Transcript Konzept für einen SFB: Zelluläre Ansätze zur Suppression

Cell Therapies – a therapeutic strategy in the 21st century
Petra Reinke
Dept. Nephrology and Internal Intensive Care
&
Berlin-Brandenburg Center of Regenerative Therapies (BCRT)
Charité, Berlin
[email protected]
September 2011 : „Institutsgebäude Süd“ hosting 5 Institutions
(BCRT / Julius-Wolf-Institut / Institut für Genetik / Research Group Nephrology & Transplantation /
Institut für medizinische Immunologie)
BCRT
Central Services
Objectives
Platform Immune System
Immune
system
Open
access
Cardiovascul.
system
Musculo
skeletal
system
(Head: Prof. Dr. P. Reinke)
Translational Technology
Polymer-based Bio-Materials
BCRT
Cell Differentiation/Characterization
immune cell therapy
- chronic infections / tumors
(effector T cells)
- undesired immune reactions
(regulatory T cells)
- ischemia/reperfusion injury
(mesenchymal stem cells)
biomarkers for personalized therapies
- development, meth. & clin. validation
delivery of methods for the other fields
- crosstalk between
stem cells <–> immune cells
- cell sorting technologies
- biomarker analyses
- biomaterial <-> immune system
CELLS (ATMPs) –
A NEW CLASS OF THERAPEUTIC TOOLS
Cells
Therapeutic
Tools
ATMPs
Gene Therapy
Tissue Eng.
conventional drugs
(small molecules)
Antibodies
Biologicals
Fusion proteins
Cytokines
BCRT Core Unit GMP
BCRT Core Unit GMP
cell therapy for reshaping immune response
- chronic infections / tumors
(protective memory/effector T cells)
- undesired immune reactions
(regulatory T cells)
- shaping intratissue inflammation
(mesenchymal stromal/stem cells)
BCRT Core Unit GMP
cell therapy for reshaping immune response
- chronic infections / tumors
(protective memory/effector T cells)
- undesired immune reactions
(regulatory T cells)
- shaping intratissue inflammation
(mesenchymal stromal/stem cells)
regulatory
effector mechanisms
mechanisms
The medical need in transplantation and autoimmunity
Problems:
• lifelong immunosuppressive therapy
• high morbidity and mortality and costs
• no improvement of long-term results
SOLUTION ?
AIM: Minimizing long-term immunosuppression
- supporting regulation
- personalized therapy (biomarker)
cell based therapeutic approach with autologous nTreg (CD 4+25+FoxP3+)





IL-2 consumption
ATP-degradation
cytolysis by secretion of granzyme B
secretion of suppressive cytokines such as TGFβ
contact-dependent mechanisms
favoring
regulation (Treg)
selective targeting
pathogenic
memory/effector cells
Particular challenges for translation to solid organ transplantation (SOT)
- high clonal size of naive alloreactive T cells
- pre-existing alloreactive memory/effector T cells
- pre-injured donor organ with enhanced immunogenicity (act.APC)
Are Tregs able to control alloreactivity under these conditions ?
How can we measure succesful regulation in order to minimize safely IS ?
FP 7 EU Grant „BIO-DrIM“ 11-2012 to 11-2017 (coordinator: Petra Reinke)
FP 7 EU Grant „ONE-Study“ 11-2010 to 11-2015 (coordinator: Ed Geissler;
WP leader biomarker & Treg production : Petra Reinke)
What are the challenges ?
Pre-clinical hurdles
-Appropriate animal models
-Basic research
-Biomarker developement and validation
Clinical and technical hurdles
-Isolation procedure
-Expansion conditions
-GMP compliance
-Allogen-specific Treg activity?
-Functional validation
-Autologous or third party?
-Single shot or repetitive?
Isolation
in vitro
expansion
Characterization
in vivo
analysis
Clinical
application
What are the challenges ?
Pre-clinical hurdles
-Appropriate animal models
-Basic research
-Biomarker developement and validation
anti-CD4 mab (non-depl)
T-cell depletion
CTLA-4Ig
LEW
DA
life-supporting
kidney Tx
tolerance
-> secondary Treg induction in vivo
Treg
no tolerance
in patients
What was wrong with our model ?
