Design of an Anti-HMGB1 Synthetic Antibody for In Vivo Ischemic/Reperfusion Injury Therapy

• Published in: Journal of the American Chemical Society 2023-10, Vol.145 (42), p.23143-23151
• Main Authors: Koide, Hiroyuki, Kiyokawa, Chiaki, Okishima, Anna, Saito, Kaito, Yoshimatsu, Keiichi, Fukuta, Tatsuya, Hoshino, Yu, Asai, Tomohiro, Nishimura, Yuri, Miura, Yoshiko, Oku, Naoto, Shea, Kenneth J.
• Format: Article
• Language: English
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/jacs.3c06799

High-mobility group box 1 (HMGB1) is a multifunctional protein. Upon injury or infection, HMGB1 is passively released from necrotic and activated dendritic cells and macrophages, where it functions as a cytokine, acting as a ligand for RAGE, a major receptor of innate immunity stimulating inflammation responses including the pathogenesis of cerebral ischemia/reperfusion (I/R) injury. Blocking the HMGB1/RAGE axis offers a therapeutic approach to treating these inflammatory conditions. Here, we describe a synthetic antibody (SA), a copolymer nanoparticle (NP) that binds HMGB1. A lightly cross-linked N-isopropylacrylamide (NIPAm) hydrogel copolymer with nanomolar affinity for HMGB1 was selected from a small library containing trisulfated 3,4,6S-GlcNAc and hydrophobic N-tert-butylacrylamide (TBAm) monomers. Competition binding experiments with heparin established that the dominant interaction between SA and HMGB1 occurs at the heparin-binding domain. In vitro studies established that anti-HMGB1-SA inhibits HMGB1-dependent ICAM-1 expression and ERK phosphorylation of HUVECs, confirming that SA binding to HMGB1 inhibits the proteins’ interaction with the RAGE receptor. Using temporary middle cerebral artery occlusion (t-MCAO) model rats, anti-HMGB1-SA was found to accumulate in the ischemic brain by crossing the blood–brain barrier. Significantly, administration of anti-HMGB1-SA to t-MCAO rats dramatically reduced brain damage caused by cerebral ischemia/reperfusion. These results establish that a statistical copolymer, selected from a small library of candidates synthesized using an “informed” selection of functional monomers, can yield a functional synthetic antibody. The knowledge gained from these experiments can facilitate the discovery, design, and development of a new category of drug.