new strategies for the prevention and treatment of graft vs. host disease (gvhd)
TRANSCRIPT
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New Strategies for the Prevention and Treatment of Graft vs. Host Disease (GVHD)
Simrit Parmar, MD
Stem Cell Transplant & Cellular Therapy
BTG2013, Hong Kong
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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
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Acute GVHD: Pathophysiology1. Recipient conditioning
2. Donor T cell activation
3. Cellular and Inflammatory Effectors
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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
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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)
DEATH
RELAPSE
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“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?”
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GVHD Prophylaxis
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“No Free Lunch” Principle
GVHD
• Relapse• Rejection• Delayed Immune
Reconstitution
GVHD
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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 allogeneicHCT in other clinical settings such as treatment of autoimmune diseases and tolerance induction for organ transplantation
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Risk of GVHD in Two Eras
Gooley et al. N. Engl. J Med 363:2091, 2010
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In vivo tracking of
light emitting donor cells
Allogeneic HCT
B
TM
BM BMBM
B
T
Bone Marrow
Splenocytes
FVB/N
WT
luc+
Balb/c
H-2q/Thy1.1H-2d/Thy1.2
CD4+
CD8+
B220+
NK1.1+
Gr-1/Mac-1+
2x105 cells/well Absolute light
emission
0.00 0.05 0.10 0.15
Luciferase 2A eGFPAct
luc+ reporter mouse
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Acute Graft-vs-Host Disease Development
Beilhack, A. et al. Blood. 2005. 106:1113
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The Evolution of acute GVHD
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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
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Regulation of Immune Function
• Critically important in health and disease
• Compartmentalization of immune responses
• Cytokines
• Regulatory T cells (Treg, NK-T, iTreg, others)
RegulationReactivity
T regulatory cell T effector cell
CD4+ T Cell Subsets
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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 alloreactiveresponses 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
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Regulatory T-cells
• Allogeneic HCT recipients with aGVHD had Tregfrequencies 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.
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Magenau et al. BBMT. 2010.
38%
63%
Circulating Tregs predict OS
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d15 Death from
GVHD
100
5000
1000
20000
1
10
100
1000
10000
0 20 40 60 0 20 40 600 20 40 60
Time [d] post BMT
Re
lative
Sig
nal In
ten
sity
0
25
50
75
100
0 20 40 60
Time [d] post BMT
Su
rviv
al [%
]
TCD BM only, n = 14
TCD BM + Tcon, n = 15
TCD BM + Tcon + Treg n = 9
Control of GVHD with Retention of GVL
TconBM only Tcon + Treg
500
5000
d5
Edinger et al. Nature Medicine 9:1144, 2003
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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
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Expanded CB Tregs show FOXP3 demethylation and suppress alloMLR
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3rd Party CB Tregs Prevent GVHD
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In vivo tracking of Treg transduced with GFP and Firefly Luciferase
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Treg Treg+PBPC
Day -1
Day 0
Day 3
dorsal
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Treg Treg+PBPC
Day -1
Day 0
Day 3
dorsal
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Treg Treg+PBPC
Day -1
Day 0
Day 3
dorsal
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Treg Treg+PBPC
Day -1
Day 0
Day 3
dorsal
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Treg Treg+PBPC
Day 3
Day 10
ventral
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Proposed phase I Clinical Trial
Treg Doses to be Studied
Dose Cohort Treg Dose
Dose Level 1 1 × 105 Tregs/kg
Dose Level 2 5 × 105 Tregs/kg
Dose Level 3 1 × 106 Tregs/kg
Dose Level 4 5 × 106 Tregs/kg
Dose Level 5 1 × 107 Tregs/kg
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Next Step: Adoptive Therapy with Treg
Day-8
Day-7
Day-6
Day-5
Day-4
Day-3
Day-2
Day-1 0 +1 +2
Day+3
Day+4
Day+6
BUTestDose
32mg/m2
Rest BU BU BU BU BMT Infusionof
Ex-vivoExpanded
TregsFLU40
mg/m2
FLU40
mg/m2
FLU40
mg/m2
FLU40
mg/m2
CY**50
mg/kg
CY**50
mg/kg
Day-6
Day-5
Day-4
Day-3
Day-2
Day-1
0
MEL BU BU BU Infusion ofEx-vivo
ExpandedTregs
BMT
FLU40
mg/m2
FLU40
mg/m2
FLU40
mg/m2
FLU40
mg/m2
MMF+Sirolimus
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Individual clinical outcome of patients who received a Treg dose > 30x105/kg
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Haploidentical Transplant Schema (Stanford)
Mel, TT, Flu +Thymoglobulin@
0 +14 +16Day -10
CD34+ cell
selected
graft
CD4+CD25+
Treg
CD4+/CD8+
Tcon
Cell
Dose
5-10 x
106/kg
105/kg
3x105/kg
106/kg
Endpoints:
Chimerism
Immune reconstitution
Acute and chronic
GVHD
EFS, OS
BB IND13923
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Selection of CD4+CD25+ Tregs (U. Perugia)
Cells (x109) 1060 (540-1370) 280 (202- 390)
%CD4CD25 3.0 (1.5-7.45) 92.4 (90-97.1)
N° cells (x 106) 330 (221-1020) 256 (185.6-365.4)
%CD4CD25high 0.3 (0.12- 0.89) 33.6 (14.4-39.6)
N° cells (x 106) 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
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>50 >100 >2000
50
100
150
200
CD4/ l
Days p
ost
BM
T
>50 >100 >2000
20
40
60
80
100
CD8/ l
Days p
ost
BM
T
Recovery of CD4+ and CD8+ T cell subpopulations
0
50
100
150
200
250
1 2 3 4 5 6 7 8 9 10 11 12
Sp
ectr
aty
pe c
om
pexit
y S
co
re Donors
Months after transplant
Co
mp
lexity s
co
re
Spectratyping
Pattern of immunoreconstitution
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Evaluable Patients
Patients with CMV reactivation
0
10
20
30
40
50
60
70
80
90
100
0-30 31-60 61-90 91-120 121-150 151-180 181-365 >365
10096
82
75
67
56
48
2928
50
34
22
9 9
1 1
0
5
10
15
20
25
30
0-30 31-60 61-90 91-120 121-150 151-180 181-365 >365
27
21
16
109
5
212
5
10 0 0 0 0
Days after transplant
Days after transplant
CMV reactivation episodes
Tregs Group
Control Group
p<0.05
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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
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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