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Immunotherapy of Leukemic Stem Cells using
Natural Killer Cell lines
by
Brent A. Williams
A thesis submitted in conformity with the requirements
for the degree of Doctor of Medical Sciences
Institute of Medical Sciences
University of Toronto
© Copyright by Brent A. Williams 2015
ii
Immunotherapy of Leukemic Stem Cell using
Natural Killer Cell lines
Brent A. Williams
Doctor of Philosphy
Institute of Medical Sciences
University of Toronto
2015
Abstract
The field of cancer immunobiology has focused on the interaction of bulk tumour cells with the
immune system. The discovery of stem cells in a number of cancers including leukemia has
raised new questions about the how the immune system interacts with these cancer stem cells and
has implications for immune based therapies. Here the natural killer cell lines NK-92 and
KHYG-1 are tested against primary acute myeloid leukemia and cell lines utilizing techniques to
address the impact on bulk and leukemic stem cells. NK-92 and KHYG-1 are both shown to
preferentially target leukemic stem cells in primary AML and cell lines using a novel clonogenic
cytotoxicity assay. Further, both cell lines can prolong survival in AML xenograft models using
primary AML and cell lines. NK-92 gene modified to express CD16 can mediate ADCC against
leukemic stem cells in vitro and in vivo. KHYG-1 can have significant enhancement against
leukemia when pretreated with anti-NKp30 and NKp44 monoclonal antibodies via reverse
ADCC, overcoming resistance of leukemic cell lines and primary AML samples. These data
support the notion that cell therapy agents can target leukemic stem cells, be enhanced by
antibodies, and lead to enhanced survival, providing a rationale for clinical trials testing this
approach in AML patients with minimal residual disease lacking a suitable allogeneic transplant
donor.
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Dedication
In loving memory of my father, Bernard K. Williams, my first mentor. While not a scientist by
trade, he taught me the structure of the atom, the theory of evolution and the power of rationale
thought.
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Acknowledgments
I would like to acknowledge my supervisor Dr. Armand Keating for taking me into the lab group
to pursue the question of how the immune system recognizes cancer stem cells and guiding the
process of discovery into uncharted regions. Further, I appreciate his decades of translational
research experience which I have been able to tap in the formulation of laboratory experiments
and models with the primary objective of designing novel therapeutic strategies for the treatment
of leukemia. Finally, I wish to acknowledge his career mentorship in pursuing my goal of
becoming an independent clinician scientist.
I would like to thank my committee members Dr. Mark Minden and Dr. Pam Ohashi for their
scientific critique of experimental design and results. They have been patient with delays and
experimental dead-ends and provided insight in surmounting difficult scientific challenges.
Their expectation of scientific rigor has elevated the quality of my work and enhanced my
approach to experimental design and translational research. Dr. Minden provided critical input
in the selection of AML cell lines and primary AML samples and their use in developing
xenogeneic leukemia models.
I must give special thanks to Dr. Xing-Hua Wang our lab manager who is a pillar of the lab
group and has provided continual support of my work. I learned most of the lab techniques
utilized in the thesis from him. Further, he generously provided his expertise in animal models
and experimentation to make some of the key discoveries presented here.
Next I would like to acknowledge the members of the Keating lab, past and present who have
provided insights and colleagial support over my PhD studies. Thanks to Sonia Montanari,
Roula Antoon, Iran Rashedi for sharing the ‘pain’ of the PhD experience with me and providing
critical insights into my work during lab meetings and other casual conversations. I would like
to acknowledge the work of Dr. Yoko Kosaka, a former post-doc, who started the NSG animal
colony for our lab and started us using a cutting edge immunodeficient animal model to develop
xenogeneic cancer models.
I have also had pleasure of having several highly motivated summer students work with me who
assisted in conducting pilot experiments. Sonam Maghera assisted in clonogenic cytotoxicity
assays that yielded puzzling results at the time, but led to much of the work done in Chapter 4 on
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reverse ADCC. Richard Cheng also assisted in evolving the clonogenic assay to explore NK cell
cytotoxicity using antibodies.
I also must acknowledge a fruitful collaboration with Dr. Jeff Leyton formerly Dr. Raymond
Reilly’s postdoctoral fellow and currently Assistant Professor at Sherbrooke University. We
both had set out to develop a therapeutic strategy to target leukemic stem cells; he using
radioconjugated anti-CD123 antibodies and myself with ADCC capable NK cell lines and anti-
CD123 antibodies. The overlap in approaches was clear and we began to discuss the optimal in
vivo readouts for novel therapeutics targeting leukemic stem cells often debating the superiority
of primary and secondary engraftment versus survival outcomes. In the end we settled on using
all methods which is reflected in this thesis and in our collaborative work using radioconjugated
antibody therapy to target leukemic stem cells which has recently been published. I must thank
Jeff for criticially reviewing early versions of chapter 3 and paying attention to the small details.
While material from this collaborative work does not appear in the thesis it allowed for me to
engage in a productive collaboration while dealing with delays in key reagents.
I would like to thank Dr. Neal DenHollander, director of the UHN HLA bank, for stimulating
immunological discussions and facilitating the link of the Princess Margaret Leukemia Bank
with the HLA bank which allowed us to address key questions of NK cell line recognition. I
would like to acknowledge Dr. Hans Hitzler, my clinical mentor in pediatric
hematology/oncology for allowing me flexibility to attend leukemia clinics in the context of a
busy research program.
I would like to thank my former research supervisor and mentor, Dr. David Hoskin, who taught
me basic immunology as an undergraduate and medical student and instilled a rigorous approach
to the scientific method and experimental design. His continual support of all my recent research
applications is greatly appreciated.
I would like to acknowledge the financial support of the National Cancer Institute Terry Fox
Foundation and Canadian Institute of Health research which provided my primary salary support
over the PhD research period. Also, additional support came from the Cell Therapy Program at
Princess Margaret Hospital, the Hospital for Sick Children, University of Toronto, Ontario
Cancer Institute, Canadian Hematology Society who have provided financial support for both
salary and operating costs.
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I would like to thank my mother and father, Bonnie and Bernie, for their enduring support of me
and my career aspirations. I would like to acknowledge my children Hayden, Ethan and Kate
who keep me balanced and engaged in the important things in life. Special thanks to Hayden and
Ethan for building a ‘lab’ under the stairs in the basement as a backup place to do experimental
work if my scientific career doesn’t work out. Finally I would like to acknowledge the love and
support of my wife, Jeannine, to accept the long years of training I have chosen. It has been
challenging and without her support this work would not have been possible.
vii
Quotation
Dans les champs de l'observation, le hasard ne favorise que les esprits prepares.
In the fields of observation, chance favors only the prepared mind.
Louis Pasteur, 1822-1895
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Table of Contents
Dedication ..................................................................................................................................... iii
Acknowledgments ........................................................................................................................ iv
Quotation ..................................................................................................................................... vii
Table of Contents ....................................................................................................................... viii
List of Tables .............................................................................................................................. xiii
List of Figures ............................................................................................................................. xiv
1 Chapter 1: Literature review ..................................................................................................... 1
1.1 Overview ............................................................................................................................. 1
1.2 Acute myeloid leukemia (AML) ......................................................................................... 1
1.2.1 Epidemiology .......................................................................................................... 1
1.2.2 Pathology ................................................................................................................ 2
1.2.3 Immunophenotype of AML .................................................................................... 5
1.2.4 Minimal residual disease (MRD) ............................................................................ 6
1.2.5 Chemotherapy for AML ......................................................................................... 8
1.2.6 Leukemia cell lines ................................................................................................. 9
1.2.7 Animal models of leukemia .................................................................................. 10
1.2.8 Cancer stem cell hypothesis .................................................................................. 14
1.2.9 Clinical relevance of LSCs ................................................................................... 17
1.3 Cytotoxicity assays ........................................................................................................... 20
1.3.1 Bulk cytotoxicity assays ....................................................................................... 20
1.3.2 Flow cytometric cytotoxicity assays ..................................................................... 21
1.3.3 Clonogenic cytotoxicity assays ............................................................................. 21
1.4 Cancer immunobiology ..................................................................................................... 24
1.4.1 Lymphocytes ......................................................................................................... 24
ix
1.4.2 NK cells ................................................................................................................ 24
1.4.3 NK cell lines ......................................................................................................... 41
1.5 Antibody therapy for cancer ............................................................................................. 43
1.6 Cell therapy for cancer ...................................................................................................... 46
1.6.1 Allogeneic hematopoietic stem cell transplantation ............................................. 46
1.6.2 Adoptive immunotherapy ..................................................................................... 47
2 Chapter 2: Hypotheses and experimental approach ................................................................. 57
2.1 Thesis aims ........................................................................................................................ 57
2.2 Hypotheses ........................................................................................................................ 57
2.2.1 Leukemic stem cells are present in cell line KG1 and are sensitive to NK-92
mediated cytotoxicity ............................................................................................ 57
2.2.2 Primary AML leukemic stem cells have greater sensitivity to NK92 than bulk
leukemia and can be targeted by CD16+NK-92 and anti-CD123 mAb
mediated ADCC in vivo ........................................................................................ 58
2.2.3 KHYG-1 has less cytotoxicity than NK-92 against leukemic targets which can
be modulated by antibody pretreatment of targets and effectors .......................... 58
3 Chapter 3: Clonogenic assays measure leukemia stem cell killing not detectable by
chromium release and flow cytometric cytotoxicity assays ..................................................... 60
3.1 Abstract ............................................................................................................................. 61
3.2 Introduction ....................................................................................................................... 62
3.3 Materials and methods ...................................................................................................... 64
3.3.1 Cell lines ............................................................................................................... 64
3.3.2 Antibodies and reagents ........................................................................................ 64
3.3.3 Chromium release assay ....................................................................................... 64
3.3.4 Flow cytometry and cell sorting ........................................................................... 65
