1. New Strategies for the Prevention and Treatment of Graft vs. Host Disease (GVHD) Simrit Parmar, MD Stem Cell Transplant & Cellular Therapy BTG2013, Hong Kong

2. Risk Factors for Acute GVHD HLA disparity Increasing age Donor and recipient gender disparity Type and status of underlying disease Amount of radiation and intensity of the transplant conditioning regimen Doses of methotrexate and cyclosporine or tacrolimus

3. Acute GVHD: Pathophysiology 1. Recipient conditioning 2. Donor T cell activation 3. Cellular and Inflammatory Effectors

4. Acute GVHD – Typically occurs around the time of engraftment. – Previously mis-defined as GVHD which occurs prior to day 100 post-transplant. – Three main organs involved: Skin: macularpapular rash GI system: Nausea / Vomiting and Diarrhea Liver Abnormalities: typically cholestatic (jaundice). – Incidence of 9-50% of sib transplants. Vigorito et al. Blood 2009

7. Acute GVHD: Survival and Relapse Grade 0 acute GVHD — hazard ratio (HR) for TRM: 1.0 Grade I — HR 1.5 (95% CI 1.2-2.0) Grade II — HR 2.5 (95% CI 2.0-3.1) Grade III — HR 5.8 (95% CI 4.4-7.5) Grade IV — HR 14.7 (95% CI 11-20) Grade 0 acute GVHD — hazard ratio (HR) for relapse 1.0 Grade I — HR 0.94 (95% CI 0.8-1.2) Grade II — HR 0.60 (95% CI 0.5-0.8) Grade III — HR 0.48 (95% CI 0.3-0.8) Grade IV — HR 0.14 (95% CI 0.02-0.99) DEATHDEATH RELAPSERELAPSE

8. “Be good or I’ll send you to transplant” “”I am telling you, by the time they get done with you, you’ll be wearing diapers” “Do you want a little vidaza or total body skin sloughing?”

9. GVHD Prophylaxis

10. “No Free Lunch” Principle GVHD Relapse Rejection Delayed Immune Reconstitution GVHD

11. Immune Function in HCT Dysfunctional immune responses are common in clinical medicine Major mechanism of disease control due to GVT reactions, yet major limitation of allogeneic HCT is GVHD Controlling GVHD could lead to use of allogeneic HCT in other clinical settings such as treatment of autoimmune diseases and tolerance induction for organ transplantation

12. Risk of GVHD in Two Eras Gooley et al. N. Engl. J Med 363:2091, 2010

13. In vivo tracking of light emitting donor cells Allogeneic HCT B T M BM B T Bone Marrow Splenocytes FVB/N WT luc+ Balb/c H-2 q /Thy1.1  H-2 d /Thy1.2 CD4+ CD8+ B220+ NK1.1+ Gr-1/Mac-1+ 2x10 5 cells/wellAbsolute light emission 0.000.050.100.15 Luciferase2AeGFP  Act luc+ reporter mouse

14. Acute Graft-vs-Host Disease Development Beilhack, A. et al. Blood. 2005. 106:1113

15. The Evolution of acute GVHD

16. Approaches to the Prevention of GVHD Pharmacologic – CNI/MTX – CNI/MTX vs Rapa/MTX Graft source – BM vs PBPC – MRD vs URD vs UCB T Cell depletion – CD34 Selection – ATG, Campath Immune regulation

17. Regulation of Immune Function Critically important in health and disease Compartmentalization of immune responses Cytokines Regulatory T cells (T reg, NK-T, iT reg, others) Regulation Reactivity T regulatory cell T effector cell CD4 + T Cell Subsets

18. CD4 + CD25 + Regulatory T Cells Major population of cells which regulate immune reactions Express transcription factor FoxP3 Deficiency or mutation of FoxP3 has autoimmune consequences in animal models and humans Cell contact-dependent suppression of alloreactive responses in mixed lymphocyte reactions (MLR) Prevent organ specific autoimmune diseases in animal models (e.g. IBD, diabetes) IL-10 and TGF-  implicated in mediating suppressive effect in vivo

19. Regulatory T-cells Allogeneic HCT recipients with aGVHD had Treg frequencies 40% less than those without aGVHD. Treg frequencies decreased linearly with acute GVHD severity. The frequency of Tregs at acute GVHD onset predicted response to therapy. Magenau et al. BBMT. 2010.

