Abstract
Development of antagonistic antibody (Ab) against interleukin-4 receptor alpha (IL-4Rα) subunit of IL-4/IL-13 receptors is a promising therapeutic strategy for T helper 2 (TH2)-mediated allergic diseases such as asthma and atopic dermatitis. Here we isolated anti-human IL-4Rα antagonistic Abs from a large yeast surface-displayed human Ab library and further engineered their complementarity-determining regions to improve the affinity using yeast display technology, finally generating a candidate Ab, 4R34.1.19. When reformatted as human IgG1 form, 4R34.1.19 specifically bound to IL-4Rα with a high affinity (KD ≈ 178 pM) and effectively blocked IL-4- and IL-13-dependent signaling in a reporter cell system at a comparable level to that of the clinically approved anti-IL-4Rα dupilumab Ab analogue. Epitope mapping by alanine scanning mutagenesis revealed that 4R34.1.19 mainly bound to IL-4 binding sites on IL-4Rα with different epitopes from those of dupilumab analogue. Further, 4R34.1.19 efficiently inhibited IL-4-dependent proliferation of T cells among human peripheral blood mononuclear cells and suppressed the differentiation of naïve CD4+ T cells from healthy donors and asthmatic patients into TH2 cells, the activities of which were comparable to those of dupilumab analogue. Our work demonstrates that both affinity and epitope are critical factors for the efficacy of anti-IL-4Rα antagonistic Abs.
Highlights
Allergic diseases such as asthma and atopic dermatitis affect a huge population globally, but a subset of severe cases are not managed efficiently[1]
The engineered human IgG1 Ab 4R34.1.19 bound to IL-4Rα with high affinity to competitively block the ligand binding to IL-4Rα and thereby suppressed both IL-4- and IL-13-dependent signaling in ex vivo assays with human immune cells at comparable levels of dupilumab analogue. 4R34.1.19 differed from dupilumab analogue in the binding epitopes and kinetics
To isolate IL-4Rα-specific Ab, the yeast library was screened against biotinylated IL-4Rα protein by one round of magnetic activated cell sorting (MACS), followed by three rounds of fluorescence activated cell sorting (FACS) by gradually decreasing antigen concentration in every round
Summary
Allergic diseases such as asthma and atopic dermatitis affect a huge population globally, but a subset of severe cases are not managed efficiently[1]. Dupilumab (DupixentTM), a humanized IgG4 Ab directed against human IL-4Rα, has been approved by U.S Food & Drug Administration for the treatment of atopic dermatitis[10,11] and is under clinical trials for treating asthma, suggesting that IL-4/IL-13 dual blocking by IL-4Rα antagonist is a plausible way for treating allergic diseases[12]. Another anti-IL-4Rα antagonistic IL-4 mutant protein, pitrakinra, is being evaluated for asthma[13]. The engineered human IgG1 Ab 4R34.1.19 bound to IL-4Rα with high affinity to competitively block the ligand binding to IL-4Rα and thereby suppressed both IL-4- and IL-13-dependent signaling in ex vivo assays with human immune cells at comparable levels of dupilumab analogue. 4R34.1.19 differed from dupilumab analogue in the binding epitopes and kinetics
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