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