New Strategies for the Prevention and Treatment of Graft vs. Host Disease (GVHD)

Simrit Parmar, MDStem Cell Transplant & Cellular Therapy

BTG2013, Hong Kong

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

Acute GVHD: Pathophysiology1. Recipient conditioning

2. Donor T cell activation

3. Cellular and Inflammatory Effectors

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

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

“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?”

GVHD Prophylaxis

“No Free Lunch” Principle

GVHD

• Relapse• Rejection• Delayed Immune Reconstitution

GVHD

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 allogeneic HCT in other clinical settings such as treatment of autoimmune diseases and tolerance induction for organ transplantation

Risk of GVHD in Two Eras

Gooley et al. N. Engl. J Med 363:2091, 2010

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 eGFPbAct

luc+ reporter mouse

Acute Graft-vs-Host Disease Development

Beilhack, A. et al. Blood. 2005. 106:1113

The Evolution of acute GVHD

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

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

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 alloreactive responses 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

Regulatory T-cells• Allogeneic HCT recipients with aGVHD had Treg

frequencies 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.

Magenau et al. BBMT. 2010.

38%

63%

Circulating Tregs predict OS

d15 Death fromGVHD

100

5000

1000

20000

Time [d] post BMT

Rel

ativ

e S

igna

l Int

ensi

ty

Sur

viva

l [%

]

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

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

Expanded CB Tregs show FOXP3 demethylation and suppress alloMLR

3rd Party CB Tregs Prevent GVHD

In vivo tracking of Treg transduced with GFP and Firefly Luciferase

Treg Treg+PBPC

Day -1

Day 0

Day 3

Day 10

dorsal

Treg Treg+PBPC

Day -1

Day 0

Day 3

Day 10

dorsal

Treg Treg+PBPC

Day -1

Day 0

Day 3

Day 10

dorsal

Treg Treg+PBPC

Day -1

Day 0

Day 3

Day 10

dorsal

Treg Treg+PBPC

Day 3

Day 10

ventral

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

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

0MEL BU BU BU Infusion of

Ex-vivoExpanded

Tregs

BMT

FLU40

mg/m2

FLU40

mg/m2

FLU40

mg/m2

FLU40

mg/m2

MMF+Sirolimus

Individual clinical outcome of patients who received a Treg dose > 30x105/kg

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

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

CD

25

CD127

CD4

FoxP3

Gate on CD4CD25+high

Gate on CD4CD25+

Fox P3+ cells 71.9 ± 15 %

ImmunomagneticSelection of

CD4+CD25+Cells

1st step:Depletion of CD8+/CD19+cells

2ndstep:Enrichment of CD25+ cells

>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

atyp

e co

mp

exit

y S

core

Donors

Months after transplant

Com

ple

xity

sco

re

Spectratyping

Pattern of immunoreconstitution

Evaluable Patients

Patients with CMV reactivation

0-30 31-60 61-90 91-120 121-150 151-180 181-365 >3650

10

20

30

40

50

60

70

80

90

100100

96

8275

67

5648

2928

50

34

22

9 91 1

0-30 31-60 61-90 91-120 121-150 151-180 181-365 >3650

5

10

15

20

25

30 27

21

16

109

5

212

5

1 0 0 0 0 0

Days after transplant

Days after transplant

CMV reactivation episodes

Tregs Group

Control Group

p<0.05

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

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