Natural Killer cells and HematopoieticStem Cell Transplantation

Jeffrey S. Miller, M.D.

University of Minnesota Cancer CenterAssociate Director of Experimental Therapeutics

Division of Heme/Onc/TransplantMinneapolis, MN

NK cells are important

• Cancer treatment and tumor surveillance

• Infection disease control• Autoimmunity• Pregnancy (placental angiogenesis)

NK cell functions• Killing targets• Produce cytokines

– Interferon-γ– Tumor necrosis factor– Many others

NK cells after transplant are increased

Percentage Donor and Recipient NK cells (TCD vs. UBM)

0.005.00

10.0015.0020.0025.0030.0035.0040.0045.0050.00

% N

K Ce

lls

= Normal DONOR

= RECIPIENT

TCD UBM

NK

Cooley et alBlood 106:4370, 2005

2DL22DS2 2DL5B 2DS3 2DL1 3DP1 2DL5A 2DS4 2DS5 2DS1 3DL23DL1

2DL42DP12DL33DL33DS1

HLA-C N80 HLA-C N80 HLA-C K80 HLA-C K80HLA-Bw4 HLA-AHLA-G

5 6 7 5 6 7 2 10 4 9 20 1 6 5 4 13

KIR:

Ligand:

No. of Alleles:

Chr. 19 determines the personality of NK cells:Killer-immunoglobulin receptor (KIR) gene locus

From Peter Parham

NKG2 family recognizes HLA-E

NK cells are very different after URD HCT

10 0 10 1 10 2 10 3 10 4

% NKG2A

10 0

10 1

10 2

10 3

10 4

%C

D56

1.44 6.9

32.159.5

10 0 10 1 10 2 10 3 10 4

% NKG2A

10 0

10 1

10 2

10 3

10 4%

CD

56

3.34 29.1

52.115.5

10 0 10 1 10 2 10 3 10 4

% KIR

10 0

10 1

10 2

10 3

10 4

% C

D56

8.99 0.82

59.731.2

10 0 10 1 10 2 10 3 10 4

% KIR

10 0

10 1

10 2

10 3

10 4

% C

D56

34.1 8.2

38.321.6

Donor Recipient

% C

D56 % KIR

% NKG2A

60% 46%

81%39%

Recipients have• < KIR• > CD56+bright

• > NKG2A• < Function

Cooley et alBlood 110:578, 2007.

NKG2A/KIR expression distinguishes populations of CD56+dim NK cells

KIR-/NKG2A- subset: 19.4 ± 2.8% of CD56+dim NK cells healthy donors (n=26)

These cells do not kill targets or make IFN, thus are hyporesponsive (immature)

100 101 102 103 104CD3 PerCP-Cy5-5-H

ADULT BLOOD.fcs

100 101 102 103 104CD3 PerCP-Cy5-5-H

ADULT BLOOD.fcs

100 101 102 103 104NK2Ga PE-H

ADULT BLOOD.fcs

100 101 102 103 104NK2Ga PE-H

ADULT BLOOD.fcs1% 5%

86% 8%

BRIGHT

100 101 102 103 104NK2Ga PE-H

ADULT BLOOD.fcs

100 101 102 103 104NK2Ga PE-H

ADULT BLOOD.fcs42% 12%

30% 16%

DIM

A B C

Cooley et al Blood 110:578, 2007.

Hypothesized NK cell development schema

CD34 + /CD7-

CD56 -CD34 + /CD7 +

CD56 -

Lymphoidcommitted progenitor

CD34-/CD7 +CD56 -

NK cellprecursor

CD56 + KIR -

NK cellcommitment

Marrow BloodStage 5 & 6

CD56+bright

KIR-CD56+dim

KIR+

MatureFc +

cytotoxic NKIFN-γ

producing NK

KIR

CD94NKNKNKNKp

NK precursors left shifted intothe blood after transplant?

HS

C=C

D34

+ /Li

n- /CD

38- LN

Stage 4

Outpatient subcutaneous IL-2 promotes in vivo NK cell expansion

100

1000

10000

Days On IL2

Abs

olut

e C

ount

per

µl

Pre-IL2 14 28 42 56

NK

T

Miller et al, Biol Blood Marrow Transplant 3:34, 1997

…but NK cells are not maximally activated

Autologous NK Administration in Cancer Patients

IL-2

PB

NK IL-2

Recovery from autologous HCT

IL-2

NK cells more activated using this approach

Conclusions� Enhanced activation of NK cells.� A matched paired analysis with our data and data

from the IBMTR showed no apparent efficacy (survival or time to disease progression).

NK cell-based autologous Immunotherapy to prevent relapse (HD, NHL, BC)

Burns et al, Bone Marrow Transplant, 32:177-186, 2003

KIR - MHC class Imatch->

No Killing

NK

KIR - MHCmismatch->Lysis occurs

NK

To Kill or

not to kill

Hypothesis: Autologous NK cell therapy failed due to inhibitory receptors that recognize MHC

apoptosis

Auto Allo

AML Transplant trials based on promoting NK cell alloreactivity

Transplant Graft Outcome

Ruggeri et alScience 3/2002

HaploidenticalKIR-L Mismatch

TCD Benefit in AML

Davies et alBlood 11/2002

URD KIR-L Mismatch

UBM No Benefit

Giebel et alBlood 8/2003

URD KIR-L Mismatch

In Vivo TCD Benefit

Pros and cons

Safer More TRMTransient PermanentCan expand in vivo (IL-2) Too risky 2o

GVHD risk

Adoptive Transfer Transplant?

How can we best exploit NK cells?

