This document discusses using ex vivo expanded and fucosylated regulatory T cells (Tregs) from third party umbilical cord blood to prevent graft-versus-host disease (GVHD) after transplantation. Experiments in mice show that fucosylated Tregs home more effectively to sites of inflammation, persist longer in vivo, and reduce GVHD symptoms and mortality more than untreated Tregs. The document proposes a clinical trial to evaluate the safety and efficacy of fucosylated Tregs for preventing GVHD in patients receiving double cord blood transplants.
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Third Party CB Tregs Fucosylation Prevents GVHD
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.
4. Acute GVHD: Pathophysiology
1. Recipient conditioning
2. Donor
T cell activation
3. Cellular and
Inflammatory Effectors
5. 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
6. 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
15. 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
23. 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
25. 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
26. 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
32. 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
33. 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
34. 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
35. Fucosylated Tregs persist for longer
duration in vivo vs. untreated Tregs
Day 7
Day 10
Day 12
FTVI-TREG TREG
36. 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
37. 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
40. 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
41. 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.