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PACT Meeting, Houston Wagner
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T Regulatory CellsT Regulatory CellsTranslational Development and Clinical Testing
John E. Wagner, M.D.University of Minnesotay
PACT ProgramBaylor College of Medicine
22 October 2012
Three dreaded words
‘You have cancer’
Leukemia
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Chemotherapy
Diagnosis ofAcute Leukemia
RemissionRemission
HLA identical siblingdonor
No sibling donor
Transplant Chemotherapy AML
G ftAlternative donor
search
Adult HSC
Factors ‐ age
‐ comorbidities
‐ donor
Graft vs
Leukemia
Cord Blood
Relapse
Phase I Agent
Barriers
Delayed Cell AcquisitionLimited Access
Adult Volunteer Donor Registries y q‐Donor attrition‐Process lag times‐Extended ‘matching’ requirements
High TRM‐High risk of aGVHD/cGVHD‐Prolonged immunodeficiency state ‐T i diti i i
Adult Volunteer Donor Registries
BMT (14,000,000 donors)
HLA A (allele level)
HLA B (allele level)
HLA C (allele level)
HLA DR (allele level)
Chance of finding a donor
50% Caucasians
Toxic conditioning regimens‐~45% unrelated marrow or PBSC
HLA DQ (not considered) 35% Hispanics
20% Afr Amer
8‐9% lower survival for each
HLA antigen mismatch
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Grades III‐IV Acute GvHD
IST
I
100%
90%
MTX + CsA + Pred (n = 948) MTX + CsA (n = 1577) Other IST (n = 979)MTX + FK-506 (n = 281)
Cutaneous GVHD
Incid
en
80%
70%
60%
50%
40%
30%
20%
0 1 4 2 8 4 2 5 6 7 0 8 4 9 8Days after Transplant GI GVHD
ce
20%
10%
0%
Tackling the ProblemsInitial Observations with UCB
Unique properties of theUnique properties of thefetal immune system
normal proliferative capacity but cytotoxicity
highly immunosuppressive
‐ T regulatory cells
Ci l ti t h bl t (IL 10)
Maternal‐Fetal ToleranceMechanisms
‐ Circulating trophoblasts (IL‐10)
Less HLA restriction
Immediately available
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Leukemia Free Survival in Children with
Acute Leukemia100
80
40
60
BM matched (n=116) 38%
CB matched (n=35) 60%
CB 1‐AG MM (n=157) 45%
CB 2‐Ag MM (n=267) 33%
20
0
g ( )
0 12 24 36 48 60
NYBC NCBP Months
Eapen et al. Lancet 2007; 369:1947‐54
Leukemia Free Survival in Adults with
Acute LeukemiaP=0.191.0
MM URDSIB
0.8
III I I I
I I IIIIII II IIII II I II
DUCB MUD
II
IIIII II
I0.6
0.4
0 2
Years post‐transplantation Brunstein and Delaney et al.
Blood 2010; 116: 4693‐4699
0 1 2 3 4 5
SIB
0.0
0.2
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Acute GVHD in Children
100
80
40
60
Grade 2‐4
BM matched (n=116), 50%
CB MM (n=454), 40%
BM MM (n=166), 70%
Days Eapen et al. Lancet 2007; 369:1947‐54
0 20 40 60 80 100
20
0
CB matched (n=35), 20%
Acute GVHD in Adults
Grade II‐IV Acute GvHD Grade III‐IV Acute GvHD
by Donor Type by Donor Type1.0
0.8
MMUD
SIBMUD 0.6
DUCB0.4
0.2
P (0.010 0
1.0
0.8
0.6
0.4
0.2
0 0
P ( 0.01
MMUD
DUCB
SIB
MUD
Brunstein and Delaney et al.
Blood 2010; 116: 4693‐4699
0.00.00 50 100 0 50 100
Days post‐transplantation Days post‐transpla ntation
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Perpetual Cycle of Translational Research
ProblemorNew
Observation Translational
Research
Clinical
Care
PatientR f l d
R&DScale UpReferrals and
EnrollmentScale‐UpValidation
Clinical
Research
Jumping over the Hurdles
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GVHD
Erythoderma and bullus formation
Prolonged Immuno‐Incompetence
HypothesisHypothesisGVHD results in tissue injury and delayed immune recovery. Therefore, elimination of GVHD will reduce tissue injury and j yenhance immune recovery, leading to reduced opportunistic infection and increased survival.
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What are T regulatory cells
specialized subpopulation of T cells
suppress T cell activation
maintain tolerance to self‐antigens.
