Poster C Linical Nk Generation Ash 2009 Js
1
Adoptive transfer of CD34+ derived NK cells in poor-prognosis AML patients Glycostem ® clinical grade NK cell generation system provides sufficient numbers of functional NK cells from UCB CD34+ cells Process is transferred into a GCP standard operating procedure. Clinical protocol (phase I/II) has been designed for testing safety and toxicity NK cells as Donor Lymphocyte Infusion (DLI) for AML patients in non transplantation setting Escalating doses will be applied in KIR-ligand mismatched setting GBGM ® can be purchased via www.clearcelltechnologies.com Effective NK cell differentiation from umbilical cord blood (UCB) derived CD34+ cells using GBGM ® Ex-vivo generated NK cells are devoid of T- and B cells and express specific NK cell antigens Stroma-free cell culture system for optimal NK cell generation Clinical Scale Generation of Functional Human Natural Killer Cells From Umbilical Cord Blood CD34-Positive Cells for Immunotherapy Jan Spanholtz , Marleen Tordoir, Carel Trilsbeek, Jos Paardekooper, Diana Eissens, Arnold van der Meer, Irma Joosten, Theo de Witte, Nicolaas Schaap, Frank Preijers and Harry Dolstra Department of Internal Medicine - Laboratory of Hematology, Radboud University Nijmegen Medical Centre, The Netherlands mailto: [email protected] Clinical grade NK cell cultures provide sufficient numbers of functional NK cells which efficiently lyse various primary AML tumor cells Efficient CD34+ cell enrichment from cryopreserved UCB units using the CliniMACS system Efficient NK cell production from cryopreserved UCB units using various bioreactors CD56 content 99%±1% using GBGM ® 10 100 1,000 10,000 100,000 1 2 3 4 5 week Fold expansion total cells Medium1 (n=3) Medium 2 (n=3) GBGM ® (n=3) Percentage CD56+ cells 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 fold using GBGM ® expansion differentiation 1 • GBGM ® + human serum (HS) • SCF, Flt3L, IL-7, TPO • Clinical grade Heparin (GAGs) 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 (GAGs) Low - dose cytokine cocktail • Low - dose cytokine cocktail • Low - dose cytokine cocktail • • GBGM ® + HS • GBGM ® + HS Novel GMP grade Glycostem Basal Growth Medium (GBGM ® ) www.glycostem.nl Clinical scale generation of allogeneic NK cell products CliniMACS selection • CD34 selection Washing & volume reduction NK cell generation + Umbilical cord blood Product release UCB unit Results thawing Results CD34 CliniMACS UCB Volume (ml) NC (x10 6 ) CD34 (%) Recovery (%) CD34 (%) CD34 (x10 6 ) Recovery (%) 1 80 368 0.88 76 52 1.5 50 2 69 469 0.92 69 77 2.0 53 3 75 653 0.47 62 70 2.3 73 4 70 819 1.04 78 92 6.3 73 5 53 583 0.36 56 54 1.7 76 6 74 829 0.30 68 65 1.7 79 7 71 440 0.45 88 64 1.7 82 8 53 403 0.49 68 73 1.4 69 9 80 248 1.04 69 88 1.3 72 range 53 - 80 248 - 819 0.3 – 1.04 56 - 88 52 - 92 1.3 – 6.3 50 - 82 0 500 1000 1500 2000 0 1 2 3 4 5 6 week CB0109 bag CB0209 bag CB0309 bag 0 20 40 60 80 100 0 1 2 3 4 5 6 week CB0109 plate CB0209 plate CB0309 plate 0 20 40 60 80 100 0 1 2 3 4 5 6 week CB0109 bag CB0209 bag CB0309 bag 0 2000 4000 6000 8000 0 1 2 3 4 5 6 week CB0109 plate CB0209 plate CB0309 plate 0 2000 4000 6000 0 1 2 3 4 5 6 week CB0709 wave CB0709 plate 0 20 40 60 80 100 0 1 2 3 4 5 6 week CB0709 wave CB0709 plate Fold expansion