Umbilical Cord Blood-Derived T Regulatory Cells

David H. McKenna, M.D.PACT Workshop -

University of Pittsburgh

May 5, 2008

Slide 1

Outline•

Overview of T regulatory (TR

) cells•

Potential for clinical application

•

Summary of efforts to isolate and expand TR

cells at the U of MN–

Peripheral blood

–

Umbilical cord blood•

Current trials

Slide 2

T Regulatory Cells•

Naturally arising TR

cells are minor population (~5-10%) of CD4+

T cells

•

Crucial for control of autoreactive

T cells in vivo; contribute to maintenance of immunologic self-tolerance

•

CD4+

and CD8+

T cell responses (auto-, allo-,

and anti-tumor-

immunity) can be inhibited by TR

cells •

Exact mechanism of suppressive effects unknown (cell-cell contact in vitro/in vivo)

Slide 3

T Regulatory Cells•

Main mechanism of suppression seems to be inhibition of transcription of IL-2 in responder cells

•

Express high levels of immune suppressive molecules (e.g., CTLA-4 and TGF-β)

•

Originally characterized in mice; more recently in humans

•

Generated through central thymic

development and poorly defined peripheral mechanisms

Slide 4

CD4+ Tregs

and immune control. Fehervari

Z and Sakaguchi

S. JCI 114:1209-17,2004.Slide 5

T Regulatory Cells•

IL-2 = key growth/survival factor for TR

cells •

TR

cells co-express high levels

of IL-2 receptor α-chain (CD25)

•

Foxp3 = master control gene for development and function of natural CD4+/CD25+

TR

cells•

Foxp3 transcription factor: controls expression of CD25 in natural CD4+/ CD25+

TR

cells, but not in activated T cells in general

Slide 6

Potential Clinical Applications

CD4+ Tregs

and immune control. Fehervari

Z and Sakaguchi

S. JCI 114:1209-17,2004.Slide 7

Clinical Application in BMT

•

TR

cells essential for ex vivo induction of tolerance•

Addition of B6 TR

cells resulted in dose-dependent suppression of allo-responses in MLR (responders = B6 CD4+/CD25-

and stimulators = irradiated bm12)

•

CD25 depletion of CD4+ T cells resulted in heightened MLR responses

•

Role for TR

cells in allo-responses in vivo (e.g., GVHD)?

Taylor PA, Noelle RJ, and Blazar BR. CD4+CD25+ immune regulatory

cells are required for induction of tolerance to alloantigen via costimulatory

blockade. J. Exp. Med. (2001); 193(11): 1311-1317.Slide 8

Depletion of CD4+CD25+

Cells Accelerates

GVHD Lethality

Dose = 105 cellsDonor = B6 T cellsRecipient = bm12 (subleth. irr.)P = 0.024

Dose = 0.5 x 105 cellsDonor = B6 T cellsRecipient = bm12 (subleth. irr.)P = 0.0068

Taylor PA, Lees CJ, and Blazar BR. The infusion of ex vivo activated and expanded CD4+CD25+

immune regulatory cells inhibits GVHD lethality. Blood (2002); 99: 3493-3499.

A)

B)

Slide 9

Depletion of CD4+CD25+

Cells Accelerates GVHD

Lethality in a Different Strain Combination

Dose = 2 x 106 T cellsDonor = B6 BM and T cellsRecipient = BALB/c (leth. irr.)P = 0.016

Taylor PA, Lees CJ, and Blazar BR. The infusion of ex vivo activated and expanded CD4+CD25+

immune regulatory cells inhibits GVHD lethality. Blood (2002); 99: 3493-3499.

Slide 10

Depletion of CD25+

Cells from a Whole T Cell

Inoculum

Accelerates GVHD in a Non-irradiated SCID GVHD Model

Dose = 106 T cellsDonor = B6 T cellsRecipient = BALB/c SCIDP = 0.021

Taylor PA, Lees CJ, and Blazar BR. The infusion of ex vivo activated and expanded CD4+CD25+

immune regulatory cells inhibits GVHD lethality. Blood (2002); 99: 3493-3499.

Slide 11

Pre-transplantation In Vivo Depletion of CD25+

Cells

Accelerates GVHD

Dose = 15 x 106 splenocytesDonor = BALB/c BM and

splenocytesRecipient = thymectomized,

leth. irr. B6P = 0.0063

Taylor PA, Lees CJ, and Blazar BR. The infusion of ex vivo activated and expanded CD4+CD25+

immune regulatory cells inhibits GVHD lethality. Blood (2002); 99: 3493-3499.

