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Updated: Jun 1, 2026
Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood
Published on: February 6, 2018
Efficient generation of useful monoclonal antibodies reactive with globotriaosylceramide using knockout mice lacking
Yuji Kondo1, Noriyo Tokuda, Keiko Furukawa
1Department of Biochemistry II, Nagoya University Graduate School of Medicine, Tsurumai, Showa-ku, Nagoya, Japan.
Abstract:
Efficient generation of useful monoclonal antibodies (mAbs) with high performance in cancer therapeutics has been expected. Generation of mAbs reactive with globotriaosylceramide (Gb3/CD77) was compared between A/J mice and Gb3/CD77 synthase-deficient (A4GalT-knockout) mice by immunizing Gb3-liposome. Specificity and functions of established antibodies were examined by ELISA, TLC- immunostaining, cytotoxicity of cancer cells and immunoblotting. Compared with results with conventional mice, better generation of mAbs with higher functions has been achieved with A4GalT-knockout mice, i.e. acquisition of IgG class antibodies, activities in antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and aggregation activity toward a Burkitt's lymphoma line Ramos. Binding of mAb k52 induced tyrosine phosphorylation of several proteins in Ramos cells. One of the strongest phosphorylation bands turned out to be c-Cbl. Pretreatment of B cell lines with mAbs resulted in the attenuation of BCR-mimicking signaling. All these results suggested that A4GalT-knockout mice are very useful to generate mAbs against globo-series glycolipids, and that suppressive signaling pathway driven by endogenous Gb3-ligand molecules might be present in B cells.
Insights
Generating monoclonal antibodies against globotriaosylceramide (Gb3/CD77) was more effective in knockout mice. These antibodies demonstrated enhanced functions, including cytotoxicity, suggesting a novel pathway in B cells.
Area of Science:
- Immunology
- Glycobiology
- Cancer Therapeutics
Background:
- Monoclonal antibodies (mAbs) are crucial for cancer therapeutics.
- Efficient generation of high-performance mAbs targeting specific cancer biomarkers is essential.
- Globotriaosylceramide (Gb3/CD77) is a potential target for cancer therapy.
Purpose of the Study:
- To compare the efficacy of generating monoclonal antibodies against globotriaosylceramide (Gb3/CD77) using conventional A/J mice versus Gb3/CD77 synthase-deficient (A4GalT-knockout) mice.
- To evaluate the specificity and functional activities of the generated mAbs in cancer models.
Main Methods:
- Immunization of A/J and A4GalT-knockout mice with Gb3-liposomes.
- Characterization of mAbs using ELISA, TLC-immunostaining, cytotoxicity assays, and immunoblotting.
- Analysis of B cell signaling pathways upon antibody binding.
Main Results:
- A4GalT-knockout mice yielded a superior generation of mAbs with enhanced functions compared to conventional mice.
- Generated IgG class antibodies exhibited significant antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and aggregation activity against Ramos Burkitt's lymphoma cells.
- mAb binding induced tyrosine phosphorylation, notably of c-Cbl, and attenuated BCR-mimicking signaling in B cell lines.
Conclusions:
- A4GalT-knockout mice are highly effective for generating monoclonal antibodies against globo-series glycolipids like Gb3/CD77.
- These findings suggest the presence of a suppressive signaling pathway in B cells mediated by endogenous Gb3-ligand molecules.
- The developed mAbs hold promise for cancer therapeutics targeting Gb3/CD77 expressing cancers.
