Structural optimization of a TNFR1-selective antagonistic TNFα mutant to create new-modality TNF-regulating biologics

Masaki Inoue,Yuta Tsuji,Chinatsu Yoshimine,Shota Enomoto,Yuki Morita,Natsuki Osaki,Masahiro Kunishige,Midori Miki,Shota Amano,Kanako Yamashita,Haruhiko Kamada,Yasuo Tsutsumi,Shin-Ichi Tsunoda

doi:10.1074/jbc.ra120.012723

Abstract

Excessive activation of the proinflammatory cytokine tumor necrosis factor-α (TNFα) is a major cause of autoimmune diseases, including rheumatoid arthritis. TNFα induces immune responses via TNF receptor 1 (TNFR1) and TNFR2. Signaling via TNFR1 induces proinflammatory responses, whereas TNFR2 signaling is suggested to suppress the pathophysiology of inflammatory diseases. Therefore, selective inhibition of TNFR1 signaling and preservation of TNFR2 signaling activities may be beneficial for managing autoimmune diseases. To this end, we developed a TNFR1-selective, antagonistic TNFα mutant (R1antTNF). Here, we developed an R1antTNF derivative, scR1antTNF-Fc, which represents a single-chain form of trimeric R1antTNF with a human IgG-Fc domain. scR1antTNF-Fc had properties similar to those of R1antTNF, including TNFR1-selective binding avidity, TNFR1 antagonistic activity, and thermal stability, and had a significantly extended plasma t1/2in vivo In a murine rheumatoid arthritis model, scR1antTNF-Fc and 40-kDa PEG-scR1antTNF (a previously reported PEGylated form) delayed the onset of collagen-induced arthritis, suppressed arthritis progression in mice, and required a reduced frequency of administration. Interestingly, with these biologic treatments, we observed an increased ratio of regulatory T cells to conventional T cells in lymph nodes compared with etanercept, a commonly used TNF inhibitor. Therefore, scR1antTNF-Fc and 40-kDa PEG-scR1antTNF indirectly induced immunosuppression. These results suggest that selective TNFR1 inhibition benefits the management of autoimmune diseases and that R1antTNF derivatives hold promise as new-modality TNF-regulating biologics.

Highlights

Biologics represented by antibody drugs have dramatically changed the way of treating autoimmune diseases, including rheumatoid arthritis, psoriasis, and ulcerative colitis
The N terminus and C terminus of the R1antTNF monomer are located on the same side as demonstrated by crystal structure data (PDB ID 2E7A), and the single-chain structure of R1antTNF was generated and properly functioned as a TNF receptor 1 (TNFR1) antagonist
The model structure of scR1antTNF-Fc demonstrated that TNFR1 antagonist proteins become bivalent by fusion with the human IgG1-Fc domain via a hinge region (Fig. 1A and Fig. S1A)

Summary

Introduction

Biologics represented by antibody drugs have dramatically changed the way of treating autoimmune diseases, including rheumatoid arthritis, psoriasis, and ulcerative colitis. To evaluate the effects of scR1antTNF-Fc treatment on the development of arthritis, we investigated the severity of CIA mice treated with saline, etanercept (1250 mg/kg), and scR1antTNF-Fc (50 mg/kg) twice a week for 3 weeks (Fig. 6A). The results of grading right front or right hind paws indicated that the arthritis states (cell infiltration, synovitis, destruction of cartilage, the juxta-articular bone involvement) were suppressed in etanercept (1250 mg/kg)- or scR1antTNF-Fc (50 mg/kg)– treated mice (Fig. 7B).

Results

Conclusion