Fig 1: Engineering of 5R65 to generate a more potent Ab, 5R65.7, and domain level epitope mapping of anti–IL-5Ra Abs. (A) The scheme of yeast scFab library construction for VL-CDR3 and VH-CDR3 of the 5R65 Ab, where the indicated residues (X) were randomly mutated using spiked oligonucleotides designed to keep the original amino acids at each position of 5R65 at a frequency of approximately 50%. (B) IL-5Ra–blocking activity of the indicated anti–IL-5Ra Abs, as determined by the effect on the proliferation of TF-1/IL-5Ra cells after stimulation with rhIL-5 (80 pM) in the presence of Abs (0 to 32 nM) for 40 h. The absolute (Abs) IC50 values of the Abs are provided. Each point of the curve represents mean ± SD. (C) Overall structure of the human IL-5Ra–IL-5 complex (Protein Data Bank ID: 3QT2), where the three domains (D1, D2, and D3) within the extracellular regions of IL-5Ra are indicated. (D) The yeast surface display scheme for domain-swapped IL-5Ra variants, where the three extracellular domains of human IL-5Ra (hD1, hD2, or hD3) were replaced with the corresponding mIL-5Ra domain (mD1, mD2, or mD3). (E) Flow-cytometric analysis of yeast cell surface expression levels of the indicated wild-type IL-5Ra and domain-swapped IL-5Ra variants as well as binding activity of the indicated anti–IL-5Ra Abs (100 nM) toward the IL-5Ra variants. Representative histograms from three independent experiments are depicted.
Fig 2: Affinity improvement of hu2B7 to generate a more potent Ab, 5R65. (A) The scheme of VH-CDR2 library construction and screening of hu2B7 in the scFab format by the yeast surface display technology. The indicated residues (N) in the VH-CDR2 of hu2B7 were diversified by means of the NHB degenerate codon. (B) Evaluation of nonspecific binding of the indicated Abs (100 nM) to four plate-immobilized antigens [dsDNA, insulin, cardiolipin, and hemocyanin] with sIL-5Ra as a control, as determined by ELISAs. Data are depicted as mean ± SD. (C) The competitive ELISA showing the percentage of sIL-5Ra (50 nM) bound to plate-immobilized IL-5-mFc in the presence of the indicated Abs (0–0.5 µM) as compared to that without the Ab competitor. Fitted curves were obtained in the GraphPad software. IC50 values of the Abs are provided. Each point of the curve represents mean ± SD. (D) IL-5Ra–blocking activity of the indicated Abs, as evidenced by the influence on the proliferation of TF-1/IL-5Ra cells after stimulation with rhIL-5 (160 pM) in the presence of Abs (5 or 20 nM) for 40 h. Data are presented as a percentage (mean ± SD) of the proliferation relative to PBS-treated samples (vehicle). Significance was tested by two-way ANOVA followed by the Newman–Keuls post hoc test. ***P < 0.001; ns, not significant versus the benralizumab analogue–treated group.
Supplier Page from Sino Biological, Inc. for Human IL-5/IL5 / Interleukin 5 Gene ORF cDNA clone in cloning vector