Slide 12

Ex Vivo Expanded and Activated CD25+

Cells Inhibit

GVHD

Dose = 2 x 106 fresh CD4+

Tcells +/-

2 x 106 activated cells

Donor = B6Recipient = non-irr., NK-depleted

BALB/c SCIDP = 0.022

Taylor PA, Lees CJ, and Blazar BR. The infusion of ex vivo activated and expanded CD4+CD25+

immune regulatory cells inhibits GVHD lethality. Blood (2002); 99: 3493-3499.

Slide 13

Non-Sorter Bead-Based Human TR

Isolation from PB

*Miltenyi

beads(low titer) x 2

PBMC (Apheresis)

CD25 positive selection

CD8,14,19,20,56 negative selection

CD4 +25+ Anti-CD3/28 beadsIL-2

CD4 positive selection

Irradiate, useas feeders

Slide 14

10 0 10 1 10 2 10 3 10 4CD4 PerCP

10 0

10 1

10 2

10 3

10 4C

D25

PE

0.39 1.41

38.359.9

10 0 10 1 10 2 10 3 10 4CD4 PerCP

10 0

10 1

10 2

10 3

10 4

CD

25 P

E

0.68 98

1.240.062

1.4%

98%

Non-Sorter Bead-Based Human TR

Isolation from PB

Pre-purification

After purification

Godfrey WR, et al. In vitro-expanded human CD4+CD25+ T-regulatory cells can markedly inhibit allogeneic dendritic cell-stimulated MLR cultures. Blood (2004); 104: 453-461.

Slide 15

CD3/28 Beads Augment CD4+25+

Cell Expansion

0

20

40

60

80

100

120

140

1 4 7 10 14 17 21 24 28 32

time-days

3/28 beadsimm-CD3

Godfrey WR, et al. In vitro-expanded human CD4+CD25+ T-regulatory cells can markedly inhibit allogeneic dendritic cell-stimulated MLR cultures. Blood (2004); 104: 453-461.

Slide 16

Purified Cultured CD4+CD25+ Cells Markedly Suppress MLRs

-MLR-1 week-1:2 suppressor:responder

-Suppression->50% (15/22=68%)->95% (7/22=32%)-More potent than fresh-CD62L+/CD27+

subsetmost potent

Godfrey WR, et al. In vitro-expanded human CD4+CD25+ T-regulatory cells can markedly inhibit allogeneic dendritic cell-stimulated MLR cultures. Blood (2004); 104: 453-461.

Slide 17

Broad-Based Inhibition of Cytokine Production

0

100

200

300

400

500

600

pg/ml

TNF-a IFN-g GM-CSF IL6 IL10

No Tregs+Tregs

Inhibition of Cytokine Accumulation During MLR

0

50

100

150

1 2 3 4 5 6 7

Days

IL2

(pg/

ml) No Tregs

+ Tregs

IL-2 Accumulation During MLR

Godfrey WR, et al. In vitro-expanded human CD4+CD25+ T-regulatory cells can markedly inhibit allogeneic dendritic cell-stimulated MLR cultures. Blood (2004); 104: 453-461.

Slide 18

UCB as a TR

Cell Source

•Would UCB be a better source of T

R

cells?•Low exposure to prior antigenic stimulus•UCB T cells have defects in T cell activation•Is the reduced GVHD risk of UCB transplants potentially due to higher TR

cell number or function?

•More distinct CD4+CD25++ population •Reduce the likelihood of isolating or expanding CD4+25-

cells?

Slide 19

Adult PB Has More CD25lo

Cells Than Cord Blood

CD25

CD4

UCB

UCB CD25+

Adult PB

Adult PB CD25+

Godfrey WR, et al. In vitro-expanded human CD4+CD25+ T-regulatory cells can markedly inhibit allogeneic dendritic cell-stimulated MLR cultures. Blood (2004); 104: 453-461.

Slide 20

Uniform Suppression by UCB TR

v. Adult PB TR

Adult CD25++Lin-Tregs

were more

rigorouslypurified than adult CD25+

*

UCB CD25+

cells havehigher CD25 and L-selectin

levels as

compared to adult cells

Godfrey WR, et al. In vitro-expanded human CD4+CD25+ T-regulatory cells can markedly inhibit allogeneic dendritic cell-stimulated MLR cultures. Blood (2004); 104: 453-461. Slide 21

Clinical Production of UCB-Derived TR

Cells

Minnesota Molecular & Cellular Therapeutics FacilityMinnesota Molecular & Cellular Therapeutics FacilityUniversity of Minnesota Cancer CenterUniversity of Minnesota Cancer Center

Cancer Center photo courtesy of Chris Cancer Center photo courtesy of Chris GregersonGregerson; ; http://http://www.phototour.minneapolis.mn.uswww.phototour.minneapolis.mn.us//

;;PhototourPhototour

of Minneapolis; MMCT Facility photo courtesy of Diane Kadidloof Minneapolis; MMCT Facility photo courtesy of Diane KadidloSlide 22

•

Enrich for CD4+/CD25+ T regulatory cells using the CliniMACS® device, CD25 MicroBeads.