Fold expansion Fold expansion NK cells (%) NK cells (%) NK cells (%) 0 0 5 95 0 0 9 91 0 4 96 0 CD3 CD19 CD15 SSC CD14 CD38 CD56 CD45 NK cells per experiment CD34+ cells fold expansion CD56+ (%) NK cells experiment CB0109 1.7x10 6 1770 63 1.9x10 9 static bag CB0209 1.4x10 6 759 80 8.6x10 8 static bag CB0309 1.3x10 6 1291 70 1.2x10 9 static bag CB0709 0,81x10 6 2861 95 2.2x10 9 bioreactor 0 20 40 60 80 100 AML1 AML2 AML3 AML4 AML5 K562 KG1a day2 day3 day1 specific lysis (%) NK cell products are analyzed for purity, showing no contamination with T- or B cells (A). The NK cell phenotype was determined by 10-color flowcytometry using the Gallios Flow Cytometer and the Kaluza software (B). A B The final NK cell products were analyzed for functionality in a CFSE based cytotoxicity assay for 18h with a 2:1 effector:target ratio. Clinical protocol (phase I/II study) AML patient >65 years treated with remission-induction chemotherapy HLA – haploidendical donor sibling orchild >18 years HLA and KIR typing KIR-ligand incompatibility Complete remission (<5% blasts in bone marrow) Consolidation chemotherapy Non-myeloablative immunosuppression Flu 30 mg/m 2 and Cy 1200 mg/m 2 at day -6, -5, -4, -3 CD34 selection(BM or UCB) NK cellinfusions(escalatingdoses) I. 3x10 6 NK cells/kg (3 patients) II.10x10 6 NK cells/kg (3 patients) III. 3x10 7 NK cells/kg (3 patients) IV.10x10 7 NK cells/kg (3 patients) Evaluation for Toxicity, NK cell survival and expansion, disease status, GVL effect Two-step protocol ex vivo NK cellexpansion (3x10 8 -1x10 10 NK cells) UCB banks Control experiments in plates Experiments in static bags Experiments in bioreactors
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Transcript of Poster C Linical Nk Generation Ash 2009 Js
Adoptive transfer of CD34+ derived NK cells in poor-prognosis AML patients
Glycostem® clinical grade NK cell generation system provides sufficient numbers of functional NK cells from UCB CD34+ cells
Process is transferred into a GCP standard operating procedure. Clinical protocol (phase I/II) has been designed for testing safety and toxicity NK cells as Donor Lymphocyte Infusion (DLI) for AML patients in non transplantation setting Escalating doses will be applied in KIR-ligand mismatched setting
GBGM® can be purchased via www.clearcelltechnologies.com
Effective NK cell differentiation from umbilical cord blood (UCB) derived CD34+ cells using GBGM®
Ex-vivo generated NK cells are devoid of T- and B cells and express specific NK cell antigens
Stroma-free cell culture system for optimal NK cell generation
Clinical Scale Generation of Functional Human Natural Killer Cells From Umbilical Cord Blood CD34-Positive Cells for Immunotherapy
Jan Spanholtz, Marleen Tordoir, Carel Trilsbeek, Jos Paardekooper, Diana Eissens, Arnold van der Meer, Irma Joosten, Theo de Witte, Nicolaas Schaap, Frank Preijers and Harry Dolstra
Department of Internal Medicine - Laboratory of Hematology, Radboud University Nijmegen Medical Centre, The Netherlandsmailto: [email protected]
Clinical grade NK cell cultures provide sufficient numbers of functional NK cells which efficiently lyse various primary AML tumor cells
Efficient CD34+ cell enrichment from cryopreserved UCB units using the CliniMACS system
