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Therapeutic Effects of TRA-8 on Rheumatoid Arthritis Synovial Fibroblasts: A Mouse Model Study

Investigating TRA-8 antibody's impact on RA synovial fibroblasts through a novel mouse model study. This research delves into TRA-8's potential in inducing apoptosis in pathogenic cells.

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Therapeutic Effects of TRA-8 on Rheumatoid Arthritis Synovial Fibroblasts: A Mouse Model Study

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  1. Decoy TRAIL - R1 R2 R3 R4 Osteoprotegerin Cell Res. 2004 Oct;14(5):359-72. TRAIL, Death Receptors, TRA-8, and Apoptosis • DR5is a pro-apoptotic molecule, which can induce cell death in a variety of cells by engaging the it ligand, TRAIL or anti-DR5 antibody. • TRA-8is a monoclonal anti-human DR5 antibody, which induces apoptosis upon binding to its receptor, DR5. TRA-8/TRAIL DR5 Apoptosis

  2. Anti-human DR5 Antibody Triggers Apoptosis in RA Synovial Fibroblasts • TRA-8 can induce apoptosis in human RA synovial fibroblasts. Rheumatoid Arthritis Synovial Fibroblasts Isotype control TRA-8 • Advantages of TRA-8 compared to TRAIL: Specificity to DR5; Long half-life; No hepatocellular toxicity. • Can TRA-8 induce apoptosis of pathogenic macrophages and T cells in RA?

  3. Creation of a mouse model with a humanized DR5 reactive with TRA-8. • 2. Control of expression through a Cre/loxp system, using both general (Ubiquitous C) and macrophage specific (Lysozyme M) expression Cre mice. • 3. Murine models of inflammatory arthritis (Collagen II-induced arthritis). Strategies Used to Determine the Arthritis Therapeutic Effects of TRA-8 in Mice

  4. Generation of human/mouse Chimeric DR5 Transgenic Mice Human DR5 Mouse DR5 Chimeric DR5 Not recognized by anti-hu DR5 Recognized by anti-hu DR5 Doesn’t trigger apoptosis in mouse cells Trigger apoptosis in mouse cells Human, mouse and chimeric DR5 Generation of hu/mo chimeric DR5 Tg mice

  5. Generation of human/mouse Chimeric DR5 Transgenic Mice Mo 3kb promoter Ubc.Cre LysM.Cre

  6. Tg DR5 Expression in Draining LN of Ubc.Cre DR5 Mice (2-mo post CII) B6 Ubc.Cre DR5 Ubc.Cre CD19+ CD4+ CD8+ CD4+ B6 Ubc.Cre DR5 Tg+ Ubc.Cre CD8+ CD19+ CD11b+ Gr1+ CD11b+ Ly6C+ CD11b-Ly6C+ CD11b+Gr1+ CD11b+ Ly6C+ Ly6C+ CD11b+ CD11b- Ly6C+ Count 0 20 40 60 80 100 Hu/mo DR5 hu/mo DR5+ (%) Induction of CIA. CIA was induced using DR5 Tg mice of C57BL/6 background that were 8- to 16-weeks old. Mice were immunized by intradermal administration of chicken Type II collagen in complete CFA, followed by injection of chicken CII in IFA on day 30 after the primary injection. To ensure a higher incidence of CII arthritis, an adenovirus expressing mouse IL-17 (2x109 pfu/mouse) was administered IV to all mice 2 days prior to the primary immunization with CII. TRA-8 treatment of CIA mice. TRA-8 (dissolved in PBS, 0.2 mg per mouse) or IgG1 isotype control was administered i.v. or i.p. twice/week starting on day 0 (early treatment) or on day 30 (late treatment) until mice were sacrificed.

  7. TRA-8 Suppresses the Development of CIA in Ubc.Cre DR5 Mice DR5 Tg+ UbcCre Isotype DR5 Tg+ UbcCre Early TRA-8 (from day 0) DR5 Tg+ UbcCre Late TRA-8 (from day 30) ** Arthritis Scores Early TRA-8 cCII cCII Late TRA-8 DR5Tg- DR5Tg+ H&E Mac-3 H&E Mac-3 Day 60 post 1 cCII

  8. TRA-8 Treatment Depletes Macrophages Question: What is the TRA-8 effect if TgDR5 expression is restricted in macrophages?

  9. M1/M2 Imbalance in Rheumatoid Arthritis - Can It Be Corrected by TRA-8? Current therapeutic targets related to macrophages Anti-Inflammatory Inflammatory Anti-GM-CSF Anti-M-CSF M-CSF IL-4 IL-13 IL-10 GM-CSF LPS IFN-γ TNF-α Imatinib mesylate Anti-TNF-α Clodoronate Liposomes Tofacitinib TNF-α CD80,86 IL-1, 6, 12, 23 IFN-I RNI ROI CXCL9,10,11 CCL2,3,4,5,15,20 iNOS Tyrosine kinase Tyrosine phosphatase IL-4,10,IL-1Ra, CD163, MR, GR CD200R TGF-β Ym1/2 Fizz1 CCL1,16,17,18,22 Arg1 CD200-Fc JAK IRF4 IRF5 RANK DR5 TRA-8 Fas IL-4R IL-10R RANKL TLR2,4 Denosumab Blocker Osteoclast Ligand or agonistic antibody Adapted from J. Li, et al 2012