3.3.5 Flow cytometric cytotoxicity assay ...................................................................... 65
3.3.6 Methylcellulose and liquid reculturing cytotoxicity assays .................................. 66
3.3.7 Animals ................................................................................................................. 67
x
3.4 Results ............................................................................................................................... 68
3.4.1 Clonogenic capacity of KG1 in vitro and in vivo ................................................. 68
3.4.2 Immunophenotyping and fractionation studies of KG1 ........................................ 71
3.4.3 Chromium release assay (CRA) and flow cytometric cytotoxicity assay of
NK-92 and chemotherapy drugs versus KG1 ....................................................... 74
3.4.4 Clonogenic and proliferation assays of NK-92 and chemotherapy drugs versus
KG1 ....................................................................................................................... 77
3.5 Discussion ......................................................................................................................... 80
4 Chapter 4: Irradiated CD16+NK-92 prolongs survival in an AML xenograft model in
combination with anti-CD123 monoclonal antibody therapy by targeting leukemic stem
cells through antibody dependent cell mediated cytotoxicity (ADCC) ................................... 84
4.1 Abstract ............................................................................................................................. 85
4.2 Introduction ....................................................................................................................... 86
4.3 Methods ............................................................................................................................. 88
4.3.1 Cell lines and primary samples ............................................................................. 88
4.3.2 Chromium release assay ....................................................................................... 88
4.3.3 ADCC chromium release assay ............................................................................ 89
4.3.4 Flow cytometry ..................................................................................................... 89
4.3.5 Cell sorting ............................................................................................................ 89
4.3.6 Methylcellulose cytotoxicity assay ....................................................................... 90
4.3.7 Animals ................................................................................................................. 90
4.3.8 Statistics ................................................................................................................ 90
4.4 Results ............................................................................................................................... 91
4.4.1 NK-92 is cytotoxic against primary AML by granule exocytosis ........................ 91
4.4.2 Chromium release assay versus methylcellulose cytotoxicity assay .................... 93
4.4.3 NK-92 preferentially kills leukemic stem cells relative to bulk leukemia ............ 94
4.4.4 Primary AML xenograft model ............................................................................ 97
xi
4.4.5 In vitro treatment of primary AML cells by irradiated NK-92 reduce
engraftment potential ............................................................................................ 99
4.4.6 Irradiated NK-92 reduce leukemic stem cell fraction in secondary
transplantation assay ........................................................................................... 101
4.4.7 NK-92 prolongs survival in a primary AML xenograft model ........................... 103
4.4.8 iCD16+NK-92 can mediate ADCC against bulk and stem cell antigens in vitro 106
4.4.9 CD16+NK-92 improves survival in an AML xenograft model with
enhancement by anti-CD123 mAb therapy ......................................................... 109
4.5 Discussion ....................................................................................................................... 113
5 Chapter 5: NK cell line killing of leukemia cells is enhanced by reverse antibody
dependent cell mediated cytotoxicity (R-ADCC) via NKp30 and NKp44 and target cell
Fcγ receptor II (CD32) ........................................................................................................... 118
5.1 Abstract ........................................................................................................................... 119
5.2 Introduction ..................................................................................................................... 120
5.3 Methods ........................................................................................................................... 122
5.3.1 Cell lines and primary samples ........................................................................... 122
5.3.2 Chromium release assay ..................................................................................... 122
5.3.3 Antibody pretreatment of NK cell effectors ....................................................... 122
5.3.4 Flow cytometry ................................................................................................... 123
5.3.5 High throughput sampling flow cytometry ......................................................... 123
5.3.6 Animals ............................................................................................................... 124
5.3.7 Statistics .............................................................................................................. 124
5.4 Results ............................................................................................................................. 125
5.4.1 NK-92 and KHYG-1 cytotoxicity against leukemia cell lines ........................... 125
5.4.2 High throughput screening flow cytometry of NK-92 and KHYG-1 surface
receptors .............................................................................................................. 127
5.4.3 Anti-Class I HLA blockade of AML targets ....................................................... 129
5.4.4 Effect of pretreating NK-92 and KHYG-1 with activating receptor specific
antibodies ............................................................................................................ 131
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5.4.5 Relationship of Fcγ receptor expression and enhancement of cytotoxicity ........ 142
5.4.6 Effect of anti-NKp30 pretreatment on NK cell line cytotoxicity against
clonogenic OCI/AML5 ....................................................................................... 147
5.4.7 In vitro effect of anti-NKp30 pretreated iKHYG-1 against OCI/AML5
capacity for leukemic progression in an NSG xenograft model ......................... 149
5.4.8 Effect of anti-NKp30 pretreatment of iKHYG-1 on therapeutic efficacy for
OCI/AML5 or primary AML xenografted mice ................................................. 152
5.5 Discussion ....................................................................................................................... 155
6 Chapter 6: General Discussion ............................................................................................... 163
6.1 Overview ......................................................................................................................... 163
6.2 Methodologic approaches to measuring the impact of immune effectors on leukemic
stem cell and bulk leukemia ............................................................................................ 164
6.3 Natural cytotoxicity of NK cell lines with and without ADCC enhancement against
leukemic stem cells ......................................................................................................... 166
6.4 Natural cytotoxicity of NK cell lines with and without reverse-ADCC enhancement
against leukemic stem cells ............................................................................................. 169
6.5 Translational relevance ................................................................................................... 172
7 Chapter 7: Conclusions and future directions ........................................................................ 174
8 Chapter 8: References ............................................................................................................ 176
Copyright Acknowledgements (pending) ................................................................................... 249
xiii
List of Tables
Table 1.1: WHO histopathological classification of AML ............................................................. 4
Table 1.2: NK cell inhibiting receptors ......................................................................................... 32
Table 1.3: NK cell activating receptors ........................................................................................ 35
Table 1.4: FDA approved therapeutic monoclonal antibodies for cancer* .................................. 45
Table 1.5: Comparison of autologous cell therapy and cell line therapy ...................................... 55
Table 3.1: Frequency of KG1 stem cell frequency using liquid culture repopulation .................. 68
Table 3.2: Frequency of KG1 stem cell frequency using two fold serial dilutions in 96 well
confluence assay (5000 to 0.3 per well) ........................................................................................ 68
Table 3.3: Frequency of KG1 stem cell frequency using cell sorting and 96 well confluence assay
(1000 to 1 per well) ....................................................................................................................... 69
Table 5.1: Differential expression of cell surface activating, inhibiting and apoptosis inducing
molecules on NK-92 and KHYG-1 ............................................................................................. 128
Table 5.2: HLA type of primary AML panel and sensitivity to NK-92 and KHYG-1 +/- HLA
blockade ...................................................................................................................................... 130
Table 5.3: Comparison of antibody pretreatment effects on KHYG-1 cytotoxicity and synergy
assessment at 0.1 µg/ml .............................................................................................................. 137
Table 5.4: Comparison of antibody pretreatment effects on KHYG-1 cytotoxicity and synergy
assessment at 0.01 µg/ml ............................................................................................................ 138
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List of Figures
Figure 1.1: NK cell degranulation by stimulation with activating ligands only ........................... 26
Figure 1.2: NK cell inhibition of degranulation by stimulation with activating and inhibitory
ligands ........................................................................................................................................... 26
Figure 1.3: NK cell degranulation by stimulation with activating and inhibitory ligands ........... 26
Figure 1.4: NK cell antibody dependent cell mediated cytotoxicity ............................................ 39
Figure 1.5: Schematic of adoptive immunotherapy using LAK cells ........................................... 48
Figure 1.6: Schematic of adoptive immunotherapy using TILs ................................................... 49
Figure 1.7: Design of CD19 CAR utilized in a clinical trial for CLL .......................................... 52
Figure 1.8: Schematic of CD19+ CAR T-cell recognition of CD19+ CLL cells ......................... 53
Figure 1.9: Schematic of adoptive immunotherapy using cell lines ............................................. 54
Figure 3.1: NOD/SCID leukemia initiating frequency of KG1 .................................................... 70
Figure 3.2: Immunophenotype of KG1 ......................................................................................... 71
Figure 3.3: Reconstitution of CD38 distribution following cell sorting KG1 .............................. 73
Figure 3.4: Chromium release assay of NK-92 against KG1 and K562 ....................................... 74
Figure 3.5: Chromium release assay versus flow cytometric cytotoxicity assay ......................... 75
Figure 3.6: Chromium release assay versus flow cytometric drug assay ..................................... 76
Figure 3.7: Liquid reculturing cytotoxicity assay ......................................................................... 77
Figure 3.8: Methylcellulose cytotoxicity assay ............................................................................ 79
Figure 4.1: Chromium release assay of NK-92 a against a primary AML sample at a range of
Effector:Target ratios with and without calcium chelation ........................................................... 91
xv
Figure 4.2: Chromium release assay of NK-92 against a panel of primary AML patient samples
at a range of Effector:Target ratios ............................................................................................... 92
Figure 4.3: Clonogenic cytotoxicity assay of NK-92 against OCI/AML2 and OCI/AML3 ........ 93
Figure 4.4: NK-92 cytotoxicity against sorted leukemic stem cells (CD34+CD38-) and bulk
leukemia (CD34+CD38+) ............................................................................................................. 94
Figure 4.5: Schematic of methylcellulose cytotoxicity assay ....................................................... 95
Figure 4.6: NK-92 against primary AML blasts using the methylcellulose cytotoxicity assay
compared to the chromium release assay ...................................................................................... 96
Figure 4.7: Primary AML immunophenotype .............................................................................. 97
