Anti-vascular endothelial growth factor receptor (VEGFR) 2 autoantibody identification in glioblastoma patient using single B cell-based antibody gene cloning

• Single B cell-based human monoclonal antibody gene PCR cloning method was developed.
• Plasma autoantibodies against VEGFR2 antigen were detected in glioblastoma patients.
• Sixty B cells bearing VEGFR2-specific human antibody were obtained by cell sorter.
• Twenty-two antibody genes were identified using direct human IgG PCR cloning.
• Two antibody clones showed VEGFR2-specific binding affinity in ELISA and SPR.

Antibody direct cloning from single B cells is simple and efficient and has been successful in antibody identification of infectious diseases. However, although a recent whole-exome sequencing revealed abundant heterogeneic mutation accumulation in cancers, identification and synthesis of autoantibodies against specific cancer-associated antigens is still difficult in cancer patients owing to the very small number of B cells producing autoantibodies. In the present study, to identify autoantibodies targeting tumor antigens, we measured the titer of autoantibodies in high-grade glioma patients’ plasma and identified two patients with elevated autoantibodies to a few transmembrane proteins. Specific B cells producing autoantibody against vascular endothelial growth factor receptor (VEGFR) 2 were immunostained with labeled protein and anti-human IgG antibody, and then collected by a single cell sorter. Finally, 22 antibody genes were successfully identified using direct IgG cloning from single B cell mRNA, and two antibody clones were found to have significant VEGFR2-specific binding affinity.The current direct human IgG gene cloning technique for identifying human antibodies derived from IgG-memory B cells avoids time-consuming procedures such as phage display-based antibody-library screening, and therefore may be applicable to identifying human autoantibodies in a variety of disorders including cancers even when antibody elevation is not detected because of a very small number of memory B cells.