1. DR5 is 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-8 is a monoclonal anti-human DR5 antibody, which induces apoptosis upon binding to its receptor, DR5. TRAIL, Death Receptors, TRA-8, and Apoptosis TRAIL - R1 R2 R3 R4 Osteoprotegerin Decoy Cell Res. 2004 Oct;14(5):359-72. TRA-8/TRAIL DR5 Apoptosis

2. TRA-8 can induce apoptosis in human RA synovial fibroblasts. Anti-human DR5 Antibody Triggers Apoptosis in 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. 3 1.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. Human DR5 Mouse DR5 Chimeric DR5 Human, mouse and chimeric DR5 Generation of human/mouse Chimeric DR5 Transgenic Mice Generation of hu/mo chimeric DR5 Tg mice Doesn’t trigger apoptosis in mouse cells Not recognized by anti-hu DR5 Recognized by anti-hu DR5 Trigger apoptosis in mouse cells

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

6. 6 CD8 + CD4 + CD19 + CD11b + Gr1 + CD11b + Ly6C + CD11b - Ly6C + Hu/mo DR5 Count B6 Ubc.Cre DR5 Ubc.Cre Ly6C+ CD11b + CD4 + CD8 + CD19 + CD11b + Gr1 + hu/mo DR5 + (%) B6 Ubc.Cre DR5 Tg + Ubc.Cre 0 20 40 60 80 100 CD11b - Ly6C + CD11b + Ly6C + 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 (2x10 9 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. Tg DR5 Expression in Draining LN of Ubc.Cre DR5 Mice (2-mo post CII)

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

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

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

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

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

12. 12 Before TRA-8 treatment DR5 Tg - DR5 Tg + After TRA-8 treatment DR5 Tg - DR5 Tg + Caspase Activity ** DR5 Tg - DR5 Tg + Before TRA-8 After TRA-8 AB50-Cy5 Intensity/Joint 25 20 15 10 5 0 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. Apoptosis Induced by TRA-8 in Joints of DR5 Transgenic LysM.Cre Mice with CIA

13. 13 TUENL + cells in the sub synovial lining layer(%) ** Total M  Fibroblasts DR5 Tg - DR5 Tg + 50 40 30 20 10 0 DR5 Tg - DR5 Tg + H&E TUNEL Mac-3 H H E E H H E H H E H H E H H E H H TU M M 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. Apoptosis Induced by TRA-8 in Joints of DR5 Transgenic LysM.Cre Mice with CIA

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

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

16. 16 1.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; 2.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; 3.TRA-8 is potential novel biologic agent for rheumatoid arthritis. Summary I QUESTION: What is the TRA-8 effect in a systemic autoimmune disease model?

17. Prominent disorders of SHP-1 (PTPN6) deficient mice: 1.Myeloid hyper-proliferation and inappropriate activation. 2.Rapidly progressive patchy dermatitis, limb necrosis, recurrent infections, and premature death consequent to hemorrhagic pneumonitis. Joint Lung Mac-3 H.E. Chimeric DR5 Transgenic x Viable Motheaten Mice (me v /me v )

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

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

20. TRA-8 Treatment Reduces Tissue Inflammation and Increases Lifespan of DR5 Tg me v /me v 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 Summary II 1.DR5 expression is upregulated in inflammatory macrophages and pathogenic CD4 T cells in DR5 Tg me v /me v Mice; 2.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. 3.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. Thank you for your support Dr. John D. Mountz and lab members Dr. Hui-Chen Hsu and lab members Division of clinical rheumatology, UAB Dr. Robert P. Kimberly Dr. S. Louis Bridges Dr. David M. Spalding Dr. W. Winn Chatham Dr. Laura Hughes Acknowledgement