NK Cells: Therapeutic Tools in Haematopoietic Transplantation Loredana Ruggeri Division of Hematolog

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NK Cells Therapeutic Tools in Haematopoietic
TransplantationLoredana RuggeriDivision of
Hematology and Clinical Immunology, University
of Perugia, Italy
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In adults with acute leukemia survival is poor

• when they have unfavorable prognosis at diagnosis
  (high blast count, unfavorable cytogenetics,
  secondary leukemia, etc.)
• when they do not achieve CR after the first
  induction cycle
• when they are in second or later remission
• An allogeneic HSC transplant is usually required

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Bone marrow donors
One Haplotype-Mismatched Family Donor
100 Immediately available
40
30
No Donor
Unrelated
Obstacles
Matched sibling
T-Replete T-Depleted BMT BMT
GvHD Rejection
30
4
High doses of T cell-depleted stem cells allow
transplants across HLA barriers
(Aversa et al. NEJM 1998)
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THE CONDITIONING REGIMEN
-
9
TBI 7.5 Gy in a single fraction (4 Gy on the
lungs)
-
8
Thiotepa 5 mg/kg
-
7
Thiotepa 5 mg/kg
-
6
Fludara 40 mg/m2
ATG 1.5-2.5 mg/kg
Patient day
-
5
Fludara 40 mg/m2
ATG 1.5-2.5 mg/kg
-
4
Fludara 40 mg/m2
ATG 1.5-2.5 mg/kg
-
3
Fludara 40 mg/m2
ATG 1.5-2.5 mg/kg
-
2
Rest
-
1
Rest
0
HSCT
6
GRAFT PROCESSING BY CLINIMACS T- and B-cell
depletion
Procedures 250
Before After
Before After Median
15.4 0.06 Range 1.7 - 64.2
0.01 - 2.92 log depletion
4.5 Median 3.06
0.35 Range 0.07 - 16.7
0.01 - 9.4 log depletion 3.2
CD3
CD3
CD20
CD20
HSCT Program University of Perugia 8/2003
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GRAFT PROCESSING BY CLINIMACS CD34 cell Recovery
Procedures 250
Before After
CD34
CD34
Median 0.77 90 Range
0.16 - 2.66 24 - 98.7
Yield 78
HSCT Program University of Perugia 8/2002
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High doses of T cell-depleted stem cells allow
engraftment across MHC barriers
Aversa et al., NEJM 1998
Graft composition CD34 10
x 106/kg CD3 2 x 104/kg
Data from NEJM study
AML ALL No 20 23 CR I
3 4 CR? II 10 11 REL
7 8
Engraftment gt 95 GvHD lt 10
No post-transplant immune suppression
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Aversa et al., NEJM 1998, updated
Haplo transplant in AML (1993-2004) EFS
MUD in AML ALWP Study 1992-2002 LFS
CR I (24)
0.500.10
CRII (37)
0.360.08
Relapse (51)
0.160.05
CR1 (n301)
48 7
CR2 (n351)
42 7
Advanced (n400)
20 6
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High doses of T cell-depleted stem cells allow
transplants across HLA barriers (gt90
engraftment, lt 10 GvHD)
Problems associated with T cell depletion a)
Leukemia relapse b) Delayed immune recovery and
TRM
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Haploidentical Transplants
NK cell alloreactivity strengthens the GvL effect
and reduces transplant-related mortality
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NK cell function balance between activating and
inhibitory receptors
NK cell
Inhibitory Receptor
Activating Receptor
MHC class I
Ligand
AutologousTarget
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Inhibitory Ig-like receptors for HLA Class I and
their ligands
Ligand
Receptor
group 2 HLA-Clys80
2DL1
group 1 HLA-Casn80
2DL2
group 1 HLA-Casn80
KIR
2DL3
Bw4 group HLA-B
3DL1
HLA-A3/A11
3DL2
Lectin-like receptor
CD94
HLA-E
NKG2A
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Missing expression in recipient of donors HLA
class I group lysis
Donor Recipient HLA
NK cell Target
HLA-C group 2 Cw2/Cw4
HLA-C group 2 Cw2
KIR2DL1
missing HLA-C group 1
HLA-C group 1 Cw1
KIR2DL 2/3
HLA-Bw4 B27
KIR3DL1
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HLA-C group mismatches HLA typing predicts donor
NK alloreactivity, donor KIR genotyping for 100
accuracy
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Donors Recipients
HLA NK repertoire HLA Target
HLA-Bw4 group mismatch After HLA and donor KIR
gene typing Assessment of donor NK repertoire
necessary
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Donor NK Cell Recognition of Missing Self
No missing self (no KIR ligand mismatch)
Missing C2
Missing C1
Missing Bw4
KIR ligand mismatches in GvH direction
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NK cloning protocol

