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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
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OBJECTIVES • NK cell tumor immunity
• Generation of CB NK cells
• Modulating the NK response
• Clinical implications
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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
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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
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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
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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
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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
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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
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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
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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
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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
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TECHNIQUES OF CB NK CELL EXPANSION
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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
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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
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GAS PERMEABLE EXPANSION FLASKS
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GAS PERMEABLE EXPANSION FLASKS
Gas permeable membrane allows for more efficient gas exchange for cells
Media automatically feeds cells by convection and diffusion
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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
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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
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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
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AFTER EXPANSION, CB NK CELLS ARE AS ACTIVE AS PB NK CELLS
Pre-Expansion Post-Expansion
Xing et al, J of Immunotherapy, 2010
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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
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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
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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
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-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
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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
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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
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ENHANCING CB NK ACTIVITY: LENALIDOMIDE
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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
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Chromium Cytotoxicity Assay
Treatment of target myeloma cells with lenalidomide enhances CB NK cytotoxicity
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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
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CAN LENALIDOMIDE REVERSE NK DYSFUNCTION?
…Can lenalidomide enhance normal NK function? Ramsay, Blood, 2012
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CB NK CELL -PROJECTS IN DEVELOPMENT
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FUCOSYLATION OF NK CELLS TO IMPROVE HOMING TO BM
√
P-, L- & E- Selectin? BM endothelial cell
A
*
* * *
* *
*
*
* *
*
*
*
* CD34+
CB NK
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NK
MM
FcR
Elotuzumab
NK CAR Potential Targets for MM • CS1 • Kappa/ lambda light chain • CD38
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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)
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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
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THANK YOU!