Blocking of the IL-33/ST2 Signaling Axis by a Single-Chain Antibody Variable Fragment (scFv) Specific to IL-33 with a Defined Epitope.

Publisher: MDPI Country of Publication: Switzerland NLM ID: 101092791 Publication Model: Electronic Cited Medium: Internet ISSN: 1422-0067 (Electronic) Linking ISSN: 14220067 NLM ISO Abbreviation: Int J Mol Sci Subsets: MEDLINE

Original Publication: Basel, Switzerland : MDPI, [2000-

Epitopes*/chemistry ; Epitopes*/immunology ; Interleukin-1 Receptor-Like 1 Protein*/chemistry ; Interleukin-1 Receptor-Like 1 Protein*/immunology ; Interleukin-33*/antagonists & inhibitors ; Interleukin-33*/chemistry ; Interleukin-33*/immunology ; Single-Chain Antibodies*/chemistry ; Single-Chain Antibodies*/immunology; MAP Kinase Signaling System/*immunology; Cell Line ; Humans

Interleukin 33 (IL-33) is an IL-1 family cytokine that plays a central role in immune system by regulating and initiating inflammatory responses. The binding of IL-33 to the suppressor of tumorigenicity 2 (ST2) receptor induces mitogen-activated protein kinases (MAPK) and nuclear factor κB (NF-κB) pathways, thereby leading to inflammatory cytokines production in type 2 helper T cells and type 2 innate lymphoid cells. To develop an antibody specific to IL-33 with a defined epitope, we characterized a single-chain antibody variable fragments (scFvs) clone specific to IL-33, C2_2E12, which was selected from a human synthetic library of scFvs using phage display. Affinity ( K d ) of C2_2E12 was determined to be 38 nM using enzyme-linked immunosorbent assay. C2_2E12 did not show cross-reactivity toward other interleukin cytokines, including closely related IL-1 family cytokines and unrelated proteins. Mutational scanning analysis revealed that the epitope of IL-33 consisted of residues 149-158 with key residues being L150 and K151 of IL-33. Structural modeling suggested that L150 and K151 residues are important for the interaction of IL-33 with C2_2E12, implicating that C2_2E12 could block the binding of ST2 to IL-33. Pull-down and in-cell assays supported that C2_2E12 can inhibit the IL-33/ST2 signaling axis. These results suggest that the scFv clone characterized here can function as a neutralizing antibody.

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NRF-2018R1A2B6004367 National Research Foundation of Korea; NRF-2017R1A5A1014560 National Research Foundation of Korea; NRF-2020M3A9E4038938 National Research Foundation of Korea; PJ01367602 Rural Development Administration

Keywords: C2_2E12; ST2 receptor; interleukin 33; scFv

0 (Epitopes)
0 (IL1RL1 protein, human)
0 (IL33 protein, human)
0 (Interleukin-1 Receptor-Like 1 Protein)
0 (Interleukin-33)
0 (Single-Chain Antibodies)

Date Created: 20200925 Date Completed: 20210224 Latest Revision: 20210224

20231215

PMC7554688

10.3390/ijms21186953

32971846