AKIHIRO SHIMOSAKA, MD

Houston, USA• Chairperson, Asian Cellular Therapy Organization,

Tokyo, Japan

• Director, R & D, Research Foundation for Community

Medicine, Utsunomiya, Japan Secretary, ISCT Asian

Region

• Honorary Professor, Hematology Institute, Peking

Union Medical College

• Chinese Academy of Medical Sciences, Tianjin, China

• Honorary Professor, School of Oncology, Peking

University, Beijing, China

• Visiting Professor, the Fourth Military Medical

University, Xi-an, China

2

Akihiro Shimosaka, Ph.D.

Asian Cellular Therapy Organization

Research Foundation for Community Medicine

Human NK cell expansion

for cancer therapy

•3

Study Start

material

Stimulation Feeder

cells

NK cell

isolatio

n

Serum Medium NK fold

expansion/timeReferences

Alici PBMC Anti-CD3 No No Human AB

serum

CellGro

SCGM

x190/3w Hum Immunol

62:1092, 2001

Campana PBMC 4-1BB&IL-15

gene-

transfected K562

K562 No Fetal

bovine

serum

CellGro

SCGM

x152/2w Cancer Res

69:4010, 2009

Childs PBMC No EBV-TM

B-cell line

Yes Human AB

serum

X-VIVO 20 x300-900/19d Cytotherapy

11:341, 2009

Multhoff PBMC Hsp-70-peptide No No No CellGro

SCGM

0.9-1.9x109 J Transl Med

7:50, 2009

Dolstra Umbical

Cord

CD34+

SCF,IL7,Flt3L,

TPO,

G-CSF,GM-CSF,

IL-6, MIP-1a, LIF

No No No GBGM x1500-6500/5w

(4.6±2.4x109)PlosONE

5:e9221, 2010

Masuyama PBMC Ab-immobilized

culture bag

No No Self-

plasma

NKGM x859/2w

(6.4±2.3x109/20ml

peripheral blood)

Culture methods for expansion of human NK cells

Protocol for human NK cell expansion

PBMCs + autologous plasma

+

Large bags containing

NKGM (1Lx2~3,) + IL-2

A small bag immobilized with two different

GMP level antibodies

•2 w

eeks

Suspended in 100ml

physiological saline

Stimulation

Expansion

NKGM

Cel

lGro

SCGM

X-V

IVO 1

0

AIM

-V

0

200

400

600

800

Fo

ld e

xpansio

n o

f N

K c

ells

NK cell expansion by various media

4

•5

Large-scale expansion of NK cells

with JM NK cell culture kits

(20 ml peripheral blood from healthy donors, n = 25)

Yield of cells (x109)

NK cell yield (x109)

Fold expansion of NK cells

: 11.4 ± 1.6 (mean ± SD)

: 6.4± 2.3

: 1101 ± 670

•6

Large-scale expansion of NK cells

with JM NK cell culture kits

Subsets of bulk NK cells（％）

NK cell

CD8 T cell

CD4 T cell

: 56.9 ± 18.6 (mean ± SD)

: 26.3 ± 15.9

: 15.9 ± 10.9

•7

0 5 10 15 20 25

0

2

4

6

8

10

12

(x 2100)

CD4+T cell

CD8+

T cell

CD3 CD56 + NK cell

addition of culture medium

Days of culture

Cell n

um

ber（ｘ１０９）

-Peripheral

blood 30ml

Selective proliferation of NK cells derived

from peripheral blood lymphocytes

•8

Selective proliferation of NK cells derived

from peripheral blood lymphocytes

- FACS analysis -

CD

56

ＣＤ３

Initial PBL

4.0x107

Proliferative

lymphocytes

1x1010

culture

for 2 w

NKNK

CD3 CD56+ NK cells 25.1% (1.0x106) 74.4% (7.4x109)-

•9

Cytotoxicity of expanded and resting NK cells

Target : K562

4h exposure

0

20

40

60

80

100

0.3:1 1:1 3:1 10:1

E:T ratio

% c

yto

toxi

city

resting

expanded

•10

Activating receptors Inhibitory receptors

Enhanced expression of activating receptors on expanded NK cells

Control IgG

Resting NK

Expanded NK

•11

CXCR3 expression of NK cells

Peripheral

resting NK

LAK (stimulation

by IL-2 alone)