In murine models, CD4+CD25+LSelhi phenotype bestdefines Tregs and protect against GVHD-associated
mortality
Taylor et al. Blood 2004;104:3804
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Tregs inhibited effector T cells expansionin GVHD target tissues
Elements of Strategic PlanningTranslational Medicine
Core competenciesCore competencies
‐ GMP cell and tissue manufacture
‐ Clinical trial design and monitoring
‐ Statistical (adverse events and efficacy)
Toxicity measures
Efficacy measures
‐ Surrogates versus clinical outcome
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As compared to peripheral blood, UCBTregs are a more distinct cell
populationPeripheral blood UCB
0.4 1.4
60 38
CD4
Godfrey et al. Blood. 2005 15;105(2):750-8. Godfrey et al. Blood. 2004
15;104(2):453-61.
UCB-Derived Regulatory T Cells arehighly and reproducibly suppressive
Godfrey et al. Blood. 2005 15;105(2):750-8.
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In humans, CD4+FoxP3+CD1 27- thephenotype better defines Tregs, is preserved
after expansion culture
McKenna et al. Cytotherapy 2010, 12 (suppl 1):19
Testing in relevant animalmodel UCB Tregs inhibit xenogeneic GI
GVHD
T cell infiltration and tissue
destruction
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Regulatory T cell Unit Expansion andActivation Culture
CD25+ l ti Fl k lt L t R lCD25+ selection Flask culture Lot Release
Gram stain neg Endotoxin < 5 EU/kg Viability ≥ 70% CD4+/CD25+ ≥ 70% CD3+/CD8+ ≤ 10% Sterility neg Mycoplasma neg Bead count < 100/3 x1 06 cells
Culture in X-VIVO 15 Human AB serum 10% , Anti-CD3/antiCD28-coated beads. Supplemented with IL-2 300 IU/mL
McKenna et al. Cytotherapy 2010, 12(suppl 1):19
UCB T Regulatory CellsProof of Concept
ld iFold ExpansionMedian 211
Range 13‐1796
N=23
14000
12000
10000
8000
6000
Brunstein C et al. Blo2010
Pre‐Expansion Post‐Expansion0
Mean4000
2000
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Phase I Treatment PlanUCB-derived Regulatory T Cells
Separation and start culture of UCB-derived UCBT
UC B-derived
UC B-derived
18 (±1) 9 8 7 6 5 4 3 2 1 0 +1 + 2 + 3 +15
Treg cells
UCB-derived Treg cells Production
Cyclophosphamide
Fludarabine
UCBTTreg cells Treg cells
Brunstein et al Blood 2011, 117:1061Brunstein et al Blood 2011, 117:1061Brunstein et al Blood 2011, 117:1061
-18 (±1) -9 -8 -7 -6 -5 -4 -3 -2 -1 0 +1 + 2 + 3 +15
MMFSi rolimusC SA
Phase I semi-log “fast-track” dose escalation:Level 1 -1 x 105/kg Level 2 -3 x 105/kg Level 3 -10 X
105/kg Level 4 -30 x 105/kg Level 5 - 30 x 105/kg (X2)Level 5ª -30 x 105/kg (X2) rapa
0 1 / 0 9
G-CSF
Infusional ToxicityEvent
CTCAE v. 3.0 Grade
1 2 3 4 5Allergic reaction
0 0 0 0 00 0 0 0 0Sinus bradycardia 1 0 0 0 0
Bleeding/hemorrhage 0 0 0 0 0
Sinus tachycardia 1 0 0 0 0
Hypertension 2 1 1 0 0
Hypotension 1 1 0 0 0
Fever 0 1 0 0 0
Rigors/chills 0 0 0 0 0
Brunstein et al Blood 2011, 117:1061Brunstein et al Blood 2011, 117:1061
Creatinine 0 0 0 0 0
Vomiting 4 0 0 0 0
Rash 0 0 0
Dyspnea 0 0 0 0 0
Hypoxia 0 0 0 0 0
Neurologic 1 1 0 0 0
Headache 0 1 0 0 0
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Sustained Donor EngraftmentHistorical Controls vs. Treg patients
Historical 86%1.0
0.8
Treg 87%
P = NS
0.6
0.4
0 2
0 7 14 21 28 35 42
Days post-UCBT
Brunstein et al Blood 2011, 117:1061
P = NS
0.0
0.2
Prevalence of mixed donor chimerismin unseparated BM at day 21
P=.06
Mixed chimeraSingle chimera
Historical Controls
Brunstein et al Blood 2011, 117:1061Brunstein et al Blood 2011, 117:1061
UCB-Derived Treg
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Grade II-IV Acute GVHDHistorical Controls vs. Treg patients
1.0 P = .0
0.8
0.6
Historical 60%
Treg 43%
0.4
Brunstein et al Blood 2011, 117:1061
0.2
0.0 0 20 40 60 80 100
Days post-UCBT
No difference in the risk Grade III-IV or chronic GVHD
Relapse at 1 yearHistorical Controls vs. Treg patients1.0
P = NS
0.8
0.6
0.4
0.2
Historical 48%
Treg 35%
Brunstein et al Blood 2011, 117:1061
0.0
0 2 4 6 8 10 12Months post-U CBT
Brunstein et al Blood 2011, 117:1061
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Viral + Fungal Infections at 100 daysHistorical Controls vs. Treg patients
1.0
0 80.8
0.6
Historical 51 %
0.4
Treg 39%
Days post-UCBT
Brunstein et al Blood 2011, 117:1061
0.2
P = NS0.0
0 20 40 60 80 100
24 hours post-Treg infusion: 97% of CD4+Foxp3- cells are HLA-A2+, 43% of CD4+Foxp3+CD127- cells are HLA-A2-
Patient #9
104
103
38.9
1000
800
600
Day 1 post transplant 4h Post-Treg infusion.
