freiburg nk2009 jan spanholtz
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Jan Spanholtz
Department of Laboratory MedicineLaboratory of Hematology
Radboud University Medical Center Nijmegen
and
Glycostem Therapeutics
Natural Killer (NK) cell immunotherapy in AMLusing CD34+ derived NK cells
Adapted from Velardi et al. Curr Opin Immunol 2008, Moretta et al. Immunol Rev 2008
D
HSC donor NK donor
AML patient
DC patient
T cells patient
No GVHD
YES GVL
Therapeutic role of alloreative NK cells
in HLA haploidentical setting
Adoptive transfer of allogeneic NK cells
Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer. Miller et al. Blood, 2005.
• Adoptive transfer of NK-enriched apheresis products after Hi-Cy/Flu conditioning therapy in AML
• Cell dose: up to 2 x 107 cells/kg
• Cell products: 40% ± 2% CD56+CD3- NK cells (range 18%-68%)
RESULTS:
• Transient NK engraftment and in vivo expansion
• Hematologic CR in 5 of 19 poor-prognosis AML patients
BUT:
• T cell administration: 2.1 ± 0.3 (range 0.5-6.5) x 105 T cells/kg (risk GVHD)
• B cell contamination: 19% ± 2% (1 patient developed EBV-lymphoma)
Low cell number
T-cell contamination
Low cytotoxicity
Need extra IL-2 stimulation
Low recovery after enrichmentNot a standardized protocol
Need for expansion system
Need extra aphaeresis
for multiple infusions
Bottlenecks to develop sufficient products for NK cell immunotherapy isolated from peripheral blood
Low cell number
Expansion and differentiation technology
for NK cell production
expansion differentiation 1
• GBGM + human serum (HS)
• SCF, Flt3L, IL-7, TPO
• Clinical grade Heparin
CD34+ cells
Day 0-9 Day 9-14
Expansion medium: Differentiation medium 2:
• SCF, IL-7, IL-2, IL-15
NK progenitors Mature NK cells
STEP 1 STEP 2
differentiation 2
Day 14-35
Differentiation medium 1:
• SCF, IL-7, Flt3L, IL-15
• Clinical grade Heparin
Low-dose cytokine cocktail• Low-dose cytokine cocktail• Low-dose cytokine cocktail•
• GBGM + HS • GBGM + HS
Formulation of a novel GMP-grade medium designated GBGM (Glycostem Basal Growth Medium)
www.glycostem.nl
GBGM significantly improved the NK cell generation process
CD56 content 99% ± 1%
using GBGM
10
100
1,000
10,000
100,000
1 2 3 4 5week
fold
exp
ansi
on
tota
l cel
lsMedium1 (n=3) Medium 2 (n=3) GBGM (n=3)
Per
cen
tag
eC
D56
+ c
ells
A
B
*
0
20
40
60
80
100
3 4 5
week
*
Medium1 (n=3) Medium 2 (n=3) GBGM (n=3)
Cell expansion ~50,000 foldusing GBGM
NK cells cultured in GBGM media display high expression of
activating NK cell receptors and mediate a strong cytotoxicity
0
20
40
60
80
100
NKG2A NKG2D NKp30 NKp44 NKp46
Po
sitiv
e ce
lls(%
)
CD56
0
200
400
600
800
1000
IFN
-re
lea
se(p
g/m
l)
0
20
40
60
80
100K562
KG1a
Sp
eci
ficly
sis
(%
)
A B
+4 h +24 h
C D
+24 h+4 h
K562
KG1a
Medium
+4 h +24 h +24 h+4 h +24 h+4 h+4 h +24 h +24 h+4 h +24 h+4 h
CD
10
7a+
NK
ce
lls(%
)
0
5
10
15
20K562
KG1a
Medium
E:T ratio 2:1
E:T ratio 2:1E:T ratio 2:1
NK cells can be efficiently generated from CB, BM
and mPB derived CD34+ cells using GBGM medium
CB
fold
exp
ansi
on
C
D56
+ c
ells
BM
0 1 2 3 4 5 6
Weeks of culture
1x10 4
8x10 3
6x10 3
4x10 3
2x10 3
10 0
Medium 1 Medium 2 GBGM
fold
exp
ansi
on
CD
56+
ce
lls
mPB
0 1 2 3 4 5 6
Weeks of culture
1x103
8x102
6x102
4x102
2x102
10 0
fold
exp
ansi
on
CD
56+
ce
lls
Medium 1 Medium 2 GBGM
0 1 2 3 4 5
Weeks of culture
1x10 5
8x10 4
6x10 4
4x10 4
2x10 4
10 0
Medium 1 Medium 2 GBGM
CB
fold
exp
ansi
on
C
D56
+ c
ells
BM
0 1 2 3 4 5 6
Weeks of culture
1x10 4
8x10 3
6x10 3
4x10 3
2x10 3
10 0
Medium 1 Medium 2 GBGM
fold
exp
ansi
on
CD
56+
ce
lls
mPB
0 1 2 3 4 5 6
Weeks of culture
1x103
8x102
6x102
4x102
2x102
10 0
fold
exp
ansi
on
CD
56+
ce
lls
Medium 1 Medium 2 GBGM
0 1 2 3 4 5
Weeks of culture
1x10 5
8x10 4
6x10 4
4x10 4
2x10 4
10 0
Medium 1 Medium 2 GBGM
Identification of immature stages of ex vivo-generated NK cells