Figure 4.8: Survival of NSG mice with primary AML versus 1st passage AML derived from BM
....................................................................................................................................................... 98
Figure 4.9: Schematic of in vitro cytotoxicity assay with in vivo engraftment readout ............... 99
Figure 4.10: In vitro cytotoxicity assay with in vivo engraftment readout ................................. 100
Figure 4.11: In vivo cytotoxic impact of iNK-92 on secondary BM engraftment of AML cells and
LSCs ............................................................................................................................................ 102
Figure 4.12: Schematic of NK-92 therapy for primary AML xenografted NSG mice ............... 103
Figure 4.13: NK-92 therapy of primary AML xenografted NSG mice ...................................... 104
Figure 4.14: iNK-92 therapy of primary AML xenografted NSG mice ..................................... 105
Figure 4.15: Immunophenotyping of CD16+NK-92, NK-92 and OCI/AML 2, 3 and 5 ............ 106
Figure 4.16: CD16+NK-92 in vitro ADCC assay against primary AML ................................... 107
Figure 4.17: CD16+NK-92 in vitro ADCC assay against OCI/AML5 ...................................... 108
Figure 4.18: Schematic of iCD16+NK-92 +/- a single anti-CD123 mAb dose for primary AML
xenografted NSG mice ................................................................................................................ 109
xvi
Figure 4.19: iCD16+NK-92 +/- a single anti-CD123 mAb dose for primary AML xenografted
NSG mice .................................................................................................................................... 110
Figure 4.20: Schematic of CD16+NK-92 +/- five doses of anti-CD123 mAb therapy for primary
AML xenografted NSG mice ...................................................................................................... 111
Figure 4.21: iCD16+NK-92 with and without 7G3 or isotype control treatment for primary AML
xenograft model .......................................................................................................................... 112
Figure 5.1: NK-92 and KHYG-1 cytotoxicity against a panel of leukemia cell lines ................ 125
Figure 5.2: NK-92 and KHYG-1 cytotoxicity against K562 with and without calcium chelation
..................................................................................................................................................... 126
Figure 5.3: KHYG-1 cytotoxicity against 4 primary AML samples +/- class I HLA blockade . 130
Figure 5.4: Effect of antibody pre-treatment of activating receptors on NK-92 and KHYG-1
cytotoxicity against leukemia cell lines ...................................................................................... 132
Figure 5.5: Effect of antibody pre-treatment of activating receptors on NK-92 and KHYG-1
cytotoxicity against primary AML samples ................................................................................ 133
Figure 5.6: Effect of antibody pre-treatment with isotype control on NK-92 and KHYG-1
cytotoxicity against leukemia cell lines ...................................................................................... 135
Figure 5.7: Effect of antibody pre-treatment with anti-NKp30 and anti-NKp44 on NK-92 and
KHYG-1 cytotoxicity against leukemia cell lines ...................................................................... 136
Figure 5.8: Effect of antibody pre-treatment with anti-NKp30 and anti-NKp44 on NK-92 and
KHYG-1 cytotoxicity against leukemia cell lines and primary AML ........................................ 139
Figure 5.9: Impact of antibody pre-treatment with anti-NKp30 and anti-NKp44 on NK-92 and
KHYG-1 cytotoxicity against esophageal cancer cell lines ........................................................ 141
Figure 5.10: Fc gamma receptor expression on leukemia cell lines (K562, KG1, KG1a,
OCI/AML3, OCI/AML5) ........................................................................................................... 143
xvii
Figure 5.11: Fc gamma receptor expression on esophageal cancer cell lines (OE-33, FLO-1,
KYAE-1, SKGT-4) ..................................................................................................................... 144
Figure 5.12: Regression analysis of CD32 expression and delta cytotoxicity of NKp30 and
NKp44 pretreated NK-92 and KHYG-1 ..................................................................................... 146
Figure 5.13: Methylcellulose cytotoxicity assay of NK-92 and KHYG-1 +/- pretreatment with
antibodies against OCI/AML5 .................................................................................................... 148
Figure 5.14: OCI/AML5 induced malignant ascites .................................................................. 150
Figure 5.15: In vitro incubation of OCI/AML5 with iKHYG-1 +/- anti-NKp30 and in vivo
proliferation in NSG mice ........................................................................................................... 151
Figure 5.16: Bone marrow engraftment of OCI/AML5 injected iv into NSG mice ................... 152
Figure 5.17: Treatment of OCI/AML5 leukemia in NSG mice with iKHYG-1 +/- NKp30
pretreatment ................................................................................................................................ 153
Figure 5.18: Treatment of primary AML xenografted NSG mice with iKHYG-1 +/- NKp30
pretreatment ................................................................................................................................ 154
Figure 6.1: Antibody-dependent cell-mediated cytotoxicity (ADCC) ....................................... 168
Figure 6.2: Reverse antibody-dependent cell-mediated cytotoxicity (R-ADCC) ....................... 170
xviii
List of Appendices
Appendix I: NK-92 HTS flow cytometry percent positivity and MFI of antigens ..................... 212
Appendix II: KHYG-1 HTS flow cytometry percent positivity and MFI of antigens ................ 221
Appendix III: OCI/AML3 HTS flow cytometry percent positivity and MFI of antigens .......... 231
Appendix IV: OCI/AML5 HTS flow cytometry percent positivity and MFI of antigens .......... 240
xix
List of Abbreviations
ADCC Antibody dependent cell mediated cytotoxicity
ALDH Aldehyde dehydrogenase
ALL Acute lymphoblastic leukemia
AML Acute myeloid leukemia
APC Allophycocyanin
ATTC American Type Culture Collection
BM Bone marrow
CAR Chimeric antigen receptor
CD Cluster of differentiation
CML Chronic myeloid leukemia
CRACC CD2-like leceptor activating cytotoxic cells
CSC Cancer stem cell
CTL Cytotoxic T lymphocyte
DAP12 DNAX activation protein of 12 kDa
DNAM-1 DNAX accessory molecule 1
EC50% Effective concentration to achieve 50% of maximal effect
EGIL European group for the immunological classification of leukaemias
EFS Event free survival
ERK2 Extracellular regulated kinase 2
FADD Fas associated death domain
FITC Fluorescein isothyocyanate
FLT3 fms-related tyrosine kinase 3
GFP Green fluorescent protein
GMCSF Granulocyte monocyte colony stimulating factor
GVL Graft versus leukemia effect
HSCT Hematopoietic stem cell transplantation
HLA Human leukocyte antigen
HSC Hematopoietic stem cell
HSCT Hematopoeitic stem cell transplant
HSP Heat shock protein
ICAM Intercellular adhesion molecule
IFN-γ Interferon gamma
Ig Immunoglobulin
IS Immunologic synapse
KIR Killer immunoglobulin like receptor
ITAM Immunoreceptor tyrosine-based activating motifs
ITD Internal tandem repeats
ITIM Immunoreceptor tyrosine-based inhibitory motifs
ITSM Immunoreceptor tyrosine-based switch motifs
LAK Lymphokine activated killer
LFA Lymphocyte function antigen
LIR Leukocyte immunoglobulin-like inhibitory receptors
LSC Leukemic stem cell
MIL Marrow infiltrating lymphocyte
mAb Monoclonal antibody
mRNA Messenger RNA
xx
MCA Methylcellulose cytotoxicity assay
MDS Myelodysplastic syndrome
MPS Myeloproliferative syndrome
MRD Minimal residual disease
MTT 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide
NCR Natural cytotoxicity receptor
NK Natural killer
NMP1 Nuclear Matrix Protein 1
DNMT3A DNA (cytosine-5)-methyltransferase 3A
NKG2D Natural killer group 2 D
NRG NOD/RAG gamma null
NOD Non-obese diabetic
NSB NOD/SCID/β2microglobulin null
NSG NOD-SCID gamma null
OCI Ontario cancer institute
OS Overall survival
PBL Peripheral blood lymphocyte
PE Phycoerythrin
PCR Polymerase chain reaction
Prkdc Protein kinase, DNA-activated, catalytic polypeptide
PML-RARα Promyelocytic myeloid leukemia-retinoic acid α receptor
PNET Primitive neuroectodermal tumour
RANK1 Receptor Activator of Nuclear Factor κ B
SCID Severe combined immunodeficiency
SP Side population
ScFv Single-chain variable fragment
Th1 T helper 1
TIL Tumour infiltrating lymphocyte
TNF Tumour necrosis factor
Treg T regulatory cell
TRAIL TNF-related apoptosis-inducing ligand
TWEAK Tumor necrosis factor receptor superfamily member 12A
WHO World Health Organization
1
1 Chapter 1: Literature review
1.1 Overview
The field of cancer immunology has focused primarily on the interaction of cells of the immune
system with bulk tumour cells. The discovery of cancer stem cells has brought into question
whether prior studies of immune effector cell interaction with cancer cells can be extrapolated to
these rare subset of cells. This poses many new biological questions regarding how the immune
system interacts, recognizes and responds to cancer stem cells. Further, there are significant
therapeutic implications to the cancer stem cell hypothesis if only a rare subpopulation of cells
has the capability to initiate, progress and recapitulate cancer after therapy. In the case of acute
myeloid leukemia (AML), the stem cell compartment comprises a rare subpopulation initially
identified as CD34+CD38- AML cells. (Lapidot, Sirard et al. 1994; Bonnet and Dick 1997)
While chemotherapy can cure a minority of AML patients, many relapse due to the presence of
minimal residual disease. Hematopoietic stem cell transplantation (HSCT) transplantation is one
curative approach for high risk or relapse AML, demonstrating the potential for cellular based
therapies to treat AML. There is a paucity of studies focused on the interaction of the cellular
immune system with cancer stem cells, leaving many avenues for novel investigation and the
potential to the develop future therapies designed to treat leukemic stem cells.
1.2 Acute myeloid leukemia (AML)
1.2.1 Epidemiology
The age-adjusted incidence rate of AML in the U.S. (1975–2011) was 3.51/100,000/year with
males having slightly higher incidence (4.36) than females (2.92).(Howlader N) These rates are
comparable with other North American and western European countries.(Jemal, Thomas et al.
2002; Deschler and Lubbert 2006) This leads to approximately 13,000 new cases of AML
annually in the United States(Ries LAG 2008) and 1,300 in Canada. The first peak in incidence
occurs in the first year of life and decreases up to age 4, and remains at this level through
childhood and early adulthood,(Gurney, Davis et al. 1996) increasing again progressively in late
adulthood(Howlader N). There is a ten-fold increase in the age-specific incidence of AML in the
population older than 65, leading to a high prevalence of cases (~40%) in this age group, with
the incidence increasing to 24/100,000/year in the over 80 age group.(Deschler and Lubbert
2
2006) AML accounts for approximately 90% of all acute leukemias in adults and 15% of cases in
children.(Lowenberg, Downing et al. 1999) The incidence of leukemia is highest in the United
States, Australia and Western Europe.(Jemal, Thomas et al. 2002) Projections of an aging world
population mapped onto the age-specific incidence rates for AML lead to the conclusion of a
increase in incidence of AML that will strain current resources available in most countries. Short
term, these demographic shifts in Western countries have led to predictions for a need to increase
the number of practicing oncologists treating all cancers by 40% from 2014 to 2020.(Yang,
Williams et al. 2014) This will be a greater issue for AML because of the sharp increase in
incidence above 65 of both de novo AML cases, as well as myelodysplastic syndrome, which
predisposes to AML. This highlights the need for targeted, minimally toxic and affordable
therapeutic options for AML patients.
1.2.2 Pathology
AML can be categorized by the both French American British (FAB) system(Bennett, Catovsky
et al. 1976) and the World Health Organiziation (WHO) systems (Vardiman, Thiele et al. 2009).
FAB categories include M0 (minimially differentiated), M1 (without maturation), M2
(granulocytic), M3 (acute promyelocytic), M4 (myelomonocytic), M4Eo (myelomonocytic with
eosinophilic differentiation) M5 (monocytic), M6 (erthyroleukemia), M7 (megakaryoblastic).
Acute promyelocytic leukemia is defined by a characteristic translocation (PML-RARα) and is
treated with a unique treatment regimen distinguishing it from other subclass of AML.
World Health Organization (WHO) classification has expanded to identify numerous subtypes of
AML (Table 1), mostly falling into the following categories: a) AML with recurrent genetic
abnormalities, b) AML with myelodysplasia-related changes, c) therapy-related myeloid
neoplasms and d) AML not otherwise categorized. AML with recurrent genetic abnormalities
may lead to better [e.g. t(8:21), inv16, t(15;17)] or worse survival outcomes (e.g. MLL
rearrangement, complex cytogenetics). Topoisomeriase II therapy (e.g. etoposide) can lead to
second malignancies that are particularly resistant to therapy. Patients with therapy related
AML or progressed from myelodysplatic syndrome (MDS) or myeloproliferative syndrome
(MPS), fare poorly. M3, or acute promyelocytic and M6, erythroleukemia and M7,
megakaryoblastic leukemia are considered distinct disease entities each requiring specific
3
therapy. The remaining categories are treated using similar protocols consisting of a backbone
of daunorubicin and cytarabine.
