Cord Blood Natural Killer Cells for Immunotherapy

Nina Shah, M.D. Assistant Professor

Department of Stem Cell Transplantation and Cellular Therapy M.D. Anderson Cancer Center

Houston, TX

OBJECTIVES • NK cell tumor immunity

• Generation of CB NK cells

• Modulating the NK response

• Clinical implications

BACKGROUND: NATURAL KILLER (NK) CELLS • Non T/B cytotoxic lymphocytes • CD56+/CD3-

• Mechanisms of action • ADCC • Killer Immunoglobulin-like Receptor (KIR)-MHC Class I mismatch

NCRs +

±

+

KIRs, CD94

2B4, NTBA

NKG2D

?

HLA I

CD48?

MICA, MICB, ULBPs

KIR-HLA I MISMATCH: “MISSING SELF” HYPOTHESIS1

1. Moretta et al, Nature Immunology, 2002

NK Cell Target Cell

Donor KIR Type (HLA type) Recipient HLA Type

2DL2/3 (C1) C2 homozygous

2DL1 (C2) C1 homozygous

2DL2/3 + 2DL1 (C1 + C2) C1 or C2 homozygous

3DL1 (Bw4) Bw4 negative

ALLOREACTIVE DONOR KIR-RECIPIENT HLA I COMBINATIONS

NK CELLS AND ALLOREACTIVITY

• Allo-reactive NK cells predict better outcome in haplo-SCT’s for AML1

• Lower relapse rate in patients transplanted in CR • Better EFS in patients transplanted in relapse or remission • Reduced risk of relapse or death

• KIR-ligand incompatibility associated with improved outcome after UCBT for acute leukemia • Improved LFS, OS • Decreased relapse rate • Results more evident for AML

1. Ruggeri et al, Blood 2007

2. Willemze et all, Leukemia, 2009

WHY DON’T NK CELLS PROMOTE GVHD?

Ruggeri et al, Blood, Cells, Mol and Dis, 2004

GVL GVHD

Ruggeri et al, Blood, Cells, Mol and Dis, 2004

ARE ALLOGENEIC NK CELLS SAFE? • Haplo-identical NK cell infusion well-tolerated without

evidence of GVHD and some transient CR’s1

• MDACC protocol 2005-0508 and 2010-0099: • AML/MDS, CML pts (15 +5 so far) • Up to 8.24 x 106 CD56+ cells/kg have been infused • No infusional toxicities, grade 3 GVH or delayed engraftment thus far

• U of AK experience2: Relapsed/refractory myeloma • N=10 • Haplo-identical KIR-mismatched NK cells with auto-HCT • No GVH • No autograft rejection • 50% nCR +CR; PFS 0-533 days

1. Miller, Blood 2005

2. Shi, Br J Haematol, 2008

WHY DON’T AUTOLOGOUS NK CELLS WORK? • Altered balance of inhibitory and activating receptors on

autologous NK cells1

• Altered ligands on tumor cells - requiring more active NK cells than at baseline2

• Change in distribution of NK cell subpopulations (LN, PB) 3 • Direct immunosuppression by tumor cell –produced

soluble factors (cytokines, ligands) 1, 4

• NK cells from MM patients express PD-15 • Increased Class I on MM cells in advanced disease6

1. Lion, Leukemia, 2012 2. Veuillen, JCI, 2012 3. Gibson, Hum Pathol, 2011 4. Reiners, Blood, 2013 5. Benson, Blood, 2010 6. Carbone, Blood, 2005

CORD BLOOD AS A SOURCE OF NK CELLS

• Peripheral blood (PB) • Requires collection

• (Mis)-Matching

• Cord blood (CB) • Immediate availability

• More flexibility in matching

• More naïve T cell repertoire

CB NK CELLS: CHALLENGES

• Ability to expand CB NK cells

• Expanded CB NK cells have appropriate phenotype

• CB NK cells are as active as PB NK cells

• Must use frozen cord blood units

TECHNIQUES OF CB NK CELL EXPANSION

CB NK CELL EXPANSION: TECHNIQUES

• CD56 selection + IL-2 • Feeder cells: K562 antigen-presenting

cells expressing Fc-IL-21 or IL-15 +IL-2 • Gas permeable culture system

IL-21

CD86

4-1BBL

CD19

FcγRI

IL-15 4-1BBL

K562-cl9-mIL21 K562-mb-IL15-41BBL

ARTIFICIAL APC’S

Courtesy of Dr. L Cooper Courtesy of Dr. D. Campana

GAS PERMEABLE EXPANSION FLASKS

GAS PERMEABLE EXPANSION FLASKS

Gas permeable membrane allows for more efficient gas exchange for cells

Media automatically feeds cells by convection and diffusion

Frozen cord Blood unit

Ficoll

MNC

Culture condition: 2(γ-irradiated)APC : 1 cord MNC

IL-2 100u/ml

GP500 bioreactor

Day 7 CD3 depletion (CliniMACS)

