Post on 23-Apr-2018
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