Additional molecular abnormalities can contribute to poor prognosis. A study of 854 patients in
United Kingdom Medical Research Council (MRC) AML trials demonstrated the presence of
FLT3 internal tandem duplicates (ITD) correlated with lower complete remission rate, higher
induction death rate, increased relapse risk, worse event-free survival (EFS) and overall survival
(OS) (p <0.001 for all).(Kottaridis, Gale et al. 2001) In another study, presence of FLT3 ITD
was the most predictive negative prognostic factor. (Meshinchi, Woods et al. 2001) FLT3 ITD
was evaluated in 91 pediatric patients with AML from CCG 2891 and those with and without
this abnormality had an eight year EFS of 7% and 44%.(Meshinchi, Woods et al. 2001). Another
important genetic abnormality with prognostic significance is mutation of neucleophosmin
(NPM1), which correlates with a chemosensitive disease and has a favourable prognosis in the
absence of other negative prognostic factors as reviewed by Falini et al.(Falini, Martelli et al.
2011). A meta-analysis conducted on ten studies with a total of 6,219 patients revealed that
CEBPA mutation conferred a favourable prognosis.(Li, Deng et al. 2014) Thus, the revised
WHO criterion has included both mutations of NPM1 and CEPBA as provisional diagnostic
categories for AML.
4
Table 1.1: WHO histopathological classification of AML
General diagnostic category Examples
Acute myeloid leukemia
with recurrent genetic
abnormalities
AML with t(8;21)(q22;q22); RUNX1-RUNX1T1
AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22); CBFB-
MYH11
APL with t(15;17)(q22;q12); PML-RARA
AML with t(9;11)(p22;q23); MLLT3-MLL
AML with t(6;9)(p23;q34); DEK-NUP214
AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2); RPN1-EVI1
AML (megakaryoblastic) with t(1;22)(p13;q13); RBM15-MKL1
Provisional entity: AML with mutated NPM1
Provisional entity: AML with mutated CEBPA
Acute myeloid leukemia
with myelodysplasia-
related changes
AML with a prior history of MDS or have specific MDS related
cytogenetic abnormalities (e.g. -5 or -7) or exhibit dysplasia in
50% or more of the cells in 2 or more myeloid lineages
Therapy-related myeloid
neoplasms
AML secondary to etoposide therapy
Acute myeloid leukemia,
not otherwise specified
AML with minimal differentiation
AML without maturation
AML with maturation
Acute myelomonocytic leukemia
Acute monoblastic/monocytic leukemia
Acute erythroid leukemia
Acute megakaryoblastic leukemia
Acute basophilic leukemia
Acute panmyelosis with myelofibrosis
*Adapted from Vardiman 2009 et al. pg 939, table 2.(Vardiman, Thiele et al. 2009)
5
1.2.3 Immunophenotype of AML
AML cells typically express antigens found on normal myeloid progenitor and differentiated
cells, such as macrophages and monocytes, with aberrant expression of other lineage markers.
AML expresses the pan-leukocyte marker CD45 and other myeloid markers such as CD11b,
CD13 and CD33. A review of 106 adult AML cases was conducted to assess immunophenotypic
variation based on FAB classification using a 22 antibody panel.(Khalidi, Medeiros et al. 1998)
The most commonly expressed antigens were CD45 (97.2%), CD33 (95.3%), and CD13
(94.3%). Lymphoid-associated antigens were expressed in approximately half of cases with the
frequency in descending order being: CD20 (17%), CD7 (16%), CD19 (9.8%), CD2 (7.5%),
CD3 (6.7%), CD5 (4.8%), and CD10 (2.9%). CD56, typically found on NK cells can also be
found on AML cells, but not on normal myeloid cells. CD56 expression in (t8;21) AML was
associated with a higher rate of relapse.(Iriyama, Hatta et al. 2013)
The European Group for the Immunological Classification of Leukaemias (EGIL) had proposed
in the past that AML be defined immunologically by the expression of 2 or more of the
following myeloid markers: myeloperoxidase, CD13, CD33, CDw65, and CD117, but only three
of FAB subtypes could be easily defined by immunological markers alone: M0, M6, M7.(Bene,
Castoldi et al. 1995) The prognostic significance of 21 antigens was evaluated in 177 adult AML
patients and no single antigen expressed on blasts predicted patient survival. However, patients
with blasts expressing all 5 myeloid markers (myeloperoxidase, CD13, CD33, CDw65, CD117)
had a higher complete remission rate (p< 0.0001), improved disease-free survival (p =0.02) and
overall survival (p =0.008), than patients whose cells expressed fewer than 5 of these markers.
(Legrand, Perrot et al. 2000) This finding suggests that more differentiated leukemia responds
better to the therapy than an undifferentiated leukemia.
6
1.2.4 Minimal residual disease (MRD)
Morphological assessment of bone marrow samples using light microscopy can only determine
when leukemic blasts are abundant and greater than the normal frequency of 5% of cells in the
bone marrow. AML patients are routinely assessed with bone marrow aspiration and molecular
testing, which may include PCR for common translocations and fusion transcripts or flow
cytometry to detect cells with leukemia associated phenotypes.(Baer 2002) Most patients can be
put into a complete morphological remission with current protocols, but many relapse within two
years due to minimal residual disease that has not been irradicated.(Burnett, Wetzler et al. 2011)
Minimal residual disease detection for AML is not part of standard of care at most institutions,
but there is considerable evidence to support measuring this parameter for prognostication and
risk stratification for dose intensification.
1.2.4.1 Polymerase chain reaction based MRD
Polymerase chain reaction (PCR) involves the amplification of a sequence of DNA using DNA
polymerase in combination with sequence specific primers and in a controlled thermocycling
program. Using reverse transcriptase a PCR reaction can also copy mRNA sequences into DNA
and subsequently amplify abnormal mRNA sequences. Both techniques can be used for initial
diagnostic purposes and MRD detection. Variations on methodology include semi-quantitative
and real-time quantitative PCR approaches. A limiting factor with this approach is that many
patients do not have a gene translocation that facilitates amplification.
T(8;21), inv(16), FLT3 internal tandem duplication (ITD) (20-30%) represent the most common
molecular abnormalities in AML amenable to detection by PCR based MRD methods, with
significant implications for relapse and overall survival.(Kottaridis, Gale et al. 2001; Meshinchi,
Woods et al. 2001; Stirewalt, Willman et al. 2001) MRD for FLT3 using PCR had a high degree
of sensitivity (0.01-0.001%).(Stirewalt, Willman et al. 2001).
AML patients with recurring reciprocal translocations can have MRD detected using reverse
transcriptase PCR (RT-PCR) to detect aberrant mRNA transcripts.(Strehl, Konig et al. 2001;
Olesen, Clausen et al. 2004) RT-PCR based MRD for acute promyelocytic leukemia (APL) with
t15;17 (PML-RARα fusion transcript) has been essential to the assessment of therapeutic
response, and sensitive approaches using ‘nested’ or ‘semi-nested’ RT-PCR have been
7
utilized.(Miller, Levine et al. 1993; Gau, Young et al. 2000; Chendamarai, Balasubramanian et
al. 2012) Sensitivity for this approach ranges from 1 in 10,000 in some instances to 1 in
1,000,000 in others.
1.2.4.2 Flow cytometry based MRD
Flow cytometry approaches to MRD detection have the advantage of being applied to a larger
proportion of AML patients using leukemia-associated phenotypes to track reduction of disease
at end of induction or consolidation. Flow cytometry techniques allow for a sensitivity in
detecting leukemic cells of approximately 1 in 10,000 (0.01%). Presence of MRD post therapy
has been shown to be predictive of survival in ALL(Dworzak 2002; Borowitz, Devidas et al.
2008; Bassan, Spinelli et al. 2009) and AML(San Miguel, Vidriales et al. 2001; Buccisano,
Maurillo et al. 2010), but MRD detection is more routinely used in risk stratification for ALL
patients in the clinical trial setting or in standard of care management. San Miguel et al. assessed
BM samples of AML patients in first morphologic remission in 126 patients with aberrant
phenotypes at diagnosis, which demonstrated low risk of relapse with MRD <1 in 10,000 bone
marrow cells with progressively higher relapse rates with each log increase in MRD.(San
Miguel, Vidriales et al. 2001) In a large multicenter study of pediatric AML, an MRD marker
was identified in 90% of 232 AML patients and those with MRD higher than 0.1% at the end of
a second induction cycle predicted relapse.(Rubnitz, Inaba et al. 2010) MRD positivity prior to
allogeneic stem cell transplantation is also highly predictive of relapse in both the myeloablative
and non-myeloablative settings.(Walter, Gyurkocza et al. 2014) Application of MRD techniques
to the detection of leukemic stem cells is a potential extension of this approach currently under
investigation.
1.2.4.3 Leukemic stem cell MRD
While MRD is clinically predictive of relapse free and overall survival, most studies do not
attempt to detect residual leukemic stem cells. However, MRD positive patients do not always
relapse, suggesting that in some cases the assay is not detecting leukemic stem cells, but rather
more differentiated progenitors or perhaps pre-leukemic stem cells(Shlush, Zandi et al. 2014). It
stands to reason that in MRD positive patients who relapse, there remains a proportion of these
detected cells which are bona fida leukemic stem cells. Several attempts have been made to
8
measure leukemic stem cell burden pre and post therapy which is discussed further in the section
on leukemic stem cells (Clinical relevance of LSCs).
1.2.5 Chemotherapy for AML
Overall survival outcomes for patients with acute myeloid leukemia (AML) have been slower to
improve over the last 20 years relative to acute lymphoblastic leukemia (ALL)(Lowenberg,
Downing et al. 1999; Burnett, Wetzler et al. 2011) indicating a need for novel therapies for this
disease. The mainstay of therapy for AML is based on a core regimen of daunorubicin and
cytarabine, and 70-85% of AML patients treated with current chemotherapy protocols are able to
achieve a morphologic remission (Hurwitz, Mounce et al. 1995) (Ribeiro, Razzouk et al. 2005),
defined as less than 5% myeloblasts in a cellular marrow with trilineage hematopoiesis.
However, despite meeting these criteria many patients relapse because of recurrence from MRD
leading to a five year survival of approximately 40% in adults(Lowenberg, Downing et al. 1999)
and 60% in children(Rubnitz 2012) with high risk groups faring much worse (<10%)
(Grimwade, Hills et al. 2010). Survival outcomes remain particularly poor for patients over the
age of 60 (Laubach and Rao 2008). In these relapsing patients, eradication of leukemic stem
cells has not occurred.
