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Multimerization is required for antigen binding activity of an engineered IgM/IgG chimeric antibody recognizing a
Kwesi Teye1, Koji Hashimoto2, Sanae Numata3
1Kurume University Institute of Cutaneous Cell Biology, Kurume, Fukuoka, Japan.
Scientific Reports
|August 17, 2017
Summary
Converting mouse IgM to IgG chimeric antibodies enhanced antigen binding. Multimerization of these engineered antibodies, through specific mutations and tailpiece additions, was key to their improved functionality for cancer research.
Area of Science:
- Immunology
- Biotechnology
- Cancer Research
Background:
- Monoclonal antibodies (mAbs) are vital research tools.
- Mouse IgM mAbs present technical challenges due to their pentameric structure.
- An IgM mAb targeting an antigen lost in skin cancer was identified.
Purpose of the Study:
- To engineer a functional IgG version of a mouse IgM mAb.
- To investigate the role of multimerization in chimeric antibody function.
- To develop a tool for characterizing a novel skin cancer antigen.
Main Methods:
- Rapid amplification of cDNA ends (RACE) PCR to obtain antibody cDNA.
- Construction of chimeric IgM/IgG antibodies with varying domain combinations.
- Gel filtration chromatography to analyze antibody multimerization.
- Site-directed mutagenesis and addition of IgM tailpiece to enhance function.
Main Results:
- Specific chimeric constructs (VH-Cµ1-Cµ2-Cγ3 and VH-Cµ1-Cµ2-Hinge-Cγ2-Cγ3) retained antigen binding.
- Functional antibodies exhibited multimerization.
- A mutation (p.309Q>C) and IgM tailpiece addition induced multimerization and significantly enhanced functionality.
- Engineered antibodies were compatible with Protein A/G binding.
Conclusions:
- Multimerization is critical for the antigen-binding activity of engineered IgM/IgG chimeric antibodies.
- The developed chimeric antibodies are suitable for further antigen characterization.
- This study provides insights into engineering functional chimeric antibodies.
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