CD3-depleted NK cells

Culture condition: 2(γ-irradiated)APC : 1 CD3 - cell

IL-2 100u/ml For another 7 days

Day 14 CD3 depletion (CliniMACS)

CD3-depleted NK cells

CD3 + cells

CD3 + cells

Flow cytometry on day7 & 14

CD56 CD16 CD3

CD19 CD14 CD45

Clinical NK Expansion From Cryopreserved Umbilical Cord Blood

Thaw

Day 0

1

10

100

1000

10000

Fresh + IL-2 Fresh + APC Frozen + APC

Fold

Exp

ansi

on o

f N

K C

ells

A

0.1

1

10

100

1000

10000

0 7 14

NK

Cel

l Exp

ansi

on, x

10

6

Days

Fresh + IL-2

Fresh + APC

B

0.1

1

10

100

1000

10000

Fold NK increase # NK Cells Produced (x 10e6)

# CD3+ Cells (x 10e6)

IL-15

IL-21

C

*

* *

n=3 n=10 n=7

P <0.01

P <0.01

* P <0.01

P <0.01

APC CB-NK expansion from fresh or cryopreserved CB units yields significantly greater fold expansion of NK cells than expansion of

CD56+ cells with IL-2 alone

Fold NK increase

Absolute NK (x 10e6)

Absolute CD3 (x 10e6)

IL-15 2659.5 883.1333333 0.872

IL-21 4093.66

6667 1471 4.495666667

100 101 102 103 104

FL2 H CD(16 56) PE

100

101

102

103

104

FL4-

H: C

D3

AP

C

39.3 0.22

7.5253

100 101 102 103 104

FL2 H CD(16 56) PE

100

101

102

103

104

FL4-

H: C

D3

AP

C

0.22 0.39

77.521.9

100 101 102 103 104

FL2 H CD(16 56) PE

100

101

102

103

104

FL4-

H: C

D3

AP

C

0.032 0.15

96.43.43

0 200 400 600 800 1000

0

200

400

600

800

1000

SS

C-H

: Sid

e S

catte

r

93.6

0 200 400 600 800 1000

0

200

400

600

800

1000

SS

C-H

: Sid

e S

catte

r

78.6

0 200 400 600 800 1000

0

200

400

600

800

1000

SS

C-H

: Sid

e S

catte

r 74.8

Day 0 Day 14 Day 7 C

D3

SSC

FSC

A

CD56

aAPC

Eomes T-bet

B

C CD16 NKp30 NKp46 NKp44 KIR2DL1/DS1 KIR2DL2/DL3 KIR3DL1

NKG2A CD94 NKG2C

IL-2 alone

AFTER EXPANSION, CB NK CELLS ARE AS ACTIVE AS PB NK CELLS

Pre-Expansion Post-Expansion

Xing et al, J of Immunotherapy, 2010

K562 RPMI 8226 Primary CD138+ cells

U266 ARP-1

A

CD138+ CD138-

B

APC-expanded NK cells synapse with tumor targets.

0

10

20

30

40

50

60

% N

K:

Targ

et S

ynap

se F

orm

atio

n

A

C

-10

0

10

20

30

40

50

60

70

80

10:1 1:1 0.1:1

% C

ytot

oxic

ity

Effecto:Target Ratio

K562

RPMI 8226

ARP-1

U266

Autologous

0

5

10

15

20

25

10:1 1:1 0.1:1

% C

ytot

oxic

ity

Effector:Target Ratio

CD138+

B

0

10

20

30

40

50

60

70

80

10:1 1:1 0.1:1

% C

ytot

oxic

ity

Effector: Target Ratio

IL-2

APC

APC-expanded NK cells are cytotoxic to various myeloma targets

0

5000

10000

15000

20000

25000

30000

35000

40000

8 15 22

ARP-1

ARP-1+ NK

Days after ARP-1 injection

Lum

ines

cenc

e in

RO

I, p

/s

0

500000000

1E+09

1.5E+09

2E+09

2.5E+09

3E+09

3.5E+09

4E+09

4 8 11 15 18 22

ARP-1

ARP-1 + NK

P <0.05 at each time point

Days after ARP-1 injection

Seru

m k

appa

, ng

/mL

d.4

d.8

d.11

d.15

d.18

d.22

ARP-1 ARP-1 +NK

P <0.01 at each time point

CB-NK cells delay development of myeloma in a NSG murine model

-8 -7 -5 0

Lenalidomide 10 mg daily

High dose melphalan, 200 mg/m2

CB NK cells

Autologous graft

PHASE I/II STUDY OF UMBILICAL CORD BLOOD-DERIVED NATURAL KILLER CELLS IN CONJUNCTION WITH HIGH DOSE CHEMOTHERAPY AND AUTOLOGOUS STEM CELL TRANSPLANT FOR PATIENTS WITH MULTIPLE MYELOMA

-2

EXPANDED CB NK CELLS ARE ACTIVE AGAINST CLL

EX CB NK

CLL-B

EX CB NK

CLL-B

EX CB NK

CLL-B

EX CB NK

CLL-B

Day -8 to -2: lenalidomide 10 mg PO daily Day -7 to -4: Fludarabine 40 mg/m2 IV daily Day -4: Melphalan 140 mg/m2 IV x1

Day -16 to -2 Identify 2 CB units

Use 20% fraction of one unit to generate

NK cells

-16 -2 0

Ex vivo NK generation from 20% fraction

Day 0

Infuse unmanipulated CB units

100% CB# 1

80% CB# 2

Day -2

Infuse expanded NK cells

Protocol 2011-0493: Pilot study of double cord blood transplantation with ex-vivo expanded cord blood derived natural killer cells to enhance the graft-versus-

leukemia effect in patients with relapsed/refractory lymphoid malignancies.

NK cell dose: 3- 7 x108 (can be obtained from the 20% fraction of the CB unit)

Principal Investigator: Chitra Hosing

ENHANCING CB NK ACTIVITY: LENALIDOMIDE

LENALIDOMIDE

• Immunomodulatory agent

• First-line novel agent in myeloma

• Direct anti-tumor effects via apopotosis

• Disrupts tumor microenvironment (anti-angiogenic and anti-osteoclastic effects)

• Immunomodulatory effects, possibly on NK cells

Chromium Cytotoxicity Assay

Treatment of target myeloma cells with lenalidomide enhances CB NK cytotoxicity

TREATMENT OF EFFECTOR AND TARGET WITH LENALIDOMIDE ENHANCES CELL KILLING OF PRIMARY CLL

0

10

20

30

40

50

60

70

10:1 1:1 0.1:1

No Revlimid

Plus Revlimid

p < 0.001 at 1:1 and 10:1

E:T Ratio

Perc

ent S

peci

fic L

ysis

CAN LENALIDOMIDE REVERSE NK DYSFUNCTION?

…Can lenalidomide enhance normal NK function? Ramsay, Blood, 2012

CB NK CELL -PROJECTS IN DEVELOPMENT

FUCOSYLATION OF NK CELLS TO IMPROVE HOMING TO BM

√

P-, L- & E- Selectin? BM endothelial cell

A

*

* * *

* *

*

*

* *

*

*

*

* CD34+

CB NK

NK

MM

FcR

Elotuzumab

NK CAR Potential Targets for MM • CS1 • Kappa/ lambda light chain • CD38

CONCLUSIONS • NK cells are a potential therapy for hematologic

malignancies

• Autologous NK cells do not provide sufficient anto-tumor activity

• CB NK cells may be a safe and effective allogeneic option to shift balance towards GVL and away from GVH

• CB NK cells can be expanded to clinical scale

• CB NK cells have activity against myeloma and CLL (and other hematologic malignancies)

ACKNOWLEDGEMENTS

Cell Therapy Laboratory Elizabeth J. Shpall Katy Rezvani Ian McNeice Beatriz Martin Antonio Simon Robinson Hong Yang Eric Yvon Amer Najjar Simrit Parmar Michael Thomas Xiaoying Liu Sufang Li Junjun Lu Van Nguyen Indreshpal Kaur

Baylor College of Medicine- Center for Cell and Gene Therapy Catherine Bollard Stephanie Ku, Benjamin Tzou

Department of Stem Cell Transplantation and Cellular Therapy Richard Champlin Chitra Hosing Muzaffar Qazilbash

Department of Pediatrics Laurence Cooper Dean Lee

Department of Lymphoma/Myeloma Robert Orlowski Michael Wang Qing Yi

Celgene Corporation

THANK YOU!