• Blood mononuclear cells are depleted of T cells
  by negative anti-CD3 immuno-magnetic selection
  (Miltenyi)
• Cells are plated under limiting-dilution
  conditions in 96-well plates (5-10 NK cells per
  well).
• Day 0, plated cells are activated with clinical
  grade IL2 (Proleukine, Chiron) in the presence of
  irradiated feeder cells (buffy coats from the
  volunteer blood donors from the Blood Bank).
• Day 6, add fresh IL-2 medium feeder cells.
• Wells containing growing cultures are
  distinguished (from wells containing only feeder
  cells) by screening plates with an inverted
  microscope. Growing cultures are split as
  necessary and supplemented with IL-2 and feeder
  cells.
• NK clones are screened for alloreactivity by
  standard 51Cr release cytotoxicity against
  recipient PHA lymphoblasts at an effector/target
  ratio of 101. Clones exhibiting 50 lysis are
  scored as alloreactive.

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Allo NK and GvL
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In vitro leukaemia killing by human alloreactive
NK clones
Autologous
Allogeneic
Allogeneic leukaemia
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Clearance of human leukemia by human alloreactive
NK clones in SCID mice
CML blastic crisis in NOD-SCID mice
non-allo NK cells, 8x106 allo NK,
105 allo NK, 2x105
allo NK, 4x105

allo NK, 8x105
Human CD45
BCR/ABL
100
non-allo NK
allo NK, 105 allo NK,
2x105 allo NK, 4x105 control
50
Survival ()
0
30
0
120
Days
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Haploidentical transplant from NK alloreactive
donor
Mega-dose stem cells from allo NK donor No
post-transplant immune suppression
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Leukemia relapse AML106 transplants from 1993
through 2004
In Relapse at Transplant (n 49)
In any CR at Transplant (n 55)
1,0
1,0
Non-allo NK (n 27)
0,8
0,8
probability
0,6
0,6
Non-allo NK (n 28)
p 0.004
0,4
0,4
Allo NK (n 21)
0,2
0,2
n.s.
Allo NK (n 28)
0,0
0,0
0
2
4
6
8
10
0
2
4
6
8
10
years
years
Ruggeri et al., Science 2002, updated 2005
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Event-Free Survival AML106 transplants from
1993 through 2004
In relapse at transplant (n 49)
In remission at transplant (n 55)
1.0
.8
.6
p 0.05
Allo NK
.4
p 0.1
.2
Non-allo NK
0.0
10
8
6
4
2
10
8
6
4
2
0
years
years
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Full-Haplotype Mismatched HSC Transplant in
High-Risk Acute Leukemia On-going Study (January
1999 December 2004)
TRANSPLANT-RELATED MORTALITY
Incidence greatest first year post-transplant. I
nfections are the main cause Extensive T cell
depletion makes patients vulnerable to infections
0.480.06
HSCT Program University of Perugia 1/2004
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MUD in AML ALWP Study 1992-2002 TRM
Advanced (n400)
50 6
42 7
CR2 (n351)
CR1 (n301)
28 7
27

UCBT versus UBMT in adults with AL Transplant
related mortality
1.0
0.8
UCBT 498
0.6
UCBT 33
Cumulative Incidence
0.4
UBMT 39 8
0.2
UBMT 26
P (Fine and Gray)0.17
P (Fine and Gray)0.24
0.0
0.0
0.5
1.0
1.5
2.0
E. Gluckman EHA, Paris July 2003
Years
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Full-Haplotype Mismatched HSC Transplant in
High-Risk Acute Leukemia Present Study (January
1999 December 2003)
Non Leukemic Deaths 36/100
Rejection
GvHD
MOF
1
N2
N2
I.P.
N3
CNS toxicity
N3
Infection N25
HSCT Program University of Perugia 1/2004
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Strategy Large numbers of donor T cell clones
are raised against whole (inactivated)
Aspergillus fumigatus or CMV. IL-2 starvation
for the first 14 days to ensure immediate
specificity. Only IFN-?-producing CD4 clones
are obtained.
Clones are screened for cross-reactivity to
recipient allo-antigens by MLR.
Non-recipient-reactive clones, presumably
devoid of GvHD potential, are pooled.
don-vs-don don-vs-host anti-CD3
IFN-? (pg/ml)
don-vs-don don-vs-host anti-CD3
IL-10 (pg/ml)
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Transfer of donor immunity to pathogens
stability of repertoire
Limiting dilution analysis of pathogen-specific T
cells in patients
Non-infused
Infused
Asp.
Frequency per 106 cells
CMV
Weeks after transplant
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NK cells and transplant-related mortality
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A role for activating NK receptors?
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Group A haplotype genes in 28 of donors Group B
haplotype genes in 72 of donors
Do donor activating KIRs influence outcome?
An analysis of 74 AML haplo recipients
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Transplantation from haplo donors carrying
activating KIR genes reduces death in remission
Multivariate
- 74 AML -
P 0.04
A haplotype genes
Death in remission
B haplotype genes
months
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Haploidentical Transplantation for AML Donor
Selection
To optimize GvL (w/o GvHD) The
donor is KIR ligand-mismatched in the GvH
direction, possesses the relevant inhibitory KIR
genes, and has alloreactive NK cells for missing
self recognition on recipient targets To
improve immune competence The donor may
need to bear activating KIR genes (group B
haplotype)
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PRE-CLINICAL MOUSE MODELS FOR NK-BASED
CONDITIONING TO ALLOGENEIC TRANSPLANT
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Hybrid resistance
Cudkowicz G and Bennett M J. Exp. Med. 1971
Parent
b/b
H-2Kb receptors (Ly49C)
inhibition
b/d mouse NK repertoire
Donor BM cells
b/b
H-2Dd receptors (Ly49A/G2)
LYSIS
Mouse alloreactive NK cells reject BM but
tolerate skin and organ grafts
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A mouse model of missing self recognition (NK
alloreactivity) in haplo transplantation
F1
Parent
b/b
b/d
Donor Recipient