PatientCord 1 HLA-A2+ Cord 2
Treg cord HLA-A2-
25 12
20 Foxp3-9
Foxp3+
100
38.910
2
101
51.2
100 101 102 10 3 104
FL2-H: CD127 PE
600
400
2.1
0100 101 102 10 3 104
FL3-H: CD4 PerCP
200
Treg cord HLA A2 9
15
610
5
0100 101 102 10 3 104
FL4-H: HLA-A2
3
0100 101 102 10 3 104
FL4-H: HLA-A2
2.57 97.4 43.4 56.6
Brunstein et al Blood 2011, 117:1061
PACT Meeting, Houston Wagner
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Kinetics of Treg Donor UnitCD4+Foxp3+CD127- cells
80
70
60
Infused (HLA-A2-) cells as percent of total CD4+Foxp3+CD127- cells
50
40
30
20
Prkrw Iv Primmo.Brunstein et al Blood 2011, 117:1061
Treginf#1
Treg inf#2
10
0
0 5 10 15 20
Figure 2. Kinetics of detection of CD4+ cell that were CD127-FoxP3+ in the peripheral blood ofpatients receiving CsA (A) sirolimus (B) or derived from the Treg UCB donor unit (C)patients receiving CsA (A), sirolimus (B) or derived from the Treg UCB donor unit (C).
ea-
r n -+ 60-
X° 4 0 -
u_5
20- II Lr
B.
PO-
0so-
° 40-
4t?, 20-
i1 C.
80
c`l
(-) so
240
OrE 201 it
A.
Prkrw Iv Primmo.
IP. 44.11 ■-2 .7
postpre - , 4h «2
post
.4 . 7 + 1 4 » 1 5 + 1 6 + 1 8 + 2 1L +14 . 1 5 + 1 6 18 .i1Pre
poetT•
.14 41€ .21
Days from UCB Transplantation Days from U CB Trans plantation Days from UCB Transplantation
ANLMoment ce
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UCB T Regulatory CellsImpact on CD4 recovery
Prkrw Iv Primmo.
UCB T Regulatory CellsContinuationPhase I UCBof TregPhase DoseI Clinical Trial
Treg dose(x105/kg)
No.Treg doses IS
HLA mismatched UCBT
Treg
1x105/kg
3x105/kg
10x 105/kg
30x105/kg
30x105/kg
sTBI 200 cGy
CV 50 mg/kg
FLU 40 mg/m2
CsA
CsA
CsA
CsA
Rapa
1
1
3
11
7
(x105/kg)
100
300
1000
3000
Brick walls are to stop the OTHER people.
21 28‐9 ‐8 ‐7 ‐5 ‐2‐6 ‐4 ‐3 ‐1 0 7 14
CsAor RAPA
MMF
G‐CSF until ANC >2500/uL
3000
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the last lectureRandy Pausch
B i k ll th f it t t k
Brick walls are to stop the OTHER people.
Brick walls are there for reasons—its not to keepYOU out; its to give you a chance to show how
badly we want to succeed.
KT64/86 CD3/28 beads
10 000
CD28
TCR CD3
Purified UCBCD4+25+
cell
10,000RADS
Brick walls are to stop the OTHER people.
