regulatory t cells and gvhd

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Novel applications of regulatory T cell therapy …

Novel applications of regulatory T cell therapy
Simrit Parmar, MD

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  • 1. Third Party Cord Blood Derived Fucosylated Regulatory T cell Prevent Graft versus Host Disease Simrit Parmar, MD
  • 2. Acute GVHD • 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
  • 3. Acute GVHD: Pathophysiology 1. Recipient conditioning 2. Donor T cell activation 3. Cellular and Inflammatory Effectors
  • 4. 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
  • 5. Regulation of Immune Function • Critically important in health and disease • Compartmentalization of immune responses • Cytokines • Regulatory T cells (Treg, NK-T, iTreg, others) T effector cell T regulatory cell CD4+ T Cell Subsets Reactivity Regulation
  • 6. 63% 38% Circulating Tregs predict OS Magenau et al. BBMT. 2010.
  • 7. Day 0 Phenotype Positive Selection Negative Selection
  • 8. Expanded Cells show Treg Phenotype
  • 9. Expanded UCB Tregs maintain Gaussian Distribution of TCR VβRepertoire
  • 10. Expanded Tregs are Functional
  • 11. Expanded UCB Tregs show FOXP3 Demethylation
  • 12. Xenogenic GVHD Mouse Model Lymphoid follicle Extra-medullary hematopoeisis mixed with histiocytes Spleen Small Intestine 400x (Apoptosis with Enteritis) Apoptotic bodies diagnostic for GvHD (arrows) Interstitial edema Apoptotic bodies Skin vein B.D. A B.D. Yellow arrows represent dead hepatocytes. Outlined area is a combination of necrotic hepatocytes, fibrosis, and some lymphocytes. Scattered dark blue nuclei within outlined area are mostly lymphocytes. Liver 40X Small Intestine Liver 40X
  • 13. Firefly luciferase labeled CB Tregs were able to proliferate in response to stimulation by PBMC
  • 14. 3rd party CB Tregs prevent GVHD CONTROL TREG PROPHYLAXIS Day -1 UCB Treg 1x107 Day -1 320 cGy Day 0 PBMC 1x107
  • 15. 3rd party CB Tregs prevent GVHD Phenotype Weight GVHD Score
  • 16. 3rd party CB Tregs prevent GVHD Circulating Inflammatory Cytokines Overall Survival
  • 17. 3rd party CB Tregs prevent GVHD PBMC PBMC+Treg
  • 18. Challenges for Clinical Translation of Treg • Treg are rare cell populations • Paucity of unique markers for isolation and availability of clinical grade reagents • Marginal functional assays in humans • Regulatory requirements
  • 19. Can we improve the Efficacy of CB Tregs?
  • 20. Enhancing selectin binding through ex vivo fucosylation Hematopoietic Fucosyltransferase (FT) VI progenitor cell Fucose Glycoprotein Fucosylated glycoprotein √ X P- & E-Selectin BM endothelium Impact on homing & engraftment? Bone marrow microvasculature
  • 21. Fucosylation of CB CD34+ cells using FT-VI enhances engraftment in NSG mice 4.5x104 CD34+ cells/mouse Fucosylated Untreated washed cb. 008 Untreated 100 101 102 103 104 CB FTVI 40.008 CLA F ITC Fucosylated 100 101 102 103 104 CLA CLA F ITC CD34
  • 22. Does Fucosylation have effect on Treg homing?
  • 23. Fucosylation efficiency of Treg FTVI Treated 0.4% 8.8% 0.3% 62% Biotin IgM Strep APC Biotin CLA Strep APC UnTreated Biotin IgM Strep APC Biotin CLA Strep APC
  • 24. Fucosylated Tregs and Selectin Pathway Binding?
  • 25. Fucosylation of Tregs leads to increased ability to bind E-selectin ligand Figure 1D Figure 1E