Efficient NK cell production from cryopreserved UCB units using various bioreactors
CD56 content 99%±1%
using GBGM®
10
100
1,000
10,000
100,000
1 2 3 4 5week
Fol
d ex
pans
ion
tota
l cel
ls
Medium1 (n=3) Medium 2 (n=3) GBGM® (n=3)
Per
cent
age
CD
56+
cel
ls
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 fold
using GBGM®
expansion differentiation 1
• GBGM® + human serum (HS)
• SCF, Flt3L, IL-7, TPO
• Clinical grade Heparin (GAGs)
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 (GAGs)
Low - dose cytokine cocktail• Low - dose cytokine cocktail• Low - dose cytokine cocktail•
• GBGM® + HS • GBGM® + HS
Novel GMP gradeGlycostem Basal Growth Medium (GBGM®)
www.glycostem.nl
Clinical scale generation of allogeneic NK cell products
CliniMACS selection
• CD34 selection
Washing &volume reductionNK cell generation
+
Umbilicalcord blood
Product release
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 1.5 50
2 69 469 0.92 69 77 2.0 53
3 75 653 0.47 62 70 2.3 73
4 70 819 1.04 78 92 6.3 73
5 53 583 0.36 56 54 1.7 76
6 74 829 0.30 68 65 1.7 79
7 71 440 0.45 88 64 1.7 82
8 53 403 0.49 68 73 1.4 69
9 80 248 1.04 69 88 1.3 72
range 53 - 80 248 - 819 0.3 – 1.04 56 - 88 52 - 92 1.3 – 6.3 50 - 82
0
500
1000
1500
2000
0 1 2 3 4 5 6week
CB0109 bag CB0209 bag CB0309 bag
0
20
40
60
80
100
0 1 2 3 4 5 6week
CB0109 plate CB0209 plate CB0309 plate
0
20
40
60
80
100
0 1 2 3 4 5 6week
CB0109 bag CB0209 bag CB0309 bag
0
2000
4000
6000
8000
0 1 2 3 4 5 6week
CB0109 plate CB0209 plate CB0309 plate
0
2000
4000
6000
0 1 2 3 4 5 6week
CB0709 wave CB0709 plate
0
20
40
60
80
100
0 1 2 3 4 5 6week
CB0709 wave CB0709 plate
Fo
ld e
xpan
sio
nF
old
exp
ansi
on
Fo
ld e
xpan
sio
n
NK
cel
ls (
%)
NK
cel
ls (
%)
NK
cel
ls (
%)
005
95 009
91 0496
0
CD3 CD19 CD15SSC
CD
14
CD
38
CD
56
CD
45
NK cells per experiment CD34+ cellsfold
expansionCD56+
(%)NK cells experiment
CB0109 1.7x106 1770 63 1.9x109 static bag
CB0209 1.4x106 759 80 8.6x108 static bag
CB0309 1.3x106 1291 70 1.2x109 static bag
CB0709 0,81x106 2861 95 2.2x109 bioreactor
0
20
40
60
80
100
AML1 AML2 AML3 AML4 AML5 K562 KG1a
day2
day3
day1
spec
ific
lysi
s (%
)
NK cell products are analyzed for
purity, showing no contamination with
T- or B cells (A).
The NK cell phenotype was determined
by 10-color flowcytometry using the
Gallios Flow Cytometer and the Kaluza
software (B).
A
B
The final NK cell products were
analyzed for functionality in a
CFSE based cytotoxicity assay
for 18h with a 2:1 effector:target
ratio.
Clinical protocol (phase I/II study)AML patient >65 years
treated with remission-induction chemotherapyHLA – haploidendical donor
sibling orchild >18 years
HLA and KIR typing
KIR-ligand incompatibility
Complete remission(<5% blasts in bone marrow)
Consolidationchemotherapy
Non-myeloablative immunosuppressionFlu 30 mg/m2 and Cy 1200 mg/m2 at day -6, -5, -4, -3
CD34 selection(BM or UCB)
NK cell infusions(escalatingdoses)I. 3x106 NK cells/kg (3 patients)II.10x106 NK cells/kg (3 patients)III. 3x107 NK cells/kg (3 patients)IV.10x107 NK cells/kg (3 patients)
Evaluation forToxicity, NK cell survival and expansion, disease status, GVL effect
Two-step protocolex vivo NK cellexpansion(3x108 -1x1010 NK cells)
UCB banks
Control experiments in plates
Experiments in static bags
Experiments in bioreactors
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