Dose intensification of induction with standard-dose cytarabine and daunorubicin at 45 or 90
mg/m2 for 3 days was assessed in a large prospective trial showing that adverse events were
similar in both arms and a significantly higher complete remission rate was achieved with higher
dosing of daunorubicin (67.6 vs 57.2%), however, OS was only significantly improved in
patients with favourable or intermediate risk cytogenetics.(Fernandez, Sun et al. 2009) The
number of cycles of consolidation post remission varies between institutions with no clear
evidence to determine the optimal number.(Rowe and Tallman 2010) One other important drug
in AML therapy is the topoisomerase II inhibitor, etoposide, which has single agent activity
against AML and can be incorporated into induction or consolidation protocols depending on the
risk category, age and cardiac status of the patient.(Ho, Brado et al. 1991)
9
1.2.6 Leukemia cell lines
Leukemia cell lines have been derived from primary human AML and stored in cell banks such
as the American Type Culture Collection (ATTC). The development of cancer cell lines has
accelerated basic and translational discoveries in oncology by providing a consistent and readily
available source of tumour cells to assay. There are numerous leukemia derived cell lines well
characterized in the literature.(Drexler 2010) Cell lines are required to have been in continuous
culture from the primary tissue beyond a year and demonstrate a degree of homogeneity. The
list below includes cell lines used in this work.
1.2.6.1 K562
K562 is a cell line derived from the pleural fluid of a CML patient in blast crisis.(Lozzio and
Lozzio 1975) It was subsequently characterized as having a near triploid karyotype with a
Philadelphia chromosome, Fc gamma receptor positivity, with a tentative assignment as
granulocytic lineage (Klein, Ben-Bassat et al. 1976) Further studies of this cell line
demonstrated expression of glycophorin, a red cell sailoglycoprotein, suggesting erythroid
lineage.(Andersson, Nilsson et al. 1979) More recent immunophenotyping of K562
demonstrated the presence of myelomonocytic (CD13, CD15, CD33, CD65) and
erythrocytic/megakaryocytic (CD9, CD41, CD61 and CD235a) lineage markers(Toba, Kishi et
al. 1996) prompting its classification as an erythrocytic/megakaryocytic cell line(Drexler 2010).
It has downregulated class I HLA expression, which makes it susceptible to NK cell-mediated
lysis, in turn making it a standard target in the assessment of NK cell cytolytic function.
However, it is not a true AML cell line, because it is derived from a CML patient in blast crisis
with mixed lineage markers.
1.2.6.2 KG1 and KG1a
KG1 is a commonly used leukemia cell line derived from a patient with erythroleukemia (M7) in
myeloblastic relapse (Koeffler and Golde 1978) reported as having an immunophenotype of
CD34+CD38+. It has demonstrated the capacity to cause leukemia in SCID mice with injection
of 107 cells, leading to bone marrow infiltration, peripheral blood leukemia and end stage disease
between 6-8 weeks.(Sawyers, Gishizky et al. 1992) KG1a is a subclone derived from KG1 with
a more primitive immunophenotype, being predominantly CD34+CD38-.(Koeffler, Billing et al.
1980) Both cell lines have fast cycling time and are are known to engraft in immunodeficient
10
mice. FcγII receptors (CD32) are expressed on KG1 and KG1a (25 and 17%) respectively.(Wu,
Markovic et al. 1996). While both KG1 and KG1a express CD34, KG1a is CD33 negative(Silla,
Chen et al. 1995), which is consistent with its more primitive CD34+CD38- immunophenotype.
1.2.6.3 OCI/AML2, OCI/AML3 and OCI/AML5
Cell lines derived from primary AML samples at the Ontario Cancer Institute have been
designated with the prefix OCI/AML and a number based on the order of development. These
are novel AML lines not available from the ATCC. OCI/AML2 was derived from a 65 year old
male with AML M4 at diagnosis. Its immunophenotype is negative for CD3 and CD14 and
positive for CD13, CD15, CD19 and CD33 (Wang, Curtis et al. 1989) It has hyperdiploid
complex cytogenetic translocations. OCI/AML3 was developed from a 57 year old male with
AML M4. The cells carry an NPM1 gene mutation (Type A) and NDMT3A R8826C
mutation(Wang, Curtis et al. 1989). It also has a hyperdiploid karyotype with complex
cytogenetics and hemizygous for RB1. OCI/AML5 was derived from a 77 year-old male with
AML M4 (relapse), with complex hyperdiploid karyotype and a doubling time between 30-50
hours.(Wang, Koistinen et al. 1991) Immunophenotypically it is negative for CD3, CD7, CD19,
CD14 and positive for CD34, CD68, TdT, HLA-DR and CD8. OCI/AML2 and OCI/AML3
grow in standard medium with 10% FBS supplementation and do not require additional cytokine
supplementation, but OCI/AML5 also requires 5637 bladder carcinoma conditioned medium
(10%) for optimal growth.
1.2.7 Animal models of leukemia
While early cancer drug discovery focused on in vitro assays, animal models have progressively
become more prevalent in the preclinical assessment of cancer therapeutics and an expectation in
most cases prior to testing in humans. However, in vitro toxicity assays remain a useful
screening tool to identify agents which may be of therapeutic benefit. These assays include
measures of mitochondrial viability (e.g. MTT assay), membrane permeability (e.g chromium
release assay) and flow cytometric approaches (e.g. forward and side scatter properties,
propidium iodide staining and annexin V staining). However, in vitro efficacy of an agent often
does not translate to efficacy in animal models of disease, due to issues of biological complexity.
Unlike in vitro assays, animal models allow for an assessment of the pharmacokinetic clearance
11
of an agent, organ toxicity and interactions with the immune system and three dimensional
aspects of tumour biology.
Syngeneic murine cancer models involve use of cancer cell lines to induce cancer in the same
strain of mouse as the original tumour. This allows for murine tumour cell lines to be
transplanted into mice that are typically unable to reject the tumour and facilitate study of
therapeutic agents in a fully immunocompetent animal model. Testing of proven clinically
active oncology drugs in these models demonstrate efficacy providing some support for their use
in cancer drug discovery.(Darro, Decaestecker et al. 2005) However, the biology of murine
cancers differ from human cancer, drawing into question their use as preclinical screening tools.
Voskoglou-Nomikos et al. conducted a review of 31 anti-cancer drugs and compared their
performance in human cell line, human xenograft, and syngeneic murine models, relative to their
efficacy at the phase II clinical trial stage in breast, non-small cell lung, ovary, and colon
cancers. Syngeneic murine models of cancer were not predictive of performance in human
clinical trials. However, human cell line and human xenograft models were predictive for non-
small cell lung cancer and ovarian cancer, and cell line models were predictive for breast
cancer.(Voskoglou-Nomikos, Pater et al. 2003) This lack of translation has been an issue,
whereby agents appeared effective in murine disease models, but did not work in humans. To
address this issue, advances in murine xenograft models were developed using genetically
immunodeficient mice permissive for human tumour growth, allowing for biological
characterization of human tumours in vivo and preclinical assessment of novel agents.
The development of murine human xenograft models dates back to the 1970s, but there have
been three main advances that have had the greatest impact(Shultz, Ishikawa et al. 2007). The
first was the discovery and characterization of protein kinase DNA-activated catalytic
polypeptide (Prkdc) mutated CB17 mice(Bosma, Custer et al. 1983). The second was back
crossing the Prkdc mutation onto a non-obese diabetic (NOD) background(Shultz, Schweitzer et
al. 1995), yielding NOD/SCID mice. The third was a null or truncated mutation in the common
interleukin-2 receptor gamma chain to yield a NOD/SCID gamma null mouse(Ito, Hiramatsu et
al. 2002; Shultz, Banuelos et al. 2003; Traggiai, Chicha et al. 2004; Ishikawa, Yasukawa et al.
2005).
12
1.2.7.1 Early xenograft models
The first attempt to xenograft human AML utilized thymectomized, irradiated mice rescued with
syngeneic bone marrow infusions that were infused sc with AML cells that formed transient
local tumours(Franks, Bishop et al. 1977) Subsequently, neonatal thymectomized mice were
irradiated and conditioned with cytarabine to facilitate growth of primary AML cells injected
subcutaneously leading to localized tumours (Palu, Selby et al. 1979). Following this, athymic
nude mice(Flanagan 1966; Segre, Nemhauser et al. 1995) which lack functional T and B cells
were utilized to study AML cell lines (MO7E and TF-1) in vivo, but required gene modification
to produce essential cytokines to allow for engraftment (Thacker and Hogge 1994). Human
AML cell lines such as HL-60(Yamada, Mori et al. 1983; Potter, Shen et al. 1984) and KG-1
expanded in these models better(Machado, Gerard et al. 1984), but tended to generate localized
myelosarcomas with minimal bone marrow engraftment(Nilsson, Giovanella et al. 1977) making
them poor leukemia models. The most significant limiting factor with these early xenograft
models was that primary leukemia samples did not engraft well.(Nara and Miyamoto 1982;
Caretto, Forni et al. 1989)
1.2.7.2 SCID mice
Severe combined immunodeficient (SCID) mice were first described in 1983 by Bosma et
al.(Bosma, Custer et al. 1983) due to autosomal recessive mutation of chromosome 16, resulting
in disruption of the protein kinase DNA-activated catalytic polypeptide (Prkdc) gene.(Blunt,
Finnie et al. 1995; Kirchgessner, Patil et al. 1995; Miller, Hogg et al. 1995; Blunt, Gell et al.
1996) Abnormal Prkdc genes lead to deactivation of a DNA recombinase enzyme, resulting in
inability to rearrange T- and B-cell antigen receptors, due to failure of coding joint
formation(Lieber, Hesse et al. 1988) in the final step of VDJ recombination(Malynn, Blackwell
et al. 1988). This causes a generalized deficiency in humoral and cellular immunity, with
incomplete penetrance, allowing for the production of only a small number of functional T- and
B-cells, resulting in severe immunodeficiency.(Bosma and Carroll 1991) The ‘leakiness’ of the
SCID mutation is strain dependent, and mice can have high levels of immunoglobulin (Ig) with
production increasing with age and some functional T- and B-cell rearrangements.(Nonoyama,
Smith et al. 1993) The SCID mouse resulted in superior engraftment of human hematopoietic
tissue(McCune, Namikawa et al. 1988), as demonstrated by the generation of long-term
production of human T-cells after injection of peripheral blood leukocytes
13
intraperitoneally(Mosier, Gulizia et al. 1988). The earliest leukemia xenograft models used
SCID mice to engraft human pre-B acute lymphoblastic leukemia primary samples(Kamel-Reid,
Letarte et al. 1989). This model was then used in an attempt to get primary AML samples to
engraft. However, SCID mice have high levels of NK cell activity, limiting xenograft potential.
1.2.7.3 NOD/SCID mice
In addition to T- and B- cell deficits, NOD/SCID mice have reduced numbers and function of
NK cells, but some activity does remain.(Shultz, Schweitzer et al. 1995) This is evident in the
fact that only 70% of all AML samples exhibit detectible engraftment in NOD/SCID mice
(Ailles, Gerhard et al. 1999) and many of these samples engraft at less than 10%, measured at 6-
12 weeks. Limitations of the NOD/SCID mouse model includes development of thymic
lymphomas by 8.5 months, radiosensitivity and residual NK cell activity, which limit
engraftment of xenogeneic tissue.(Shultz, Schweitzer et al. 1995)
1.2.7.4 NOD/SCID Interleukin-2 receptor gamma null mice
The IL-R gamma chain(IL-2Rγc) is common to the cytokine receptors IL-2, IL-4, IL-7, IL-9, IL-
15 and IL-21 and is essential to multiple immune functions (reviewed by Sugamura)(Sugamura,
Asao et al. 1996). Deficiencies in IL-2Rγc alone lead to severe T- and B-cell deficiencies and a
blockade of NK cell development.(Cao, Shores et al. 1995; DiSanto, Muller et al. 1995; Ohbo,
Suda et al. 1996). The development of IL-2Rγc null mice back-crossed onto the NOD/SCID
background(Ito, Hiramatsu et al. 2002; Ishikawa, Yasukawa et al. 2005; Shultz, Lyons et al.
