STAT3-Specific Single Domain Nanobody Inhibits Expansion of Pathogenic Th17 Responses and Suppresses Uveitis in Mice.

Evaristus C Mbanefo,Sahar A Alhakeem,Ming Yan,Cheng-Rong Yu,Charles E Egwuagu,Yingyos Jittayasothorn,Sunanda Singh,Ashutosh Parihar,Minkyung Kang

doi:10.3389/fimmu.2021.724609

Abstract

STAT3 activates transcription of genes that regulate cell growth, differentiation, and survival of mammalian cells. Genetic deletion of Stat3 in T cells has been shown to abrogate Th17 differentiation, suggesting that STAT3 is a potential therapeutic target for Th17-mediated diseases. However, a major impediment to therapeutic targeting of intracellular proteins such as STAT3 is the lack of efficient methods for delivering STAT3 inhibitors into cells. In this study, we developed a novel antibody (SBT-100) comprised of the variable (V) region of a STAT3-specific heavy chain molecule and demonstrate that this 15 kDa STAT3-specific nanobody enters human and mouse cells, and induced suppression of STAT3 activation and lymphocyte proliferation in a concentration-dependent manner. To investigate whether SBT-100 would be effective in suppressing inflammation in vivo, we induced experimental autoimmune uveitis (EAU) in C57BL/6J mice by active immunization with peptide from the ocular autoantigen, interphotoreceptor retinoid binding protein (IRBP651-670). Analysis of the retina by fundoscopy, histological examination, or optical coherence tomography showed that treatment of the mice with SBT-100 suppressed uveitis by inhibiting expansion of pathogenic Th17 cells that mediate EAU. Electroretinographic (ERG) recordings of dark and light adapted a- and b-waves showed that SBT-100 treatment rescued mice from developing significant visual impairment observed in untreated EAU mice. Adoptive transfer of activated IRBP-specific T cells from untreated EAU mice induced EAU, while EAU was significantly attenuated in mice that received IRBP-specific T cells from SBT-100 treated mice. Taken together, these results demonstrate efficacy of SBT-100 in mice and suggests its therapeutic potential for human autoimmune diseases.

Highlights

Cytokines such as IFN-g, IL-2, IL-4, IL-6, IL-10, IL-21, IL-23, IL-27, and IL-35 that regulate immune responses and autoimmune diseases mediate their biological activities through the activation of the Janus Kinase (JAK)/signal transducer and activator of transcription factor (STAT) pathway [1, 2]
Previous studies have shown that activation of STAT3 pathway regulates T cell proliferation and Th17 differentiation while loss of STAT3 in T cells prevents development of central nervous system (CNS) autoimmune diseases [7, 8, 15]
Uveitis is a diverse group of potentially sight-threatening intraocular inflammatory diseases that is characterized by repeated cycles of remission and recurrent intraocular inflammation, and visual handicap is of significant public health importance as it affects the patient’s quality of life

Summary

Introduction

Cytokines such as IFN-g, IL-2, IL-4, IL-6, IL-10, IL-21, IL-23, IL-27, and IL-35 that regulate immune responses and autoimmune diseases mediate their biological activities through the activation of the Janus Kinase (JAK)/STAT pathway [1, 2]. This evolutionary conserved signal transduction pathway is orchestrated by the 4 Janus kinases (Jak, Jak, Jak, Tyk2) and the 7-member signal transducer and activator of transcription factor (STAT) family of proteins, STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6. Mice with targeted deletion of STAT3 in CD4+ T cells are resistant to development of experimental autoimmune uveitis (EAU) and experimental autoimmune encephalomyelitis (EAE) [7, 8], indicating that STAT3 is a potential therapeutic target for these central nervous system (CNS) autoimmune diseases and other autoinflammatory diseases

Methods

Results

Conclusion