•

Culture in X-VIVO 15 supplemented with 10% human AB serum, L-glutamine, n-acetylcysteine

and 2.5 mL

gentamicin

in a tissue

culture flask.

•

Anti-CD3/antiCD28-coated beads (provided by Carl June and Bruce Levine, University of Pennsylvania).

•

The cell culture is supplemented with 300 IU/mL

IL-2 starting on the third day of culture.

•

Cells are cultured as described above in an appropriately sized culture flask(s)/cell factory for 18 (±1) days.

•

Lot release testing

Slide 23

Slide 24

UMBILICAL CORD BLOOD TRANSPLANT WITH CO-

INFUSION OF UCB-DERIVED T REGULATORY CELLS

Hypothesis

We hypothesize that the infusion of CD4+CD25+ Treg cells is safe and will not be associated with excessive toxicity.

Slide 25

Regulatory T cell unit and UCB graft selection

HLA A & B: Ag levelHLA DRB1: Allele level

UCB4/6

4/6

UCB

4/6TregUCBTreg

4/6

ABO Compatible

Slide 26

Study Design

Phase I semi-log dose escalation:–

1 x 105/kg

–

3 x 105/kg–

10 X 105/kg and

–

30 x 105/kg

Planned Accrual: 12 to 24 patients

Slide 27

Endpoints

Primary:

Determine the MTD of UCB-derived Treg cells.

Secondary :1.

Determine speed of neutrophil and platelet recovery at day 42.

2. Determine incidence of ‘double chimerism’

(e.g.,

engraftment of both UCB units) at day 21.3.

Estimate the risk of severe grade III-IV acute GVHD at day 100.

4. Estimate the risk of chronic GVHD at 1 year.

5. Estimate probability of survival at 100 days and 1 year.

Slide 28

Stopping Rules

•

Graft Failure•

Grade III-IV Acute GVHD

•

Transplant Related Mortality

Slide 29

Mon

itor

ing

UCB-derivedTreg cells Production

-19 -18 -17 -16 -15 -14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3

MMFCSA

G-CSF

Double UCBT

UCB-derivedTreg cells

Treatment PlanUCB-derived Regulatory T Cells

FLU

CY

FLU

CY

FLU

1320 cGyTBI

Slide 30

Mon

itor

Nonmyeloablative UCB-Derived Treg Study

Mon

itor

ing

UCB-derivedTreg cells Production

-18 …. -6 -5 -4 -3 -2 -1 0 1 2 3….. 15 16 17

UCBT

UCB-derivedTreg cells

Conditioning

MMFCSA

G-CSF

Slide 31

Patient Activities

Laboratory Test Days

Molecular Diagnostics Laboratory

Engraftment1(±2)* 7(±2)*

14(±2)*28(±2) 60(±7)100(±7)

CancerCenter

CellTherapy CoreLaboratory

Cytokine levels

Flow cytometry

* Research only samplesSlide 32

CytokinesIFNγIL-7IL-15TGFβIL-10IL-17

Flow CytometryCD4CD25Foxp3CD19 or 20CD27CD62LCD127CD45RACD45RO

Slide 33

Conclusion•

Double UCB is efficacious but we can improve on engraftment and GVHD

•

Animal data shown potent effect of ex vivo expanded activated Tregs on GVHD and engraftment

•

Clinical scale production shows consistent expansion and function

•

Clinical protocols are accruing patients

Slide 34

Future Directions

•TR

cells will undergo extensive phase I testing in the next few years in a variety of clinical settings (e.g., BMT)

Will TR

cells be well-tolerated and efficacious insuppressing GVHD and BM graft rejection?

To what extent will efficacy be dependent upon the clinicalvenue to be used for testing of TR

?

How will immune suppressive agents alter TR

efficacy?

Will infection risk or tumor recurrence be increased?

Slide 35

Acknowledgements

Contributors/Collaborators:Angela Panoskaltsis-MortariCarl June/Bruce Levine Jon Serody

Research:Bruce BlazarPatricia Taylor (Murine studies)Wayne Godfrey/Steph Porter/Keli Hippen (Human studies)

Translational/Clinical:Bruce BlazarClaudio BrunsteinSue FautschSteph PorterClinical Cell Therapy LaboratoryJohn WagnerJeff MillerMargaret MacMillan

NHLBI-sponsored PACT Group

Slide 36

Acknowledgements

Diane KadidloDarin SumstadNancy BostromLisa VanOrsowSheryl AdamsEileen EmrickNancy ColeyCindy StanawayAmy CullinanLien LeJenny Kordosky

MCT QA & Support Staff

Slide 37