  10. Generation of Chimeric DR5 Transgenic LysM.Cre Mice Mo 3kb promoter Ubc.Cre LysM.Cre

  11. TRA-8 Treatment Eliminates Inflammatory Macrophages in DR5 Transgenic LysM.Cre CIA Mice

  12. 25 ** 20 15 AB50-Cy5 Intensity/Joint 10 5 0 DR5 Tg-DR5 Tg+ DR5 Tg-DR5 Tg+ Before TRA-8 After TRA-8 Apoptosis Induced by TRA-8 in Joints of DR5 Transgenic LysM.Cre Mice with CIA Caspase Activity Before TRA-8 treatment DR5 Tg- DR5 Tg+ After TRA-8 treatment DR5 Tg- DR5 Tg+ Measurement method: Mice were induced to develop CIA. At 8 week after the primary cCII injection, Mice were then treated with TRA-8 (0.2 mg on day 0 and day 3) and apoptosis imaging using AB50-Cy5 was performed on the same mice on day 6 after initiating TRA-8 treatment.

  13. H&E TUNEL Mac-3 H H E H E E H M H H DR5 Tg- DR5 Tg+ E H H H E E M TU H H H DR5 Tg- DR5 Tg+ 50 ** 40 30 ** TUENL+ cells in the sub synovial lining layer(%) 20 ** 10 0 Total Mf Fibroblasts Apoptosis Induced by TRA-8 in Joints of DR5 Transgenic LysM.Cre Mice with CIA Measurement method: Mice were induced to develop CIA. At 8 week after the primary cCII injection, Mice were then treated with TRA-8 (0.2 mg on day 0 and day 3) and were sacrificed for in situ staining of joint apoptosis (TUNEL) and joint macrophage (Mac-3) infiltration.

  14. DR5 Tg- Isotype DR5 Tg+ Isotype 12 DR5 Tg- TRA-8 DR5 Tg+ TRA-8 8 4 0 12 ** 8 Arthritis score Arthritis score 4 0 10 20 30 40 50 60 70 10 20 30 40 50 60 70 cCII cCII cCII cCII DR5 Tg TRA-8 Treatment DR5 Tg+ TRA-8 Treatment H&E Mac-3 H&E Mac-3 TRAP TRAP 20 X 10 X E TR H M H H E E TRA-8 Treatment Ameliorates the Severity of CIA in DR5 Transgenic LysM.Cre Mice TRA-8

  15. Immuno-modulatory Effects of TRA-8 in DR5 Transgenic LysM.Cre Mice with CIA

  16. Summary I • In human/mouse DR5 Tg Ubc.Cre mice with CIA, Tg DR5 is expressed on CD11b+ macrophages and CD4+ T cells; In DR5 Tg LysM.Cre mice, Tg DR5 is restrictively expressed on macrophages; • TRA-8 (anti-human DR5) treatment results in depletion of pathogenic macrophages and Th17 cells, increased Treg cells in draining LNs of DR5 Tg CIA mice; • TRA-8 is potential novel biologic agent for rheumatoid arthritis. QUESTION: What is the TRA-8 effect in a systemic autoimmune disease model?

  17. Chimeric DR5 Transgenic x Viable Motheaten Mice (mev/mev) H.E. Mac-3 Joint Joint Lung • Prominent disorders of SHP-1 (PTPN6) deficient mice: • Myeloid hyper-proliferation and inappropriate activation. • Rapidly progressive patchy dermatitis, limb necrosis, recurrent infections, and premature death consequent to hemorrhagic pneumonitis.

  18. DR5 expression and TRA-8 Induced Depletion of Inflammatory Macrophages in DR5 Tg mev/mev Mice

  19. DR5 expression and TRA-8 Induced Depletion of Pathogenic CD4 T Cells in DR5 Tg mev/mev Mice

  20. TRA-8 Treatment Reduces Tissue Inflammation and Increases Lifespan of DR5 Tg mev/mev Mice

  21. TRA-8 Eliminates Inflammatory M1 Macrophages and CD4 T Cells in Human RA Patients with Low PTPN6 M1 signature genes Th17 signature genes

  22. Summary II Summary • DR5 expression is upregulated in inflammatory macrophages and pathogenic CD4 T cells in DR5 Tg mev/mev Mice; • TRA-8 selectively eliminates IRF5+ IL-23+ pathogenic macrophages and IL-17+ GM-CSF+ CD4 T cells in both human and mouse autoimmune conditions, resulting in immune homeostasis and resolution of inflammation. • Anti-human DR5 (TRA-8) can deplete inflammatory cell populations that result from a hyperactive GM-CSF/IRF5 AXIS, and it is a novel biologic agent for RA and other autoimmune diseases. β

  23. Acknowledgement Division of clinical rheumatology, UAB Dr. John D. Mountz and lab members Dr. Hui-Chen Hsu and lab members Dr. Robert P. Kimberly Dr. S. Louis Bridges Dr. David M. Spalding Dr. W. Winn Chatham Dr. Laura Hughes Thank you for your support

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