Challenges in humans:
- marginal donor organs
- co-infections of recipients
- immunological experience
Need for preclinical models adapted to clinical challenges
Pascher et al. 2006, Pratschke et al. 2009, Siepert et al. 2012
Need for advanced preclinical models
that are more predicting for clinical challenges
?
Treg in vitro
Treg in
conventional
SOT models
Treg in
advanced
SOT models
?
Treg in
SOT
patients
Need for advanced preclinical models that are more predicting
for clinical challenges
DA
anti-CD mAb (non-depl)
tolerance
CTLA4Ig
T-cell depletion
LEW
life-supporting
kidney Tx
Treg
models adapted to the clinical challenges: tolerance protocols failed
brain prolonged memory co-infection
death ischemia T cells
(CMV)
DA
anti-CD4 mAb (non-depl.)
CTLA4Ig
T-cell depletion
LEW
life-supporting
kidney Tx
no or
reduced
tolerance !
Treg
Pascher et al. 2006, Pratschke et al. 2009, Siepert et al. 2012
Can Treg mediate tolerance in the challenging „memory“ model ?
A
DA
LEW
>150d
T cell depletion (>90%)
B
DA
LEW
<10d
T cell depletion (>90%)
DA-specific effector/memory 1/1000, d-7 or d-150
C
DA
LEW
T cell depletion (>90%)
DA-specific effector/memory 1/1000, d-7 or d-150
D
DA
35d
Treg d3 or d20
LEW
T cell depletion (>90%)
DA-specific effector/memory 1/1000, d-7 or d-150
>150d
Treg d3 or d20
CNI up to day 10
Siepert et al.; Am J Transplant 2012
Treg block most effectively LIP of GFP+ alloreactive Tmem
Frequency of GFP+ Memory T cells in peripheral blood
2,0
1,8
Tmem cells + T-cell depletion (GST: 13.7 d)
GFP+ Memory T cells/ MNC [%]
1,6
Tmem + depletion + CNI d0-9 (GST: 57d)
1,4
Tmem + depletion + CNI d0-150 (GST: >150d)
1,2
Tmem + depletion + CNI d0-9 + Treg d4 (GST: >150d)
1,0
0,8
0,6
0,4
0,2
0,0
pre- Tx
5
21
49
70
100
150
Time post Transplantation [d]
Treg induce long-lasting intragraft “tolerance signature” :
high Toag1; high Foxp3 ; low IL-6; intermediate CD3
d 150 post-Tx (intragraft RT-PCR)
1
CD3 (infiltrates)
0,1
TOAG1 (immune silence)
Expression/ b-Actin
0,1
0,01
0,01
0,001
0,001
0,1
IL6 (innate immunity)
0,01
0,01
Foxp3 (regulation)
0,001
0,001
0,0001
0,0001
0,00001
0,00001
Tmem + depletion
+ mTOR d3-150
Tmem + depletion
+ CNI d0-150
Tmem + depletion
+ CNI d0-9 + Treg d4
What are the challenges ?
Pre-clinical hurdles
-Appropriate animal models
-Basic research
-Biomarker developement and validation
Clinical and technical hurdles
-Isolation procedure
-Expansion conditions
-GMP compliance
-Allogen-specific Treg activity?
-Functional validation
-Autologous or third party?
-Single shot or repetitive?
Isolation
in vitro
expansion
Characterization
in vivo
analysis
Clinical
application
What are the challenges ?
Pre-clinical hurdles
-Appropriate animal models
-Basic research
-Biomarker developement and validation
Clinical and technical hurdles
-Isolation procedure
-Expansion conditions
-GMP compliance
-Allogen-specific Treg activity?
-Functional validation
-Autologous or third party?
-Single shot or repetitive?
Isolation
in vitro
expansion
Characterization
in vivo
analysis
Clinical
application
Manufacturing Approach
starting material
35 – 50 ml blood
separation
of cell subpopulation
starting cell nb.
