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Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells
Published on: June 5, 2015
Structural insight into a matured humanized monoclonal antibody HuA21 against HER2-overexpressing cancer cells
Zhenyi Wang1, Liansheng Cheng2, Gongrui Guo1
1Hefei National Laboratory for Physical Sciences at the Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, People's Republic of China.
Abstract:
HER2, a member of the epidermal growth factor receptor (EGFR) family, has been associated with human breast, ovarian and gastric cancers. Anti-HER2 monoclonal antibodies (mAbs) have demonstrated clinical efficacy for HER2-overexpressing breast cancer. A chimeric antibody chA21 that specifically inhibits the growth of HER2-overexpressing cancer cells both in vitro and in vivo has previously been developed. To reduce a potential human anti-mouse immune response, the humanized antibody HuA21 was developed and was further subjected to affinity maturation by phage display on the basis of chA21. Here, the crystal structure of HuA21-scFv in complex with the extracellular domain of HER2 is reported, which demonstrates that HuA21 binds almost the same epitope as chA21 and also provides insight into how substitutions in HuA21 improve the binding affinity compared with chA21, which could facilitate structure-based optimization in the future. Furthermore, the effects of HuA21 variants with constant domains of different lengths were explored and it was noticed that the deletion of constant domain 1 could improve the inhibition efficacy in a cell-proliferation assay, possibly functioning via increased internalization, which might guide the design of other monoclonal antibodies.
Insights
Humanized anti-HER2 antibody HuA21 shows improved binding affinity and efficacy against HER2-overexpressing cancers. Structural analysis reveals key substitutions and suggests domain deletions may enhance antibody internalization and therapeutic potential.
Area of Science:
- Oncology
- Immunology
- Structural Biology
Background:
- HER2 (human epidermal growth factor receptor 2) is implicated in breast, ovarian, and gastric cancers.
- Anti-HER2 monoclonal antibodies (mAbs) show clinical efficacy in HER2-overexpressing breast cancer.
- Previous development of chimeric antibody chA21 targeting HER2.
Purpose of the Study:
- To determine the crystal structure of the humanized antibody HuA21 in complex with HER2.
- To understand the structural basis for HuA21's improved binding affinity compared to chA21.
- To explore the impact of HuA21 constant domain length on inhibitory efficacy.
Main Methods:
- X-ray crystallography to determine the structure of HuA21-scFv complexed with the extracellular domain of HER2.
- Phage display for affinity maturation of the antibody.
- Cell-proliferation assays to evaluate the efficacy of HuA21 variants.
Main Results:
- HuA21 binds to the same epitope as chA21 on HER2.
- Structural insights reveal substitutions enhancing HuA21's binding affinity.
- Deletion of constant domain 1 in HuA21 variants improved inhibition efficacy, potentially via increased internalization.
Conclusions:
- HuA21 retains the epitope specificity of chA21 while exhibiting enhanced binding affinity.
- Structural information facilitates future structure-based optimization of anti-HER2 antibodies.
- Modifying antibody constant domains, such as through deletion, can improve therapeutic efficacy and guide future antibody design.
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