Design of Targeted B Cell Killing Agents

Alexey V Stepanov,Sergey M Deyev,Ivan V Smirnov,Alexey A Belogurov,Natalia A Ponomarenko,Alexander G Gabibov,Alexander G Tonevitsky,Leonid V Kozlov,Olga G Shamborant,Oleg A Stremovskiy,Dmitry S Balabashin,Alain Friboulet,Lidia P Sashchenko,Sergey E Dmitriev,Anna M Bichucher

doi:10.1371/journal.pone.0020991

Abstract

B cells play an important role in the pathogenesis of both systemic and organ-specific autoimmune diseases. Autoreactive B cells not only produce autoantibodies, but also are capable to efficiently present specific autoantigens to T cells. Furthermore, B cells can secrete proinflammatory cytokines and amplify the vicious process of self-destruction. B cell-directed therapy is a potentially important approach for treatment of various autoimmune diseases. The depletion of B cells by anti-CD20/19 monoclonal antibody Retuximab® used in autoimmune diseases therapy leads to systemic side effects and should be significantly improved. In this study we designed a repertoire of genetically engineered B cell killers that specifically affected one kind of cells carrying a respective B cell receptor. We constructed immunotoxins (ITs), fused with c-myc epitope as a model targeting sequence, based on barnase, Pseudomonas toxin, Shiga-like toxin E.coli and Fc domain of human antibody IgGγ1. C-MYC hybridoma cell line producing anti-c-myc IgG was chosen as a model for targeted cell depletion. C-myc sequence fused with toxins provided addressed delivery of the toxic agent to the target cells. We demonstrated functional activity of designed ITs in vitro and showed recognition of the fusion molecules by antibodies produced by targeted hybridoma. To study specificity of the proposed B cells killing molecules, we tested a set of created ITs ex vivo, using C-MYC and irrelevant hybridoma cell lines. Pseudomonas-containing IT showed one of the highest cytotoxic effects on the model cells, however, possessed promiscuous specificity. Shiga-like toxin construct demonstrated mild both cytotoxicity and specificity. Barnase and Fc-containing ITs revealed excellent balance between their legibility and toxic properties. Moreover, barnase and Fc molecules fused with c-myc epitope were able to selectively deplete c-myc-specific B cells and decrease production of anti-c-myc antibodies in culture of native splenocytes, suggesting their highest therapeutic potential as targeted B cell killing agents.

Highlights

Findings of the last two decades suggest that B cells play a significant role in autoimmune diseases [1,2]
Autoreactive B cells may act as antigen-presenting cells in autoimmune diabetes and multiple sclerosis [10], being one of the major cytokine producers in autoimmune disorders [11]
B cells producing Abs to self-antigens act as pathogenic entities, but recent findings indicate that distinct B cell subsets may have regulatory functions by suppressing unwanted autoaggressive T cells response, and ameliorate the progression of autoimmune diseases [12,13,14]

Summary

Introduction

Findings of the last two decades suggest that B cells play a significant role in autoimmune diseases [1,2]. It may include several pathways, such as production of antibodies (Abs) towards selfantigens, immune complex formation [3], or cytokine and chemokine secretion by autoreactive B cells [4,5,6]. B cells producing Abs to self-antigens act as pathogenic entities, but recent findings indicate that distinct B cell subsets may have regulatory functions by suppressing unwanted autoaggressive T cells response, and ameliorate the progression of autoimmune diseases [12,13,14]. Similar to the T cells [15], B cells may be considered as an important target for the immune intervention

Methods

Results

Conclusion