20. 38% 63% Circulating Tregs predict OS

21. d15 Death from GVHD 100 5000 1000 20000 Time [d] post BMT Relative Signal Intensity Survival [%] TCD BM only, n = 14 TCD BM + Tcon, n = 15 TCD BM + Tcon + Treg n = 9 Control of GVHD with Retention of GVL TconBM onlyTcon + Treg 500 5000 d5 Edinger et al. Nature Medicine 9:1144, 2003

22. Challenges for Clinical Translation of T reg T reg are rare cell populations Paucity of unique markers for isolation and availability of clinical grade reagents Marginal functional assays in humans Regulatory requirements

23. Expanded CB Tregs show FOXP3 demethylation and suppress alloMLR

24. 3 rd Party CB Tregs Prevent GVHD

25. In vivo tracking of Treg transduced with GFP and Firefly Luciferase

26. Treg Treg+PBPC Day -1 Day 0 Day 3 Day 10 dorsal

27. Treg Treg+PBPC Day -1 Day 0 Day 3 Day 10 dorsal

28. Treg Treg+PBPC Day -1 Day 0 Day 3 Day 10 dorsal

29. Treg Treg+PBPC Day -1 Day 0 Day 3 Day 10 dorsal

30. Treg Treg+PBPC Day 3 Day 10 ventral

31. Proposed phase I Clinical Trial Treg Doses to be Studied Dose CohortTreg Dose Dose Level 11 × 10 5 Tregs/kg Dose Level 25 × 10 5 Tregs/kg Dose Level 31 × 10 6 Tregs/kg Dose Level 45 × 10 6 Tregs/kg Dose Level 51 × 10 7 Tregs/kg

32. Next Step: Adoptive Therapy with Treg Day -8 Day -7 Day -6 Day -5 Day -4 Day -3 Day -2 Day- 10+1+2 Day +3 Day +4 Day +6 BU Test Dose 32mg/m 2 RestBU BMT Infusion of Ex-vivo Expanded Tregs FLU 40 mg/m 2 FLU 40 mg/ m2 FLU 40 mg/ m2 FLU 40 mg/ m2 CY** 50 mg/k g CY** 50 mg/k g Day -6 Day -5 Day -4 Day -3 Day -2 Day-1 0 MELBU Infusion of Ex-vivo Expanded Tregs BMT FLU 40 mg/m2 FLU 40 mg/m2 FLU 40 mg/m2 FLU 40 mg/m2 MMF+Sirolimus

34. Individual clinical outcome of patients who received a Treg dose > 30x10 5 /kg

36. Haploidentical Transplant Schema (Stanford) Mel, TT, Flu + Thymoglobulin @ 0+14+16Day-10 CD34 + cell selected graft CD4 + CD25 + T reg CD4 + /CD8 + T con Cell Dose 5-10 x 10 6 /kg 10 5 /kg 3x10 5 /kg 10 6 /kg Endpoints: Chimerism Immune reconstitution Acute and chronic GVHD EFS, OS BB IND13923

38. Selection of CD4 + CD25 + Tregs (U. Perugia) Cells (x10 9 ) 1060 (540-1370) 280 (202- 390) %CD4CD25 3.0 (1.5-7.45) 92.4 (90-97.1) N° cells (x 10 6 ) 330 (221-1020) 256 (185.6-365.4) %CD4CD25 high 0.3 (0.12- 0.89) 33.6 (14.4-39.6) N° cells (x 10 6 ) 36.12 (19.98 - 84) 68.6 (20.9-143) Starting fraction Final fraction CD25 CD127 CD4 FoxP3 Gate on CD4CD25 +high Gate on CD4CD25 + Fox P3 + cells 71.9 ± 15 % Immunomagnetic Selection of CD4 + CD25 + Cells 1st step: Depletion of CD8+/CD19+cells 2ndstep: Enrichment of CD25+ cells

39. Recovery of CD4 + and CD8 + T cell subpopulations Donors Months after transplant Complexity score Spectratyping Pattern of immunoreconstitution

40. Evaluable Patients Patients with CMV reactivation Days after transplant CMV reactivation episodes Tregs Group Control Group p<0.05

41. Outcomes – U. of Perugia Event-Free Survival 12/26 (46%) Regimen Related Toxicities: – Veno-occlusive disease (3) – Multi-organ failure (1) Acute GVHD grade III-IV (2) Serious infections (7) Relapse (AML 1) Median follow-up 18.5 months (range 16.1-27.6) D’Ianni et al. Blood 2011

42. Conclusions GVHD remains the most significant complication following allogeneic HCT Murine studies have demonstrated that immune regulatory mechanisms play a significant role in controlling dysfunctional immune responses including GVHD Clinical translation is ongoing with promising early results