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