2005), led to a significant improvement in the engraftment of human hematopoietic tissue
(benign and malignant), as they are fully deficient in T-, B- and NK cells. However, these mice
retain some elements of innate immunity in the myeloid cell compartment (ie neutrophils,
monocytes and macrophage). A study of leukemic cell engraftment was undertaken in 3 strains
of immunodeficient mice: NOD/SCID, NOD/SCID/β2microglobulin null (NSB) and
NOD/SCID/IL-2Rγc null (NSG).(Agliano, Martin-Padura et al. 2008) Engraftment of five
primary AML samples in the three murine strains revealed that only 2/5 samples engrafted in
NOD/SCID or NSB mice, whicle 5/5 engrafted in NSG mice.
Another similar study demonstrated a 6-fold increase in bone marrow engraftment of human
HSC in NSG mice relative to NOD/SCID mice.(Shultz, Lyons et al. 2005) Also, NOD/SCID
14
mice develop thymic lymphomas(Christianson, Greiner et al. 1997), and NSB mice(Shultz,
Ishikawa et al. 2007) have accelerated lymphomagenesis, leading to significantly shorter
lifespans than NSG mice, which creates confounding issues when assaying human malignant
cells.
1.2.8 Cancer stem cell hypothesis
John Bennett was the first to clearly describe a patient with leukemia and implicating it as a
disease of the blood, which he termed “leucocythaemia”.(Bennett 1845) Rudolph Virchow soon
after described another patient with leukemia, who had a very high white blood cell count, and
termed this condition, “Leukämie”, derived from two Greek words; leukos (white) and
haima(blood). (Virchow 1856) In addition, to this important diagnostic clinical discovery,
Virchow also made the observation of tumour pleimorphism, postulated that, ‘cells came from
cells’, and that cancer cells were derived from normal cells. Studies by Furth et al. using inbred
mouse strains and syngeneic leukemic cell lines demonstrated that the tumour initiating
frequency was between 1 and 100.(Furth 1935) More remarkable, was their subsequent finding
that a leukemia could be transmitted with a single cell to a murine host.(Furth, Kahn et al. 1937)
However, the underlying basis for the heterogeneity of leukemia, with apparently rare tumour
initiating cells, was not considered.
The demonstration of normal hematopoietic stem cells by Till and McCulloch,(McCulloch and
Till 1960) led them to explore the underlying process that determined cell fate decision to self-
renew or differentiate. They proposed a stochastic model based on the frequencies of HSCs as
detected by the colony forming unit-spleen (CFU-S) assay, given that the number colonies
derived from serially transplanted CFU-S approximated a gamma distribution.(Till, McCulloch
et al. 1964). They acknowledged that their study had been demonstrated in only one
hematopoietic stem cell model and remained open to the possibility of non-stochastic
mechanisms in stem cell fate decision-making. These concepts were extended to tumour models,
with a debate forming between a stochastic and stem cell model of tumour progression. The
concept of a cancer stem cell in leukemia was proposed by Bruce et al. (Bruce and Ash 1963)
and provided an alternative to the stochastic model to explain rare tumour initiating cells. One
additional feature of this theory was that cancer cells could differentiate along a hierarchy
15
somewhat analogous to normal hematopoietic stem cells. Early cell cycle kinetic studies by
Clarkson et al. in cell line and murine models of acute leukemia demonstrated that the majority
of leukemic blasts were postmitotic. Further, two additional populations were identified; a fast
and slow cycling group representing the minority of cells.(Clarkson 1969) The authors proposed
that the slow cycling fraction represented a stem cell population analogous to a hematopoietic
stem cell. LSCs in AML were the first identified cancer stem cell population, which were shown
to be enriched in the CD34+ CD38- fraction of whole blasts, as measured by the ability to
engraft in the bone marrow of SCID mice. Further, these cells possessed the capacity to
differentiate into more mature blasts (CD34+CD38+ AML cells).(Lapidot, Sirard et al. 1994;
Bonnet and Dick 1997)
A stem cell hierarchy for AML was demonstrated and cancer stem cells have subsequently been
demonstrated in a variety of cancers, including brain tumors(Singh, Clarke et al. 2003; Singh,
Hawkins et al. 2004), breast cancer(Al-Hajj, Wicha et al. 2003), multiple myeloma(Matsui, Huff
et al. 2004) and colon cancer(O'Brien, Pollett et al. 2007; Ricci-Vitiani, Lombardi et al. 2007).
Some cell lines have demonstrated cancer stem cells within cell C6 glioma cell lines and
multiple myeloma cell lines.(Kondo, Setoguchi et al. 2004; Matsui, Huff et al. 2004)
A recent report of dual LSC populations in a large number of primary AML samples further
refine the CD34+CD38- compartment as follows: Lin-CD34+CD38-CD90-CD45RA+ and Lin-
CD34+CD38-CD90-CD45RA-.(Goardon, Marchi et al. 2011) There are other non-
immunophenotypically based functional definitions which have enriched AML LSCs, including
side population (SP) cells(Wulf, Wang et al. 2001) and aldehyde dehydrogenase (ALDH)
expressing cells(Cheung, Wan et al. 2007). Colon cancer-initiating cells have been
identified(O'Brien, Kreso et al. 2009), and recent evidence indicates that they are sensitive to NK
cell-mediated killing due to low HLA expression, and increased expression of NKp30 and
NKp44 ligands, while differentiated colon cancer cells are less so.(Tallerico, Todaro et al. 2013)
1.2.8.1 Leukemia stem cell markers
While LSCs were postulated following the discovery of normal hematopoietic stem cells by Till
and McCulloch(McCulloch and Till 1960), until cell surface markers and cell sorting techniques
developed to distinguish and separate putative populations, it could not be confirmed
16
experimentally. While there have been many reported LSC markers a few have been of
particular relevance and will be described here.
1.2.8.1.1 CD123
CD123 is present on ~99% of leukemic stem cells(Jordan, Upchurch et al. 2000), but is not
expressed on normal hematopoietic stem cells(Huang, Chen et al. 1999), making it an attractive
therapeutic target. CD123 is the interleukin 3 receptor alpha chain, or low affinity receptor. It is
a type I transmembrane glycoprotein. The alpha chain is shared by the IL-5 and granulocyte
monocyte-colony stimulating factor (GM-CSF) receptors. However, when coupled to the
interleukin 3 receptor beta chain (CDw131), the binding infinity increases dramatically,
facilitating signal transduction from low concentrations of IL-3. IL-3 is important in driving
myeloid differentiation and can activate STAT5. IL3R knockout mice do not have major
hematologic impairment, but have reduced granulcotye-monocyte colony forming capacity
(CFU-GM). CD123 is expressed on committed hematopoietic progenitors and mediates
differentiation and proliferation. Variable levels of CD123 have been reported on CD34+ HSCs,
but it is low on HSCs. CD123 is expressed on cells of the hematopoietic system (monocytes,
neutrophils, basophils, eosinophils, megakaryocytes and erythroid precursors, mast cells,
macrophages, some B lymphocytes) and non-hematopoietic tissue (Leyding cells of the testis,
placenta, and brain).(Moretti, Lanza et al. 2001)
1.2.8.1.2 CD34
CD34 is a cell surface glycoprotein that as been widely used as a marker to identify and isolate
hematopoietic stem cells (Berenson, Andrews et al. 1988; Andrews, Singer et al. 1989; Ema,
Suda et al. 1990) and progenitors. It also marks vascular endothelial cells(Baumhueter, Dybdal
et al. 1994) other tissue-specific stem cells, including muscle satellite cells and epidermal
precursors, as well as mast cells and eosinophils.(Nielsen and McNagny 2008) The definitive
function of CD34 has not been determined, but has been proposed to promote the proliferation
while blocking differentiation of progenitor cells.(Krause, Fackler et al. 1996) Further, CD34
may have roles in chemotaxis and in asymmetric cell division. CD34-knockout mice have a
delay in both erythroid and myeloid differentiation, with reduced colony-forming activity of
hematopoietic progenitors derived from bone marrow, and inability to be cultured ex vivo with
standard cytokines support.(Cheng, Baumhueter et al. 1996) However, CD34-knockout mice
17
develop normally and have normal hematopoiesis in the adult mouse. CD34 selection has been
shown to enrich LSCs in primary AML samples.(Lapidot, Sirard et al. 1994)
1.2.8.1.3 CD38
While not a stem cell marker per se, the absence of CD38 expression has been used to further
enrich CD34+ hematopoietic or leukemic stem cell populations, and it is therefore an important
differentiation marker that can distinguish stem cells from progenitors.(Lapidot, Sirard et al.
1994) Human CD38 is made up of a single chain of 300 amino acids with a molecular weight of
45 kDa and is expressed by hematopoietic and non-hematopoietic cells, including NK cells and
monocytes. Other CD38+ cells include smooth and striated muscle cells, renal tubules, retinal
gangliar cells and cornea (reviewed in Malavasi)(Malavasi, Deaglio et al. 2008). CD38 is
involved in signal transduction, cell adhesion and calcium signaling. The binding to the ligand
CD31, initiates a signaling cascade that includes phosphorylation of sequential intracellular
targets and increases cytoplasmic Ca2+
levels, mediating different biological events, depending
on the cell type (e.g., activation, proliferation, apoptosis, cytokine secretion and homing). While
absence of CD38 was used to establish classic LSC definitions, subsequent work demonstrated
the LSCs can exist in the CD34+CD38+ compartment of a significant number of primary AML
samples.(Taussig, Miraki-Moud et al. 2008)
1.2.9 Clinical relevance of LSCs
Most clinical studies of leukemic stem cells (LSCs) have focused on the CD34+CD38- definition
with or without addition of CD123 or CLL-1(van Rhenen, Feller et al. 2005; van Rhenen,
Moshaver et al. 2007; Witte, Ahlers et al. 2011). The latter two antigens were discovered
through use of flow cytometric analysis of CD34+CD38- AML cells using panels of CD markers
rather than from biological studies in immunodeficient mice. While some studies have
demonstrated that higher NOD/SCID engraftment capacity correlates with worse survival
outcomes(Pearce, Taussig et al. 2006), this does not provide evidence to exclusively support the
cancer stem cell hypothesis because a stochastic model of tumour initiation and maintenance
could also explain these findings. A more recent study demonstrated that patient’s whose whole
AML samples had a gene expression profile similar to LSCs or HSCs had worse survival
outcomes, and this could be used to better risk stratify patients independent of known prognostic
factors.(Eppert, Takenaka et al. 2011) While this provides some evidence that leukemic stem
18
cell signatures influence clinical outcome, it does not definitively show that treatment of LSCs is
required for cure. This is consistent with earlier studies showing that less differentiated
leukemia, as determined by immunophenotype, has a worse prognosis.(Legrand, Perrot et al.