Expanded NK(Our method)

CX

CR

3

CD16

5% 15% 98%

NK

•12

CXCR3+ NK cells

1. Type1-immune response

Induction of Th1 and CTL

via NK-stimulated DC(DC1)

High level secretion of IFN-gMigration to inflamed tissues

1. Infiltration to tumor sites

augmented by chemokines

2. Recruitment to lymph nodes

Two different functions of CXCR3-expressing NK cells

•13

IFN-g production of NK cells purified from expanded NK cells

compared to CD3-stimulated T lymphocytes

Stimulation of 2 x 104 cells

for 36 h with IL-12 (10 ng/ml)

+ IL-18 (10 ng/ml)

nil IL-12 nil IL-120

1000

2000

3000NK cellCD3-stimulated T cell

+IL-18 +IL-18

IFN

-g (

pg/m

l)

•14

NKG2D and MICA/B

NKG2D+ lymphocytes

NK cell

CD8 T cell

gd T cell

stimulation

Killing

Normal cell

NK

MICA/B (or ULBP)

Malignant cell

NK

NKG2D

binding

Perforin

NKG2D, an activating receptor of NK cells,

binds to MICA/B expressed on malignant cells,

resulting in NK cell activation and killing.

•15

Enhanced expression of NKG2D on expanded NK

CD16

NK

G2D

30 15847 98

Resting PBL Expanded NK

Stimulation

NK NK

& Culture

MFI

CD8 T CD8 T

•16

Re-expression of CD3 z chains of expanded NK cells

PBMCPBMC Expanded NK cells

CD16

CD

3 z

chain

Patient with cancerHealthy subject

NK NKNK

Although expression of CD3 zeta chains which is necessary to induce

ADCC activity is often low in NK cells of cancer patients, expanded NK

cells re-express CD3 zeta chains.

Large-scale expansion of NK cells(20 ml peripheral blood from healthy donors, n = 25)

8

10

12

14

16

Yie

ld o

f cells (

x 10

9)

NK CD8 CD40

20

40

60

80

100

Pro

po

rtio

n o

f e

xpa

nd

ed

ce

lls (

%)

0

5

10

15N

K c

ell

yie

ld (x

10

9)

0

1000

2000

3000

Fo

ld e

xpansio

n o

f N

K c

ells

: Cryopreserved

18

JM NK Cell Expansion Kit

to culture large number of NK cells from peripheral blood

19

Characteristics of JM human NK cell culture method

1. Simple and safe: only use patient PBMC, autologous plasma

without fetal bovine serum or accessory cells

2. High Expansion : 860-fold (n = 25, normal subjects)

One of other methods : 150-fold (Cancer Res 69:4010, 2009)

3. Highly Active NK cell :

Enhanced expression of activating molecules, such as

NKG2D with high level cytotoxicity and IFN-g production

20

Experiences of JM method cultured NK cell

therapy for cancer patients

All patients were at stage III/IV and failed to

conventional therapy

21

Cancer patients treated in New City Osaki ClinicTotal 541 cases （2004.7~2012.11)

•22

Increase of NK cell activity in patients with cancer

after NK cell therapy

NK cell activity

The increase rate １２７％ ５０％ ５％

Normal range

“Low” (<２５％) “Normal” (２５～４０％) “High” (４0％<)

NK

cell

activity （％）

before after before after before after0

10

20

30

40

50

60

70

0

10

20

30

40

50

60

70

0

10

20

30

40

50

60

70

Patients were divided in 3 groups based on the ranges of NK cell

cytotoxicity before NK cell therapy.

