‘You have cancer’ - PACT GROUP › system › files › cagt_wkshp_wagner_7.pdf · John E. Wagner, M.D. University of Minnesota PACT Program Baylor College of Medicine 22 October

PACT Meeting, Houston Wagner

1

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


2

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


3

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


4

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


5

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


6

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


7

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.


8

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.


11

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


12

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


13

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


14

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


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%


0.0

0 2 4 6 8 10 12Months post-U CBT



16

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


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



17

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


18

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


19

the last lectureRandy Pausch

B i k ll th f it t t k


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



20

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)


21

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.


22

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


23

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.)

‘You have cancer’ - PACT GROUP › system › files › cagt_wkshp_wagner_7.pdf · John E. Wagner, M.D. University of Minnesota PACT Program Baylor College of Medicine 22 October

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