Related Donor Haploidentical NK Infusions After High Dose Chemotherapy

TCDIL-2

PBNK

HD Rx

IL-2

Cy 60 mg/kg x 2Flu 25 mg/m2 x 5

10 MU QOD x 6

2-8 x 107 MNC/kg

Patients and eligibility

• Poor prognosis AML (n=19)– Primary refractory disease– Relapsed disease not in CR after 1 or

more cycles of standard re-induction therapy

– Secondary AML from MDS– Relapsed AML > 3 months after HCT.

• No active infections

KIR Ligand mismatched donor correlates with achieving AML CR (5 of 19=26%)

KIR L MMn=4

KIR L Matchn=15

KIR L MM KIR L Match0

10

20

30

40

50

60

70

80

% A

chie

ving

CR

P=0.04

CR patients had higher numbers of functional NK cells after haplo NK cells

CR non-CR0

10

20

30

40

50

60

70

80%

NK

cel

lsp=0.027

% NK cells at Day 14-28

NK cells did not expand with lower dose preparative regimens

Miller et al, Blood 105:3051, 2005

In vivo expansion of haploidenticalNK cells in AML

B-act

HLA-B7

100

1 No

Don

or

D1

D2

D7

D14

H2O

D28

0.1

0.01

0.00

1

10

After cell infusions

Donor Specific HLA-A31

ß-actinPB

CD

56+

BM

CD

56+

BM

CD

3+H

20

PB C

D3+

PB C

D19

+

100%

10%

1% 0.1%

0.01

%

0.00

1%

No

Don

or

Circulating donor cells were functional NK cells 14 days after Haplo NK cell infusions

92%

20 6.6 2.2 .75 .250

20

40

60

80

100 K562Raji

Effector:Target Ratio%

Spe

cific

Lys

is

Verified by VNTR and G-banding

Hi-Cy/Flu induces in vivo expansion of donor cells (all patients by prep)

0 1 2 7 14 28

0

10

20

30 Renal - FluAML Hi-Cy/Flu

Day after NK cell infusion

% D

onor

Hi-Cy/Flu induces endogenous IL-15 which correlates with in vivo NK cell expansion

28147210Pre0

20

40

60

80

100

120

Day after cell infusion

IL-1

5 C

once

ntra

tion

(pg/

ml) **

Cy/FluFlu

Interpretation of cytokine data

• Every time we give lymphodepletingchemotherapy (±TBI), we see a sustained surge in endogenous IL-7 and IL-15

• May explain high fevers when adding exogenous IL-2 in this setting.

Questions

• Why NK cells don’t expand in everyone?

• Would other cell sources be superior to adult blood NK cells?

Adoptive Transfer Transplant+

Hypothesis

The best strategy may be to combine adoptive transfer and in vivo expansion followed by HCT

The best of both worlds?

Umbilical Cord Blood

– 100-150 ml cord blood

– Usually discarded

– High concentration of hematopoietic and NK cell progenitors

– Stem cell source for related donor transplant

Cord blood is rich in NK cell precursors

100 101 102 103 104CD3 PerCP

FRESH UCB

100 101 102 103 104CD3 PerCP

FRESH UCB

10 0 10 1 10 2 10 3 10 4CD7 PE

FRESH UCB

10 0 10 1 10 2 10 3 10 4CD7 PE

FRESH UCB20.0% 2.3%

8.4%

**Percents shown are based on the total CD3 depleted product as the denominator

Testing thispopulation

clinically

-15 -14 -13-16-19 -18 -17 0-12

Full Prep Triple UCB strategy: UCB NK + double UCB Transplant for patients with refractory AML

14

BMBx

28

BMBx

TIME

UCB 3

HLA

<2

agm

m

HLA < 2 ag mm TNC >1.5 x 107/kg

UCB 2

HLA < 2 ag mm TNC >1.5 x 107/kg

TBIFLU

CY

FLU

CY

FLU

-1

BMBx

-25

BMBx

Patient Eligibility• Age 2-45 years• Refractory AML

IL-2 x 6 doses for In vivo NK expansion

CsA (day -1 to 6 months)

MMF (day -1 to day 35 unless GVHD)

UCB 1

CD3 deplete IL-2 Overnight

(1000 U/ml)

THAW

HLA < 2 ag mm TNC >1.5 x 107/kg THAW

KIR-L MM if possible

Pablo Rubinstein

New York Blood Center

Conclusions• NK cells are important in cancer therapy

and transplant.• Better methods to optimally activate NK

cells are still needed for refractory AML patients.

• KIR genotyping may be of value in selecting donors in addition to HLA-typing.

Acknowledgements• Miller Lab

– Purvi Gada– Veronika Bachenova– Valarie McCullar (Research)– Gong Yun– Karen Peterson– Michelle Pitt– Todd Lenvik– Becky Haack– Feng Xiao– Sue Fautsch (Translational)– Sarah McNearney– Rosanna Warden– Kirsten Malvey– Liz Narten– Michelle Gleason– Ginny Kohl

• HLA typing lab - Harriet Noreen• NMDP/CIBMTR (Confer, Klein, Wang, Spellman, Maiers)• U of MN Faculty

– Phil McGlave– Arne Slungaard– Linda Burns– John Wagner– Claudio Brunstein– Bruce Blazar– Dave McKenna (GMP Facility)– Chap Le/Tracy Bergemann (Biostat)– Dan Weisdorf, Sarah Cooley, MD

• Stanford– Peter Parham

• Univ of Washington– Dan Geraghty

• Children’s Hospital Oakland– Beth Trachtenberg

• London– Steve Marsh

NK PPG working group