NK
Ly49A/G2 (H2Dd rec.) allo NK Ly49C/I (H2Db
rec.) non-allo NK
50
Ly49C/I (H-2Kbrec.)
lysis
25
Ly49A/G2 (H-2Ddrec.)
0
0
5
10
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Donor alloreactive NK cells ablate host T cells
and allow reduced-intensity haplo transplants
Donor
H-2d/b
Non-myeloablative regimens in use for human
matched transplants 1) Fludara 180mg/m2 Bu
8mg/kg 2) Fludara 120mg/m2 2 GyTBI 3) Fludara
120mg/m2 CTX 120 mg/kg 4) Fludara 120mg/m2
Melph 120 mg/m2
H-2b
Recipient
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Post-engraftment donor alloreactive NK cell
infusions convert mixed chimerism to full-donor
chimerism
Flu 120 mg/m2
b/d BM
Post-engraftment donor allo-NK cell infusion
b/b
allo NK
host
donor
Donor-reactive (V?11-V?5) host CD4 splenocytes
Before BMT
After BMT
100 donor
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Donor alloreactive NK cells might allow T
cell-replete mismatched BMT
Donor
H-2d
alloNK
Killing of host APC
alloNK20x106 allog. T cells
Gut DCs
H-2b
5x105 allog. T cells
Recipient
CTR TBI TBI non-alloNK
allo NK
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After NK conditioning and T cell replete BMT
rapidly reconstituting donor APCs present
pathogens to donor T cells
Donor H-2d DC Day 6100-fold
Donor H-2d T cells
Recipient
Response to pathogens e.g.Asp/Cand?
43
NK-based T-replete BMT resistance to early
infection and immunity to high-virulence challenge
T-depleted
NK-based T-replete
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Alloreactive NK cell conditioning and thymus
cellularity
d
d
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Conclusions
Pre-transplant infusion of alloreactive NK cells
allows a reduced-intensity (toxicity)
conditioning regimens. Post-transplant infusion
of alloreactive NK cells converts mixed to
full-donor chimerism.
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Pre-transplant infusion of alloreactive NK cells
improves immunity to infections

• 1) allows early transfer of functional
• immunity without GvHD (T replete
• BMT fast donor-type DC recovery)
• 2) protects from thymic GvHD
• 3) boosts donor-type thymocyte
• reconstitution

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Blood. 2005 Apr 15105(8)3051-7.   Successful
adoptive transfer and in vivo expansion of human
haploidentical NK cells in patients with
cancer. Miller JS, Soignier Y,
Panoskaltsis-Mortari A, McNearney SA, Yun GH,
Fautsch SK, McKenna D, Le C, Defor TE, Burns LJ,
Orchard PJ, Blazar BR, Wagner JE, Slungaard A,
Weisdorf DJ, Okazaki IJ, McGlave PB.
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The Weitzman Institute Yair Reisner Yale
University Warren Shlomchik University of
Minnesota Bruce Blazar Stella Davies University
of Basel Georg Holländer Stanford
University Karina McQeen Peter Parham University
of Genoa Lorenzo Moretta Pediatric Hematology,
Pavia Franco Locatelli Rita Maccario
Velardi lab Loredana Ruggeri Marusca
Capanni Antonella Mancusi Katia
Perruccio Emanuela Burchielli Antonella Tosti
Elena Urbani Fabiana Topini

Div. of Hematology and Clinical Immunology, Univ.
of Perugia
Direction of Transplant Program Massimo F.
Martelli
Graft processing Antonio Tabilio Franca
Falzetti Roberta Iacucci Tiziana Zei
Cytogenetics Cristina Mecucci Roberta Lastarza
BMT unit Franco Aversa Alessandra
Carotti Stelvio Ballanti Adelmo Terenzi Rita
Felicini
Mol. diagnostics Mauro Di Ianni