2000)
Only two clinical studies to date have studied the clinical relevance of functionally validated
immunophenotypic LSC definitions established using immunodeficient murine models. The first
was a study of 92 AML patients that showed a high burden of CD34+CD38- AML cells at
diagnosis correlated with poor survival and conventional MRD positivity after the third cycle of
chemotherapy(van Rhenen, Feller et al. 2005). A similar retrospective study in 17 pediatric
AML patients from the BFM clinical trial group also demonstrated a high CD34+CD38- cell
burden led to increased relapse and worse survival outcomes.(Witte, Ahlers et al. 2011) While
this provides an additional level of evidence to support the leukemia stem cell hypothesis, it is
not definitive. CD34+CD38- LSCs are chemo-resistant due to prolonged G0 status(Guan,
Gerhard et al. 2003) and drug efflux potential(Costello, Mallet et al. 2000; Wulf, Wang et al.
2001), and are presumed to be responsible for recapitulating leukemic disease.
The demonstration that a high burden of CD34+CD38- AML cells at diagnosis correlates with
worse survival outcome does not directly confirm that these cells are the subset that leads to
relapse post therapy. To directly demonstrate this requires assessing the response of these cells
to chemotherapy and demonstrating that the residual CD34+CD38- cell burden post-therapy is
correlated with relapse and survival. This approach has been utilized for prognosticating in pre-
B ALL, where MRD post induction is predictive of survival(Dworzak 2002; Borowitz, Devidas
et al. 2008; Bassan, Spinelli et al. 2009), and is currently used in risk stratification in numerous
major clinical trial groups worldwide (e.g. Children’s Oncology Group and BFM Group). This
ability to risk stratify and intensify therapy rationally has contributed to improved outcomes in
the treatment of ALL. Studies designed to assess the prognostic value of MRD have taken bone
marrow and peripheral blood samples for MRD at day 8, 14 and 29 (e.g. chemotherapy clinical
trial COG AALL 0331), while other studies have utilized later time points including at the end of
therapy. There is no published study that has risk stratified patients using either CD34+CD38-
burden at diagnosis or post-therapy, and dose intensified the higher risk group either with
additional chemotherapy or allogeneic bone marrow transplantation.
19
1.2.9.1 Strategies to detect LSCs in patients
In AML both PCR and flow cytometry can be used to assess for MRD at various time points
during and post therapy. While PCR testing may be more sensitive (~1 in 105) than flow
cytometry (~1 in 104)(Buccisano, Maurillo et al. 2009), it is an assessment of bulk residual
disease and cannot be used to differentiate leukemic stem cells from blasts. Flow cytometry on
the other hand, assesses cells on a single cell basis, allowing for examination of residual LSCs in
peripheral blood and bone marrow. LSCs can be detected easily in peripheral blood using CD34
and CD38 staining, but in bone marrow detection is confounded by normal HSCs, which also are
immunophenotypically CD34+CD38-.
The two clinical studies evaluating LSC burden at diagnosis used different gating strategies to
identify LSCs(van Rhenen, Feller et al. 2005; Witte, Ahlers et al. 2011). In a follow-up study by
van Rhenen et al., LSCs could be detected in 55 patients with either AML or high risk
myelodysplastic syndrome in remission using several 4 and 5 color staining panels. This study
established that LSCs can be distinguished from HSCs in normal bone marrow by lineage marker
expression (e.g. CD2, CD5, CD7, CD19, CD11b, CD22 and CD56), lineage marker
asynchronous antigen expression (e.g. CD13-CD33+), lineage marker overexpression (e.g.
CD33++) and underexpression (e.g. HLA-DR low). Further, the LSC specific markers
CD123(Jordan, Upchurch et al. 2000) and CLL-1(van Rhenen, van Dongen et al. 2007) were
also demonstrated to effectively differentiate LSCs from HSCs. CD123 may be present at low
levels on normal HSCs and can upregulate post chemotherapy, confounding its use as a LSC
marker immediately after therapy for some patients (i.e. post induction). CLL-1 does not
upregulate post chemotherapy, but is not as frequently or highly expressed on LSCs as
CD123(van Rhenen, Moshaver et al. 2007). CLL-1 is restricted to the hematopoietic lineage, in
particular to myeloid cells present in peripheral blood and bone marrow, is absent on HSCs, but
highly expressed on LSCs. Recently, it was reported that Tim3 provides another marker that can
differentiate HSCs from LSCs (Jan, Chao et al. 2011). Tim3 is a Th1-specific cell surface
protein that regulates macrophage activation, and is expressed on LSCs. CD96 has also been
identified as a LSC marker.(Hosen, Park et al. 2007)
20
1.3 Cytotoxicity assays
The concept that rare cancer stem cells initiate and propagate tumours have profound
implications for the interpretation of cytotoxicity assays where the assumption of tumour
homogeneity has been made. Typical cytotoxicity assays test a snap shot assessment of bulk
tumour cells to determine whether a tumour has signs of apoptosis (e.g. annexin V positivity),
mitochondrial damage (e.g. MTT assessment of mitochondrial activity), cell permeability (e.g.
51CrO4 release assay and PI staining in flow cytometry), or some other readout deemed indicative
of viability or cell death. Leukemic stem cells have been demonstrated to have resistance to
chemotherapeutic agents relative to bulk leukemia (Costello, Mallet et al. 2000), emphasizing the
importance of measure cytotoxicity against the leukemic stem cell.
Where a cancer stem cell has been identified and validated in vivo for a particular cancer using
specific cell surface markers, cell sorting can be used in conjunction with one of the previously
mentioned approaches to address toxicity against the cancer stem cell population. However,
most primary cancers and cell lines do not have an identifiable cancer stem cell population that
can be generalized, making this approach problematic. Even where cancer stem cell definitions
have been validated and one can use conventional cytotoxicity assays, functional assessments
against their proliferative and clonogenic capacity are important parameters to measure when
evaluating novel cancer therapeutics.
1.3.1 Bulk cytotoxicity assays
Most cytotoxicity assays have focused on the impact on bulk tumour populations. The classic
MTT assay measures the mitochondrial enzyme activity as a measure of viability and is used
commonly in quantitation of drug sensitivity of tumour cells. However, the assay is confounded
when testing mixtures of immune effectors and targets that cannot easily be separated,
confounding the viability readouts. This is one reason that the chromium release assay has been
commonly used to evaluate the impact of immune effectors on tumour targets. The basic
principle involves labelling tumour targets with 51
CrO4 prior to co-incubation with immune
effectors and measuring the released chromium relative to spontaneous and maximum release
values.(Brunner, Mauel et al. 1968) While newer types of assays have been developed, the
majority are designed to measure cytotoxicity against bulk tumour. The calcein AM assay is one
such approach that uses a non-radiolabelled dye to facilitate identification of viable and dead
21
cells.(Neri, Mariani et al. 2001) The colorimetric change can be detected using a
spectrophotometer. However, the assay is more labor intensive and prone to signal-noise issues.
1.3.2 Flow cytometric cytotoxicity assays
Flow cytometry based cytotoxicity assays have focused on the use of propidium iodide which
can enter dead cells with porous membranes and is easily detectable.(Jones and Senft 1985)
Additional viability or dead cell stains can be used individually or in combination to allow for
more fine discrimination of viability at the single cell level. Annexin V detection is another
means, which allows for the detection of early apoptosis which leads to flipping of membrane
phospholipids enabling Annexin V to bind. Annexin V can be conjugated to standard
fluorochromes such as FITC to facilitate use in flow cytometric assays to detect early apoptotic
cells.(Anthony, McKelvie et al. 1998) The advantage of this approach is to identify a greater
proportion of dead cells than with PI. Combining PI and Annexin V can also be used in time
course studies to show progression of apoptosis, which begins with Annexin V positivity
followed by PI positivity. Necrosis and apoptosis can be identified using this approach by
tracking the position of cells in a two-by-two plot. Necrosis tends to proceed directly to PI
positivity, without conversion to Annexin V positivity. However, flow cytometric assays have
been typically used to measure cytotoxicity at one time point, either percent or cumulative
cytotoxicity. Percent cytotoxicity is a snap shot at one time of the proportion of cells assayed.
While this is typically extrapolated to the entire cell population, this neglects the possibility of
cellular breakdown after death, leading to an underestimate of cytotoxicity. This can be
controlled for using counting beads in all tubes, enabling a more accurate measure of
cytotoxicity. The longer a cytotoxicity assay runs, the more relevant cellular disintegration
becomes and the greater the underestimate of cytotoxicity. The percent cytotoxicity from flow
cytometric assays has yielded equivalent cytotoxic readouts as the chromium release
assay.(Ozdemir, Ravindranath et al. 2003)
1.3.3 Clonogenic cytotoxicity assays
The utility of the aforementioned standard methodologies to assess cytotoxic agents against
tumor targets have been questioned by a growing body of literature supporting rare cancer stem
cells in a number of cancers.(Lapidot, Sirard et al. 1994; Bonnet and Dick 1997; Al-Hajj, Wicha
et al. 2003; Singh, Hawkins et al. 2004; O'Brien, Pollett et al. 2007; Ricci-Vitiani, Lombardi et
22
al. 2007) If only a small subfraction of tumour cells have true unlimited proliferative potential
then extrapolating the results of bulk tumour assays to the cancer stem cell fraction may not be
reflective of toxicity against the cancer stem cell fraction. Therefore, attempting to utilize
methods to address the impact of therapeutic agents against CSCs is highly relevant to the
development of curative strategies. It also has profound implications for studies of cytotoxicity
against bulk tumour samples, which have focused on bulk tumour assays and have more
confounding issues due to mixed effector and target populations. Although clonogenic readouts
have been used in the past to assess chemotherapy drugs(Curtis, Minden et al. 1995; Jacobs and
Wood 2005) they have rarely applied to evaluation of immune effectors.