  • 26. Homing pattern of fucosylated Treg vs non-fucosylated Treg FTVI
  • 27. CB CD25 selection CD25+ cells eGFP-FFluc retro-viral transduction Continually culture for 11 days Experiment procedure Treg expressing eGFP-FFluc No FT-VI treatment FT-VI treatment Treg FT-Treg
  • 28. Experiment procedure No FT-VI treatment CB CD25 selection CD25+ cells eGFP-FFluc retro-viral transduction Continually culture for 11 days Treg expressing eGFP-FFluc FT-VI treatment Treg FT-Treg Day -2 Day -1 Day 0 XRT Treg/FT-Treg PBPC cell dose: 107/mouse
  • 29. Day -1 Day 0 Day 3 Day 4 Day 5 In vivo Bio-distribution of Fucosylated Tregs vs. Untreated Tregs FTVI-TREG TREG Treg: HLA-A2 negative/ GFP PBPC: HLA-A2 positive
  • 30. Fucosylated Tregs persist for longer duration in vivo vs. untreated Tregs Day 7 Day 10 Day 12 FTVI-TREG TREG
  • 31. Lesser number of GVHD causing CD45+ lymphocytes are present in Fucosylated Treg Recipients LNs FT-TREG+PBPC TREG+PBPC BM 10.4673 50.8491 Axillary LN 1.93 93.4065 Mesenteric LN 1.13 42.1731 Spleen 37.5158 94.4055 Inguinal LN 13.2 51.1461 Cervical LN 0 11.6
  • 32. Are Fucosylated Tregs more Effective? Fucosylated Tregs 1 x 10e6 Untreated Tregs 1 x 10e6 PBPC 1 x 10e7 PBPC 1 x 10e7 Day -2 320 cGy Day -1 Day 0 Monitor for weight, GVHD score and survival
  • 33. Fucosylated Treg recipient maintain weight for longer duration (Actual weight – baseline weight)/ baseline weight
  • 34. Fucosylated Treg Recipients have significantly longer survival compared to Untreated Tregs
  • 35. Proposed Clinical Trial Day -8 Day -7 Day -6 Day -5 Day -4 Day -1 Day 0 Flu Flu Flu Flu TBI Treg 40mg/m2 40mg/m2 40mg/m2 40mg/m2 infusion CB Infusion Cy 50mg/kg + MESNA Flu, fludarabine; Cy, cyclophosphamide Fucosylated Tregs Immune Prophylaxis with MMF + Sirolimus
  • 36. Timeline Year 1 2 3 Quarter 1 2 3 4 1 2 3 4 1 2 3 4 41 Specific Aim 1 1. Validate clinical grade Treg expansion with CD3/28 beads in presence of interleukin-2 and subsequent fucosylation in the GMP laboratory 2. FDA IND application and IRB approval of the clinical protocol 3. Enrollment of first 5 patients onto the clinical protocol 4. Enrollment of the next cohort of 7 patients 5. Enrollment of the last cohort of 8 patients 6. Assess Engraftment and Chimerism Analysis 7. Perform immune correlatives and study GVHD biomarkers and circulating inflammatory cytokines Specific Aim 2 1. Elucidate the homing pattern of fucosyltransferase-VI vs. VII treated Tregs in a xenogenic GVHD mouse model. 2. Determine the role of the selectin ligands PSGL-1 in FT-VI vs. FT-VII-mediated CB Treg homing to the sites of inflammation in the NSG GVHD model. Manuscript 1: Third party Cord Blood Derived Ex Vivo Expanded and Fucosylated Regulatory T Cells Effectively Prevent GVHD Manuscript 2: Saftey and Efficacy of Fucosylated Tregs in patients undergoing Double Cord Blood Transplant. Future Plans: Based on the clinical outcome of the role fucosylated Tregs in preventing GVHD in the double cord blood transplant setting, we plan to extend our clinical trial to include other high risk transplants including mismatched unrelated donor and haplo-identical transplant. In addition, if the preclinical data with fucosyltransferase VII appears to be promising, we will work with the Targazyme company to generate clinical grade FT-VII enzyme for the purpose of a pilot study.