Humanization of an anti-p185HER2 antibody for human cancer therapy.

TLDR

The murine monoclonal antibody mumAb4D5 targets p185HER2 and inhibits proliferation of overexpressing tumor cells, but its therapeutic potential is limited by anti‑mouse responses and poor effector functions. The authors aimed to generate a humanized antibody, humAb4D5‑1, comprising only the antigen‑binding loops of mumAb4D5 with human variable framework residues and IgG1 constant domains. Humanization was achieved in a single step by gene‑conversion mutagenesis using 311‑mer and 361‑mer preassembled oligonucleotides for the light and heavy chains, respectively. The humAb4D5‑8 variant binds p185HER2 250‑fold more tightly than humAb4D5‑1 and 3‑fold tighter than mumAb4D5, blocks SK‑BR‑3 proliferation with potency comparable to the murine antibody, and induces stronger ADCC against SK‑BR‑3 while sparing WI‑38 cells.

Abstract

The murine monoclonal antibody mumAb4D5, directed against human epidermal growth factor receptor 2 (p185HER2), specifically inhibits proliferation of human tumor cells overexpressing p185HER2. However, the efficacy of mumAb4D5 in human cancer therapy is likely to be limited by a human anti-mouse antibody response and lack of effector functions. A "humanized" antibody, humAb4D5-1, containing only the antigen binding loops from mumAb4D5 and human variable region framework residues plus IgG1 constant domains was constructed. Light- and heavy-chain variable regions were simultaneously humanized in one step by "gene conversion mutagenesis" using 311-mer and 361-mer preassembled oligonucleotides, respectively. The humAb4D5-1 variant does not block the proliferation of human breast carcinoma SK-BR-3 cells, which overexpress p185HER2, despite tight antigen binding (Kd = 25 nM). One of seven additional humanized variants designed by molecular modeling (humAb4D5-8) binds the p185HER2 antigen 250-fold and 3-fold more tightly than humAb4D5-1 and mumAb4D5, respectively. In addition, humAb4D5-8 has potency comparable to the murine antibody in blocking SK-BR-3 cell proliferation. Furthermore, humAb4D5-8 is much more efficient in supporting antibody-dependent cellular cytotoxicity against SK-BR-3 cells than mumAb4D5, but it does not efficiently kill WI-38 cells, which express p185HER2 at lower levels.