The murine mAb CC49 specifically recognizes a tumor-associated glycoprotein (TAG)-72, which is expressed on the majority of human carcinomas. This Ab has potential applications in the diagnosis and treatment of human carcinomas. However, patients receiving murine CC49 generate human anti-murine Ab (HAMA) responses, preventing repeated administration of the Ab for effective treatment. To minimize the HAMA response, two versions of humanized CC49 (HuCC49) were developed: (a) HuCC49 and (b) HuCC49V10 (V10). HuCC49 was developed by grafting the CC49 CDRs, while V10 was generated by grafting only the specificity determining residues (SDRs) of the CC49 onto the frameworks of the human Abs. During the generation of both HuCC49 and V10, a few murine framework residues that were believed to be essential for the integrity of the Ag-binding site were retained. However, the indispensability of these residues for the Ag-binding activity of CC49 has not been experimentally validated. In this study, an array of V10 variants were generated by replacing, by site-specific mutagenesis, the murine framework residues that were retained in the humanized Ab with their counterparts in the human templates. The variants were tested for their (a) Ag-binding activity and (b) reactivity to sera from patients who were previously administered murine CC49 in a clinical trial. One such variant, V59, compared to the parental V10, shows a significant decrease in its reactivity to the anti-variable region Abs present in the patients' sera, while it binds to the TAG-72 Ag with a slightly higher affinity. Variant 59, which is expected to be minimally immunogenic because of its low sera reactivity, is a potentially useful clinical reagent against human carcinomas. In this study, we show for the first time that experimental validation rather than reliance on the protein data bank (PDB) should be the criterion for the indispensability of framework residues for the humanization of any murine Ab to retain its Ag-binding property and reduce its immunogenicity in patients.