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.
Highlights
Monoclonal antibodies4 are increasingly being used as therapeutic agents for cancer and other diseases
A humanized CC49 antibody (HuCC49) has been constructed by complementarity-determining regions (CDRs) grafting while retaining the buried framework regions (FRs) residues that affect the structure of the antibody and those involved in VL-VH interaction or antigen binding [25]
The sequence comparison between the CC49 VL and DPK24-JK4 revealed that only three positions (Leu27b, Gly27f, and Gln29) in the light chain CDR1 (LCDR1), one position (Ala53) in the LCDR2, and one position (Tyr94) in the LCDR3 were different, whereas 17 positions in the FRs were different
Summary
Monoclonal antibodies (mAbs)4 are increasingly being used as therapeutic agents for cancer and other diseases. The antibody showed specific tumor targeting and anti-tumor therapeutic effects in athymic mice bearing human adenocarcinoma xenografts expressing TAG-72.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have