1x106 nTregs
depletion of CD8+ T cells
enrichment CD4+25++
cell expansion
max. 2x108 nTregs
for application
anti-CD3/28 beads
+ IL-2
+ mTOR-inhibitor
blood PBMC
isolated Treg
step 1 (3-4 hrs):
Isolation of Treg
expanded Treg
step 2 (2-3 wks):
Expansion of Treg
> 100 fold
depletion of
CD8+ T cells
enrichment
CD4+25++
by CliniMacs
anti-CD3/28 beads
+ IL-2
+ mTOR-inhibitor
blood PBMC
isolated Treg
step 1 (3-4 hrs):
Isolation of Treg
depletion of
CD8+ T cells
enrichment
CD4+25++
by CliniMacs
expanded Treg
step 2 (2-3 wks):
Expansion of Treg
anti-CD3/28 beads
+ IL-2
+ mTOR-inhibitor
Novel method;
TCR sequencing by next-generation sequencing
> 100 fold
Babel N et al. submitted
blood PBMC
step 1 (3-4 hrs):
Isolation of Treg
isolated Treg
expanded Treg
step 2 (2-3 wks):
Expansion of Treg
TCR repertoire pattern before/after expansion
(>100,000 sequences / line analysed)
fresh expanded
TCR analysis by next-generation sequencing:
1. TCR repertoire of nTreg and Tconv is completely different (almost no overlap)
2. Expansion does not alter polyclonal TCR repertoire of nTreg
GMP adapted isolation/expansion of human Treg –
% CD45RA+ in Tregs
- feasible for healthy donors
Decreasing CD45RA+
naive Treg with increasing age
End-stage kidney disease (ESKD) patients
have less
CD45RA+CD62L+ naive Treg
but more
CD45RA-CD62L+ central memory Treg
compared to age-matched controls
More (central)memory Treg in ESKD patients
Are (central)-memory Treg functionally
comparable to naive Treg and useful for therapy ?
NTx patients (years post Tx)
(Central) memory nTreg are very potent Treg
FoxP3+
Helios+
*
*
Naïve
Treg
Treg cm
Treg central memory (cm)
Treg em
- FoxP3/Helios/CTLA4 ++
- high suppressive capacity
- FoxP3 TSDR demethylated
- homing receptors for tissue infiltration but also lymphnodes (CD62L)
=> highly potent nTreg
Which protocols are suitable for testing Treg ?
3 CENTERS (GER, UK)
Controls (18-24 living donations)
- IL2R-Ab induction therapy
- Tacrolimus, MMF, Steroid
Treg
DC
Treatment (48 living donations)
- Tacrolimus, MMF, Steroid
- Treg (day 7)
Tr1
Mreg
Biomarker:
- safety: allo-Ab, viral load, virus
spec. T cell
- efficacy: tolerance profile
(TR-PCR)
- TCR deep sequencing
Clinical Trials using Tregs
Target
Treg Source
Trial
Status
GvHD
UBCB
Phase I
Completed (Italy)
GvHD
Peripheral
3rd party
Phase I
Completed (US)
DM I
Autologous
Phase I
Recruiting (US)
KTx
Autologous
Phase I
CTA Q2 2013
GvHD
Ind. autologous
Phase II
Recruiting (US)
Foxp3
How to monitor success / failure
of Treg therapy
R6
2
%
R5
CD25
1. Toag-1 Molecular marker of „silenced“ adaptive immunity (Sawitzki et al. AJT 2007)
• useful to identify pre-transplant activation state (= individual risk)
• down-regulation post-Tx = predicting failure of therapy
• recovery of Toag-1 post therapy = successful therapy (reprogramming tolerance)
2. Donor-specific Memory T cells (Elispot) (Nickel et al. Transplantation 2005)
• useful to identify high-risk patients before Tx => high levels not suitable for
strong weaning or even tolerance protocols
• stably low levels post-Tx = successful control of alloreactivity
3. Molecular signature of tolerance (Sagoo P et al. JCI 2010)
• signature associated with spontaneous tolerance
• might be useful for indentifying patients that can be partly weaned after
successful Treg therapy
What are the challenges ? And where we are ?
Pre-clinical hurdles
- Appropriate animal models
- Basic research on Treg biology
- Biomarker developement and validation
Clinical and technical hurdles
- Isolation procedure
- Expansion conditions
- GMP compliance
- Functional validation
- Clinical Study Protocol
- Clinical Study
- Manufactoring Authorization acc.to EMA/FDA rules
- Autologous or third party?
- Single shot or repetitive?
Isolation
in vitro
expansion
Characterization
in vivo
analysis
Clinical
application
I have to thank…..
Support:
BMFB
DFG (SFB 650 & TR-SFB 36)
EU FP7 (The One Study)
T-cell Europe
special thanks to
clinicians, patients and nurses