This article describes the humanization of a murine monoclonal antibody, mumAb4D5, which targets the human epidermal growth factor receptor 2 (p185HER2), for use in human cancer therapy. The original antibody, while effective in inhibiting the growth of p185HER2-overexpressing tumor cells, is limited by its immunogenicity and lack of effector functions. To address these issues, a humanized antibody, humAb4D5-1, was developed by replacing the antigen-binding loops of mumAb4D5 with human variable region framework residues and IgG1 constant domains. This humanized antibody was constructed using a gene conversion mutagenesis strategy, which allowed for the simultaneous humanization of both the heavy and light chains of the antibody. Among the various humanized variants created, humAb4D5-8 was found to be the most effective. It binds the p185HER2 antigen 250-fold more tightly than humAb4D5-1 and has comparable antiproliferative activity against SK-BR-3 cells. Additionally, humAb4D5-8 is much more efficient in supporting antibody-dependent cellular cytotoxicity (ADCC) against SK-BR-3 cells compared to the murine antibody, although it does not efficiently kill WI-38 cells, which express p185HER2 at lower levels. The study highlights the importance of molecular modeling in the humanization process, as it allowed for the identification of critical framework residues that influence antigen binding and antibody function. The humanized antibody humAb4D5-8 shows promise for clinical use in treating cancers overexpressing p185HER2, as it is less immunogenic and more effective in supporting ADCC. The results demonstrate the potential of humanized antibodies in improving the therapeutic efficacy of monoclonal antibodies in human cancer therapy.This article describes the humanization of a murine monoclonal antibody, mumAb4D5, which targets the human epidermal growth factor receptor 2 (p185HER2), for use in human cancer therapy. The original antibody, while effective in inhibiting the growth of p185HER2-overexpressing tumor cells, is limited by its immunogenicity and lack of effector functions. To address these issues, a humanized antibody, humAb4D5-1, was developed by replacing the antigen-binding loops of mumAb4D5 with human variable region framework residues and IgG1 constant domains. This humanized antibody was constructed using a gene conversion mutagenesis strategy, which allowed for the simultaneous humanization of both the heavy and light chains of the antibody. Among the various humanized variants created, humAb4D5-8 was found to be the most effective. It binds the p185HER2 antigen 250-fold more tightly than humAb4D5-1 and has comparable antiproliferative activity against SK-BR-3 cells. Additionally, humAb4D5-8 is much more efficient in supporting antibody-dependent cellular cytotoxicity (ADCC) against SK-BR-3 cells compared to the murine antibody, although it does not efficiently kill WI-38 cells, which express p185HER2 at lower levels. The study highlights the importance of molecular modeling in the humanization process, as it allowed for the identification of critical framework residues that influence antigen binding and antibody function. The humanized antibody humAb4D5-8 shows promise for clinical use in treating cancers overexpressing p185HER2, as it is less immunogenic and more effective in supporting ADCC. The results demonstrate the potential of humanized antibodies in improving the therapeutic efficacy of monoclonal antibodies in human cancer therapy.