**Soo Bin Park, Sun-Jick Kim, Sang Woo Cho, Cheol Yong Choi and Sangho Lee \***

Department of Biological Sciences, Sungkyunkwan University, Suwon 16419, Korea; stephanie7007@daum.net (S.B.P.); godmouse@skku.edu (S.-J.K.); aksgdlwkd@skku.edu (S.W.C.); choicy@skku.ac.kr (C.Y.C.)

**\*** Correspondence: sangholee@skku.edu; Tel.: +82-31-290-5913

Received: 28 August 2020; Accepted: 16 September 2020; Published: 22 September 2020

**Abstract:** 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.

**Keywords:** interleukin 33; ST2 receptor; scFv; C2\_2E12