23

Case 3 55 y.o., FemaleDiagnosis: Breast cancer local recurrence and multiple liver metastases

Chemotherapy: S-1NK cell therapy: x 10

Before NK cell therapy After NK cell therapy

(5 mo. later)

24

Before NK cell therapy After NK cell therapy

(5 mo. later)

Case 3 55 y.o., FemaleDiagnosis: Breast cancer local recurrence and multiple liver metastases

Chemotherapy: S-1NK cell therapy: x 10 。

May, 2011 August, 2011

Case 2 Lung cancer 84 y.o. Male

Diagnosis: Lung cancer

Chemotherapy: none

NK cell therapy: x 6, at a 2-week interval

Case 3 56 y.o. male

Pancreatic tail cancer with multiple liver

metastases and peritoneal dissemination

10 infusions

Lymphocytes : 156.1x109

NK cells : 89.6x109

from a total of 280 ml peripheral blood

0 10 20 30 400

5000

10000

15000

20000

25000

0

50

100

150

200

250CA19-9

CEA

S-1 (day 1-14/3 weeks)GEM (day 1, 8 and 15/4 weeks)

NK cell therapy

GEM (day 1 and 8/3 weeks)

(weeks)

CA

19-9

(U

/ml) C

EA

(ng

/ml)

0 10 20 30 400

20

40

60

80

NK

acti

vit

y (

%)

0 10 20 30 400

200

400

600

800

1000 NKG2D+ NKNKG2D+ T

Nu

mb

er

of

cells

in p

eri

ph

era

l b

loo

d (

/

l)

CT scanNK cell therapy

(w)

(w)

NK and CD8 T cells having NKG2D are activated through specific recognition with MICA/B molecules

widely expressed on malignant cells, and eventually exert cytotoxicity.

Just before NK therapy 3 months later

17 months after chemotherapy

12 months after NK cell therapy

Overall survival time

CT scan

Profile of patients with pancreatic cancer

NK cell therapy

(infusions*)

0~3 4~

Number of patients 20 33

Mean age (y), M/F 65, 12/8 64, 20/13

PS 0-2

3-4

8

1230

3

Stage IVa / IVb 3/17 12/19

+ Resection 3 8

+ Chemotherapy 12 29

* usually performed at a 2-week interval

29

Ref.１．Jpn J Clin Oncol 38:755, 2008２．Cancer Chemother Pharmacol 61:615, 2008 ３．ASCO, Abstract #4550、2007 ４．Jpn J Clin Oncol 39:49, 2009

1-year survival rate of patients with pancreatic cancer

（%, median）

Gemcitabine＋Ｓ－１

３３

Gemcitabine ２６４ １）

Ｓ－１ ４０ ２）

５４ ３）

２２ ４）Gemcitabine＋Ｓ－１

CasesChemotherapy

２５

１９

１２

NK therapy

NK therapy

NK therapy

NK therapyPS 2-3

ＩＶｂ

without resection/PS 0-1

+GEM/S- 1(88%)

30

Overall survival time of patients with pancreatic cancer

（month, median）

Gemcitabine＋Ｓ－１

３３

Gemcitabine ２６４ １）

Ｓ－１ ４０ ２）

５４ ３）

２２ ４）Gemcitabine＋Ｓ－１

CasesChemotherapy

２５

１９

１２

NK therapy

NK therapy

NK therapy

NK therapyPS 2-3

Ｓｔａｇｅ ＩＶｂ

without resection/PS 0-1

+GEM/S- 1(88%)(range:4~52)

(range:4~25)

(range:4~52)

(range:5~37)

Ref.１．Jpn J Clin Oncol 38:755, 2008２．Cancer Chemother Pharmacol 61:615, 2008 ３．ASCO, Abstract #4550、2007 ４．Jpn J Clin Oncol 39:49, 2009