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Anti‐CD3
Anti‐CD28
AntiCD3/28
Expanding cord bloodTreg with aAPCs
ConsiderationsAnti‐CD3/28
beads
CD25bright Cultureor 18‐21days
Ti i f ti l tiAnti ‐CD3 Anti ‐CD28
Purity of starting population
Culture Devices
Maximum number of restimulations
Timing of restimulations (cell sizing)
aAPC:T cell ratios
Hippen Translational Project
Anti CD3 AntiCD28
UCB T Regulatory Cells50 million fold expansion
Anti CD3 loaded KT64/86 aAPCAnti CD3 loaded KT64/86 aAPC which express high affinity Fc receptor and CD86 (CD28 ligand)
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Re‐stimulationRe‐stimulationRe‐stimulationDay 0 5 10 15 20 25
KT64/86 IL‐2 KT64/86 Harvest±Rapamycin Re‐stimulation d13 ± 1 d24 ± 1
Phase II single
re‐stim.
Initial End
UCB nTreg
(x109)
KT64/86
(x109)
Volume
(liters)
UCB nTreg
(x109)
Volume
(liters)
Without Rapamycin
Primary stim. 0.005 0.01 0.01 2 4
Re‐stim. (R1) 2 4 4 50 96
Total 50 4 100
With Rapamycin
Primary stim. 0.005 0.01 0.01 0.4 0.8
Re‐stim. (R1) 0.4 0.8 0.8 18 36
Total 18 1 37
Re‐stimulationRe‐stimulation
Day 0 5 10 15 20 25
KT64/86 IL‐2 KT64/86 Harvest
± Rapamycin Re‐stimulation d13 ± 1 d24 ± 1
Benefits from expansion with Rapamycin
1.Almost no chance of Foxp3‐ cell outgrowth
2.Decreased numbers of IL‐2 and IFNsecreting cells.
3.While decreased numbers of Treg are generated, fewer KT and less media also needed.
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UCB Treg Bank
Day 0 5 10 15 20 25 30 35 40
KT64/86 IL‐2 KT64/86 KT64/86 FreezeRapamycin Re‐stim (R1) d13 ± 1 Re‐stim. (R2) d24 ± 1 d36 ± 1
0 2 4 6 8 10 12
Thaw HarvestKT64/86 re‐stim (R3) d11±1RapamycinIL‐2
1 Average size after first stimulation "0 4 x 109("1 liters) primary restimulation will require "0 81.Average size after first stimulation 0.4 x 10 ( 1 liters), primary re‐stimulation will require 0.8 x109 KT64/86 cells.2.Average yield after 1 re‐stimulation "18 x 109 cells. However, only 2x109 would be kept becausesecondary re‐stimulation will be capped @ 96 liters, so only use 2x109 cells for 2º re‐stimulation, would only require 4 x 109 KT64/86 cells.3.Total yield after secondary re‐stim. "50 x 109 cells. Freeze in aliquots of 2 x 109 each ("25 total),we freeze in cryotubes at <50x106/ml... use bags instead?4.Frozen nTreg aliquots (2 billion cells) arethawed (recovery "50%), re‐stimulated (R4) withKT64/86 (4 x 109 each), and cultured an additional "11 days.
1 Treg:1 Teff Mix for preventionof GVHD
CD3 HSCGoal CD3‐ HSC
UCB 3Reduce opportunistic infection
15 x 106 CD3
Tcells +15 x 106Treg Cells
Goal
Enhance immune reconstitution
UCB 1
UCB 2
T progenitors
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Acknowledgements
U of M Adult BMTMukta AroraVeronika
Treg Research Team
Bruce R. BlazarCETIJill AugheyA HVeronika
Bachanova LindaBurnsSarah Cooley DanKaufman BrianMcClune PhilipMcGlaveeffrey Miller
Arne SlungaardMarcie TomblynGreg VercellottiErica Warlick
Qing CaoJulie Curtsinger
Keli L. HippenCarl H. June Bruce L. Levine
David H. McKennaJMargaret McMillan Jeffrey S. Miller James L .Riley
D i
Anne HopperElizabeth Kerr JudyWitte
MCTDiane Kadidlo
StatsTodd Defor
CTO
Andromachi ScaravadouPablo Rubinstein
Erica Warlick
Dan WeisdorfDarin
Sumstadt
Claudio Brunstein
CTOMarilee Larkin
BMT Office TimKrepski KathyFrench NacyKonstantinides
NCI PPGP01 CA065493
Phil McGlave (P.I.)