This study describes the humanization of a murine monoclonal antibody, mumAb4D5, which targets the human epidermal growth factor receptor 2 (p185HER2). The humanized antibody, humAb4D5-1, was constructed by replacing the antigen-binding loops of mumAb4D5 with human variable region framework residues and IgG1 constant domains. This approach reduced the immunogenicity of the antibody and improved its potential for clinical use. The humanized antibody was generated using a gene conversion mutagenesis strategy with preassembled oligonucleotides, allowing for the simultaneous humanization of both heavy- and light-chain variable regions. The study evaluated several humanized variants of the antibody, including humAb4D5-8, which showed significantly higher binding affinity for p185HER2 compared to the original murine antibody. HumAb4D5-8 also demonstrated comparable antiproliferative activity against SK-BR-3 breast cancer cells. Additionally, humAb4D5-8 was more effective in supporting antibody-dependent cellular cytotoxicity (ADCC) against SK-BR-3 cells than the murine antibody, although it was less effective in killing 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 results suggest that humAb4D5-8 is a promising candidate for clinical use in the treatment of cancers overexpressing p185HER2. The study also emphasizes the need for further structural studies to fully understand the role of specific residues in antibody function. Overall, the humanization of mumAb4D5 represents a significant advancement in the development of humanized antibodies for cancer therapy.This study describes the humanization of a murine monoclonal antibody, mumAb4D5, which targets the human epidermal growth factor receptor 2 (p185HER2). The humanized antibody, humAb4D5-1, was constructed by replacing the antigen-binding loops of mumAb4D5 with human variable region framework residues and IgG1 constant domains. This approach reduced the immunogenicity of the antibody and improved its potential for clinical use. The humanized antibody was generated using a gene conversion mutagenesis strategy with preassembled oligonucleotides, allowing for the simultaneous humanization of both heavy- and light-chain variable regions. The study evaluated several humanized variants of the antibody, including humAb4D5-8, which showed significantly higher binding affinity for p185HER2 compared to the original murine antibody. HumAb4D5-8 also demonstrated comparable antiproliferative activity against SK-BR-3 breast cancer cells. Additionally, humAb4D5-8 was more effective in supporting antibody-dependent cellular cytotoxicity (ADCC) against SK-BR-3 cells than the murine antibody, although it was less effective in killing 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 results suggest that humAb4D5-8 is a promising candidate for clinical use in the treatment of cancers overexpressing p185HER2. The study also emphasizes the need for further structural studies to fully understand the role of specific residues in antibody function. Overall, the humanization of mumAb4D5 represents a significant advancement in the development of humanized antibodies for cancer therapy.