Kaluza™ Analysis Software
Gallios™ Flow Cytometer
Acquisition of KIR repertoire of ex vivo-generated NK cells
GMP-based production of allogeneic NK cell products
CliniMACS selection
• CD34 selection
Washing &volume reductionNK cell generation
+
Umbilical
cord bloodProduct release
Fresh vs. frozen material Titer plates vs. bags
Experimental variables during up-scaling procedure
• CD34+ selection from frozen UCB
• Container, surfaces
• Medium
• Cytokines
• Cell inoculation
• Medium refreshment
• Cell density
Efficient CD34+ cell enrichment from cryopreserved UCB
units using the CliniMACS system
UCB unit Results thawing Results CD34 CliniMACS
UCB Volume (ml) NC (x106) CD34 (%) Recovery (%) CD34 (%) CD34 (x106) Recovery (%)
1 80 368 0.88 76 52 1.47 50
2 69 469 0.92 69 77 1.99 53
3 75 653 0.47 62 70 2.36 73
4 70 819 1.04 78 92 6.34 73
5 53 583 0.36 56 54 1.74 76
6 74 829 0.30 68 65 1.70 79
7 71 440 0.45 88 64 1.70 82
8 53 403 0.49 68 73 1.42 69
9 80 248 1.04 69 88 1.32 72
range 53-80 248-819 0.3-1.04 56-88 52-92 1.32-6.34 50-82
0
500
1000
1500
2000
0 1 2 3 4 5 6week
CB0109 bag CB0209 bag CB0309 bag
fold
exp
an
sio
n
0
20
40
60
80
100
0 1 2 3 4 5 6
NK
ce
lls (%
)
week
CB0109 bag CB0209 bag CB0309 bag
Mean expansion ~1,300 fold
Mean purity71% ± 9%
0
2000
4000
6000
8000
0 1 2 3 4 5 6week
CB0109 plate CB0209 plate CB0309 plate
fold
exp
an
sio
n
0
20
40
60
80
100
0 1 2 3 4 5 6
NK
ce
lls (%
)
week
CB0109 plate CB0209 plate CB0309 plate
Mean purity97% ± 2%
Mean expansion ~3,500 fold
Efficient NK cell production using culture bags from
frozen UCB samples
0
2000
4000
6000
0 1 2 3 4 5 6
fold
exp
an
sio
n
week
CB0709 wave CB0709 plate
0%
20%
40%
60%
80%
100%
0 1 2 3 4 5 6
NK
ce
lls (%
)
week
CB0709 wave CB0709 plate
Efficient NK cell differentiation using the Wave Bioreactor
during the differentiation phase
0
05
95 0
09
91 0
496
01
8613
0
CD
56
CD
34
CD
38
CD
14
CD117 CD3 CD19 CD15SSC
CD
45
NK cell products are devoid of T- and B- cells
Bag cultures provide sufficient numbers of functional NK cells
showing cytotoxicity against various primary AML
NK cells per experiment
fold expansion
CD34+ cellsCD56+
(%)NK cells
CB0109 1770 1.7x106 63 1.9x109
CB0209 759 1.4x106 80 8.6x108
CB0309 1291 1.3x106 70 1.2x109
CB0709 2861 0,81x106 95 2.2x109
0
20
40
60
80
100
AML1 AML2 AML3 AML4 AML5 K562 KG1a
day2
day3
day1
spe
cific
lysi
s (%
)
Alloreactive potential of a natural killer-cell subset as
a criterion for donor selection
C1/C1 Donor cell
- +
KIR2DL1
NK cell fromC1/C1 donor
Tolerance Killing
C2/C2 Recipient tumor cell
HLA-C1
+
-
+
KIR-Ligand mismatched NK cell infusion
for elderly AML patients Nijmegen
Phase I/II study: 12 patients treated with escalating NK cell doses
AML patientC1/C1
C2/C2 High Cy/Fluconditioning
NK cell infusion
UCB unitC2/C2C1/C2C1/C1
GVL reaction
• Studying of human NK cell development (Poster Dorit Reiche)
• Submission clinical protocol to ethical review board (CCMO)
• Up-scaling of UCB-derived NK cells in closed system (Wave Bioreactor)
• CD133+ selection vs. CD34+ selection
• Develop clinical-grade in vivo monitoring method (19F MRI)
• Adoptive transfer studies in Rag2-/-c-/- mice bearing human AML
• Monitoring and follow up of NK cell study
PostDoc position available!!
Work in progress and Future perspectives
Financial support:
Acknowledgements
Department of Laboratory MedicineLaboratory of Hematology
& Department of HematologyRadboud University Nijmegen Medical Centre
Marleen Tordoir
Carel Trilsbeek
Jos Paardekoper
Bijan Moshaver
Frank Preijers
Michel Schaap
Theo de Witte
Harry Dolstra
Department of Laboratory MedicineLaboratory of Medical Immunology
Radboud University Nijmegen Medical Centre
Diana Eissens
Arnold van der Meer
Irma Joosten
Glycostem Therapeutics
Dirk Groenewegen
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