction of a human antimouse antibody response (1, 2). To reduce the immunogenicity of murine antibodies in humans, chimeric antibodies with mouse variable regions and human constant regions were initially constructed (3). Although chimeric antibodies proved to be less immunogenic than murine mAbs, human anti-chimeric antibody responses have been observed (4). To further reduce the immunogenicity of the mouse variable regions, a humanized antibody has been constructed by grafting the complementarity-determining regions (CDRs) of a murine mAb onto the human framework regions (FRs) by a procedure commonly referred to as CDR grafting (5). Simple grafting CDRs, however, often decreased the affinity, because some FR residues directly contact the antigen or support the conformation of the CDR loops (6, 7). Therefore, humanized antibody is currently constructed primarily by CDR grafting, while retaining those rodent FR residues that influence antigen-binding activity (8). Since the FR residues often differ from one humanized antibody to another, the identification of key rodent FR residues is a crucial part of the humanization (9). Clinical studies have indicated that such humanized antibodies are...

Engineering the variable region of therapeutic IgG antibodies

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Thermodynamic Consequences of Mutations in Vernier Zone Residues of a Humanized Anti-human Epidermal Growth Factor Receptor Murine Antibody, 528

“…However, grafting of the six CDRs of murine antibodies onto appropriate frameworks of human antibodies often results in reduced affinity or specificity for the target antigen (10,14,16). The pioneering work of Foote and Winter (21) has suggested that antibody residues in the ␤-sheet framework underlying the CDRs play a critical role in adjustment of the loop structures of the CDRs.…”

Applications of immunochemistry in human health: advances in vaccinology and antibody design (IUPAC Technical Report)

“…The next approach was, therefore, the use of specificity-determining residue (SDR) grafting, in which only the SDRs (not whole CDRs) are grafted onto the germline antibodies. These SDR-grafted humanized antibodies indeed showed lower immunogenicity than CDR-grafted humanized antibodies [379][380][381][382].…”