31

Short-term survivors ( n = ８)

died within 5 months after NK cell therapy

not effective

Long-term survivors (n = 1３)

survived over 10 months after NK cell therapy

effective

Comparison of immunological changes

between short and long term survivors

in pancreas cancer patients

32

Changes of number of NK cells in peripheral blood

after NK cell therapy

0

200

400

600

800

1000

Healthysubjects(n=25)

Short termsurvivors

(n=8)

Long termsurvivors(n=13)

pre post pre post

Num

ber

of N

K c

ells

in p

erip

hera

l bl

ood

(/l)ns p<0.005

33

Changes of number of NKG2D+ cells in peripheral blood

after NK cell therapy

0

400

800

1200

1600

Healthysubjects(n=25)

Short termsurvivors

(n=8)

pre post

Long termsurvivors(n=13)

pre post

Num

ber

of N

KG

2D+

cells

(/

l)

p<0.005ns

Adverse effects

Adverse effect Notes % of

patients

Relation to NK

cell therapy

Fever (37.5oC~) transient 17 yes

Liver dysfunction increase in LDH,

ALP, etc.

18 no

Anemia decrease in Hb 8 no

Renal dysfunction slight increase

in Cr level

3 no

Dyspnea exacerbation of

COPD

1 possible

Vomiting 1 no

All adverse effects observed in 100 patients (ID:1 ~100) received NK cell therapy.

•35

Application of NK cell therapy

- ADCC –Antibody Dependent Cellular Cytotoxicity

Cancer cell specific attack by NK cells through ADCC pathway

CD16 : FcgRIII

Activating receptor

dependent on CD3 z expression

NK cell

CD16

Cancer cell

antibody

Tumor

antigen

killing

Antibody therapy

Breast cancer : trastuzumab

Malignant lymphoma: rituximab

•36

ADCC activity of expanded NK cells

Ab alone

Target

Daudi 1x105/well

Effector

CD4 12 %

CD8 44 %

NK 43 %

Antibody

Rituximab 5 g/ml

16 h culture

0.8 4 200

20

40

60

80

100w/o Ritux.

w/ Ritux.

E/T ratio

% C

ell

lysis

NK Cell Therapy of CancerDr. Dario Campana, Singapore

Allogeneic hematopoietic

stem cells transplant

Infusion of NK cells

Donor KIR profiles predictive of

alloreactivity are associated

with lower relapse rates

Selection of donor with

alloreactive KIR profile

Immunosuppressive but non-

myeloablative conditioning

5/19 patients with high-risk

AML achieved complete

remission (Miller et al. Blood 2005)

Infused NK cells persist for at

least 1 week (Miller et al. Blood

2005; Rubnitz et al. J Clin Oncol 2010)

No GvHD

NK Cell Expansion from:

Peripheral blood of healthy donors, children with acute lymphoblastic leukemia in remission, patients with multiple myeloma, patients with gastric cancer

Cord blood

Liver lymphocytes

Clinical Use of Expanded NK Cells

Acute Myeloid Leukemia

Solid tumors

Acute Lymphoblastic Leukemia

Conclusion

1. This novel method is feasible for expansion of

human NK cells.

2. NK cell therapy presented here is safe for

cancer patients.

3. Further study is necessary to confirm the clinical

effects of NK cell therapy on cancer.

•40

41

Enhancement of NK cell killing

• Through ADCC activity in combination with antibody-based therapy

for cancer.

• In the allogeneic setting to avoid the negative signals.

Enhancement of CTL induction

• With cancer vaccine therapy (peptide, DC, DNA) through dendritic

cell-NK cell cross-talk.

Replace T-cell donor lymphocyte infusion

To manage MRD, relapse and uncontrollable infection

Future challenges of NK-cell based immunotherapy

AcknowledgmentDr. Junichi Masuyama

New Osaki Clinic, Tokyo

Dr. Dario Campana

Department of Pediatrics, Yong Loo Lin School of

Medicine, National University of Singapore