Journal: Cellular and Molecular Immunology

Article Title: Streamlined human antibody generation and optimization by exploiting designed immunoglobulin loci in a B cell line

doi: 10.1038/s41423-020-0440-9

Figure Lengend Snippet: Isolation of VEGF-A-neutralizing mAbs. a The sorting experiment to enhance the affinities of the anti-hVEGF cells. The C018 cells cultured without TSA were fluorescently stained with anti-hIgG and hVEGF. The cells showing higher reactivity to the antigen than the major population (gate “P3”) were singly sorted (left). The reactivity of the clones before and after affinity maturation was analyzed by flow cytometry (blue, parental C018; red, C018AM-20, the representative clone after affinity maturation) (right). b Bar chart representing the effects of anti-VEGF-A mAbs on the proliferation of HUVECs against the Ab(−) control. VEGF-A and serially diluted anti-hVEGF-A mAbs were preincubated and added to the HUVEC culture. The number of cells was counted after two days. P values represent significant differences between the cells cultured in the presence of anti-VEGF mAbs and identical concentrations of the negative control (anti-TNFα) mAb; error bars represent ±s.d. ( n = 4). * P < 0.05, ** P < 0.01, *** P < 0.001. c SPR analysis of the anti-hVEGF mAbs. Serially diluted hVEGF-A was applied to the anti-hVEGF-A mAb immobilized on biosensor chips. d Summary of antibody binding constants. Association rate (ka) and dissociation rate (kd) constants were determined by SPR analysis. Dissociation constant (KD) values were calculated as kd/ka. e The sequences of the anti-hVEGF-A V H regions containing VH3-23-derived sequences. The sequences of B021, B015, C018 and C018AM-20 are compared with the original synthetic VDJ sequence designed for SCLs. The colored boxes indicate the original V genes: V3-23 (green), D3-3 (purple), JH4b (brown). The red and blue horizontal bars represent GC tracts (corresponding pseudogenes are described beside the bars) and SHM, respectively

Article Snippet: Recombinant human VEGF R2/KDR Fc chimera protein (R&D Systems) was immobilized onto MaxiSorp 384-well plates at 62.5 ng/well at 4 °C overnight.

Techniques: Isolation, Cell Culture, Staining, Clone Assay, Flow Cytometry, Control, Negative Control, Binding Assay, Derivative Assay, Sequencing