The clonogenic assay was developed by Puck and Marcus in 1956, initially as a system to grow
HeLa cells(Puck, Marcus et al. 1956) and then to grow epithelial cells from normal human tissue
(liver, conjunctiva, kidney and appendix)(Cieciura, Marcus et al. 1956). This involved the use of
semi-solid medium supplemented with growth factors and nutrients to grow colonies derived
from single cells. This predated the discovery of hematopoietic stem cells in 1961 by Till and
McCulloch using the in vivo spleen colony forming assay.(Till and McCulloch 1961) An
effective means to generate murine bone marrow derived colonies was done in 1966 using agar
plates with mouse kidney or embryonic feeder layers.(Bradley and Metcalf 1966) The first
attempt to grow primary tumours in semi-solid medium was by McCulloch’s research group with
murine myeloma cells, which grew quantifiable colonies.(Park, Bergsagel et al. 1971)
Subsequently, they applied this approach to study sensitivity of murine and human hematopoietic
stem cells to chemotherapy drugs.(Ogawa, Bergsagel et al. 1973) Further applications of an
agar based colony forming assay for primary acute myeloid leukemia samples was demonstrated
to predict clinical response and resistance.(Park, Amare et al. 1980)
Currently, clonogenic assays typically involve growing the cells either in methylcellulose or agar
supplemented with various growth factors and cytokines. Proliferation of a subset of cells results
in the appearance of colonies that can be enumerated typically at 10-14 days. The value of
clonogenic assays is that it allows assessment of single cells with high proliferative capacity.
Tumour stem and progenitor cells have the ability to form colonies in a semisolid state matrix
consisted of either agar or methylcellulose providing a readout for testing cytotoxic agents.
However, limitations of clonogenic assays include low frequency of clonogenic cells, two log
range for cytotoxicity evaluation, and clump artifacts for some types of cells.(Hoffman 1991)
23
Clonogenic chemotherapy assays are good at predicting patients likely be resistant to a particular
chemotherapy agent, but not as accurate at determining those who will be sensitive. Clonogenic
assays have the potential of individualizing patient treatment as they can predict clinical
responses, but have not been incorporated into standard of care for any given cancer, possibly
because of the inability to grow a high proportion of primary tumours using this approach.
Moreover, the sensitivity of clonogenic leukemic cells to chemotherapy predicts the clinical
response in AML.(Park, Amare et al. 1980) These data suggest that clonogenicity is a clinically
relevant parameter. While clonogenic cells are not cancer stem cells per se, they include a subset
of malignant progenitors and stem cells with significant proliferative capacity and the ability to
contribute to disease progression. Inhibition of colony formation, therefore, is an important
measure of cytotoxicity that assays single cells and is a reasonable surrogate for toxicity against
cancer stem cells.
Few studies have examined the in vitro sensitivity of cancer stem cells to immune effector
killing. In one study, lymphokine activated killer (LAK) cells and allogeneic lymphocytes were
shown to exert a modest cytotoxic effect on AML cancer stem cells (CD34+ CD38-) that were
intrinsically resistant to the chemotherapeutic agent, daunorubicin.(Costello, Mallet et al. 2000)
This was done by sorting the cells and performing a chromium release assay. A limitation of that
study was that the LAK cells were a mixed population of IL-2 activated T and NK cells, making
it difficult to discern the contribution of either cell type.
Another study using the clonogenic assay to examine the role of KIR mismatched NK cells
against primary AML showed a reduction in colonies.(Langenkamp, Siegler et al. 2009) This
study also utilized the chromium release assay and reported secondary replating of clonogenic
cells. Similarly, autologous activated marrow-infiltrating T-lymphocytes (MILs) have
demonstrated superior in vitro cytotoxicity against clonogenic MM cells compared to activated
peripheral blood lymphocytes (PBLs) isolated from the same patient. Normal hematopoietic
progenitors were not affected by MILs or PBLs, providing pre-clinical evidence for the safety of
this therapy.(Noonan, Matsui et al. 2005) However, a direct comparison between bulk tumor and
clonogenic cell killing was not considered in this work. Further, neither study utilized
appropriate controls to differentiate killing during the 4 hour co-incubation versus over the 2
week low density incubation in methylcellulose, making a comparison of methods difficult to
interpret.
24
1.4 Cancer immunobiology
1.4.1 Lymphocytes
The cellular immune system comprises lymphocytes and myeloid cells, which evolved primarily
to protect organisms from microbial infection. However, the immune system has also
demonstrated the capacity to prevent cancer from developing, as evidenced by the increased rates
of leukemia and lymphoma in patients and animals with severe combined immunodeficiency.
These findings support the notion of immune tumour surveillance, and biological studies confirm
the anti-tumour activities of NK cells and T-cells. This capacity may have initially evolved in
the context of virally driven lymphoproliferative disorders and cancers. Both adaptive and innate
cellular immune systems can play a role in tumour immune surveillance and/or anti-tumour
responses. Primarily, these responses are mediated by lymphocytes. The lymphocyte subset
includes B-, T- and NK cells of which the latter two play important roles in tumour immune
surveillance.(Swann and Smyth 2007) T- and NK cells both contain cytotoxic granules
containing perforin and granzymes that can be exocytosed onto target cells, leading to apoptosis.
In addition, they can kill via death ligands on their surface such as Fas Ligand or TRAIL.
1.4.2 NK cells
Natural killer (NK) cells were discovered in the mouse by Herberman et al. and Kiessling et al.
as a population of spleen-derived non-T, non-B lymphocytes with cytolytic activity.(Herberman,
Nunn et al. 1975; Herberman, Nunn et al. 1975; Kiessling, Klein et al. 1975; Kiessling, Klein et
al. 1975) Subsequently, an analogous population was discovered in humans also termed NK
cells(Jondal and Pross 1975; Pross and Jondal 1975). Deficiency of NK cells in humans is rare
and can predispose to fatal infection.(Orange 2006) While murine NK cells are similar to
human, there are unique species-specific receptors. In humans, NK cells are classically defined
as CD3-CD19-CD56+ lymphocytes with variable expression of CD16. Cytolytic NK cells with
high perforin content express low levels of CD56, while cytokine secreting or ‘helper’ type NK
cells express high degrees of CD56 and often low levels of CD16. One proposed NK maturation
process involves differentiation from a CD56bright
CD16neg
cell predominantly in the BM, to a
CD56+CD16+ cell present in BM and SPL and finally a CD56dim
CD16+ cell predominantly in
the spleen.(Armas 2009)
25
NK cells have had have been postulated to play a role in immune tumour surveillance(Trinchieri
1989) and play a complementary role to T-cells as many virally infected or tumour cells
downregulate class I HLA as means to evade detection and destruction by the immune system.
NK cells are able to recognize and destroy cells that have downregulated HLA class I molecules
on their surface. This enables NK cells to destroy virally-infected cells or tumor cells
unrecognized by T cells, which rely on antigen presentation in the context of HLA class I.
Therefore, when cells downregulate HLA class I, they are susceptible to NK cell recognition and
cytolysis, as described by the “missing self” hypothesis.(Karre, Ljunggren et al. 1986) This
function was later attributed to the Ly49, which when engaged by H-2Dd in C57BL/6 mice
inhibited NK cell function in an MHC class I specific manner.(Karlhofer, Ribaudo et al. 1992)
Further, Karlhofer et al. noted that absence of MHC I ligand on non-malignant tissue did not
result in NK cell cytotoxicity, implicating other NK recognition mechanisms. Subsequently,
analogous inhibitory receptors were discovered in the human(Moretta, Bottino et al. 1990;
Moretta, Tambussi et al. 1990) with identification of their ligands as class I HLA being
determined later.(Dohring and Colonna 1996) These human HLA specific receptors were
collectively termed inhibitory killer immunoglobulin-like receptors (KIRs).
A balance of inhibitory and activating signals determines whether an NK cell kills its
target(Sutlu and Alici 2009). This process is depicted in simplified form with only one
activating and inhibiting receptor (Figure 1.1, Figure 1.2, Figure 1.3), but NK cell signaling is an
integrative process involving many activating and inhibiting receptors working in concert to
provide specificity to its recognition capability. While the ‘missing self’ hypothesis explains
how NK cell autoreactivity can prevented by inhibitory receptors that respond to self MHC
(mouse) or HLA (human), it does not explain how activation occurs in the context of this
regulatory mechanism. The concept of ‘induced self’ involves the upregulation of stress related
antigens such as heat shock proteins, which can occur following infection or malignant
transformation of a cell. In the case of NKG2D ligands, when upregulated, these can override
Ly49 mediated inhibition as reviewed by Malarkannan et al.(Malarkannan 2006) This supports
the notion of NK regulation as an integration of signaling through different activating and
inhibiting receptors which detect ‘induced’ and/or ‘missing’ self, allowing for recognition of
‘altered self’. The balance of signaling depends on the nature and density of NK receptors and
target cell ligands.
26
Figure 1.1: NK cell degranulation by stimulation with activating ligands only NK cells can be stimulated through a number of different activating receptors that may be
present on targets. In the presence an activating ligand (L) only granule exocytosis would be
triggered resulting in cytolysis.
Figure 1.2: NK cell inhibition of degranulation by stimulation with activating and
inhibitory ligands
In the presence of both an inhibitory and activating ligand (L) on a target cell, granule exocytosis
would be inhibited resulting in no cytolysis, provided that the effects of the inhibitory receptor
predominate.
Figure 1.3: NK cell degranulation by stimulation with activating and inhibitory ligands
In the presence of high concentration of activating ligand (L) on the target cell relative to
inhibiting ligand, granule exocytosis could be triggered resulting in cytolysis, despite
engagement of the inhibiting receptor.
27
1.4.2.1 Granule exocytosis
The major mechanism of NK cell cytotoxicity is via exocytosis of granules containing perforin
and granzymes, first characterized by Podack et al., whereby they isolated T-cell granules,
identified protein components and demonstrated cytolysis(Podack and Konigsberg 1984)
(reviewed by Henkart et al.)(Henkart 1985). Following an encounter with another cell, an NK
cell will form an immunological synapse (IS).(McCann, Vanherberghen et al. 2003) If there is
sufficient activation signals on the potential target cell and lack of inhibitory signals, this will
trigger a cytolytic response. This requires cytoskeletal rearrangement and re-orientated of the
granules to the IS, followed by fusion into the synapse, and subsequent contact of granule
contents with the target cell membrane.
Granules contain perforin, first described by Dennart and Podack et al., which can create pores in
membranes of target cells following cytolytic effector degranulation.(Dennert and Podack 1983;
Podack and Dennert 1983), and have a structure similar to complement component
C9(Lichtenheld, Olsen et al. 1988). Serine proteases termed granzymes are also contained
within granules, with Granzyme A being the first characterized member of this family in T cell
granules.(Masson, Zamai et al. 1986; Masson and Tschopp 1987) Granzymes are facilitated
entry by perforin, where they are able to initiate apotosis by both caspase-dependent and
independent pathways. Perforin is a 70 Kda protein that requires free calcium and neutral pH to
optimally integrate into the target membrane.
Perforin knockout mice have been developed that have normal numbers of CD8+ T cells and NK
cells, but lack cytolytic function against allogeneic, virally infected, or tumour targets.(Kagi,
Ledermann et al. 1994) Further, perforin knockout mice have impaired control of tumour
progression in three different models of cancer (syngeneic tumour cell line, carcinogen exposure
and oncogenic virus), implicating T and NK cells in tumour immune surveillance.(van den
Broek, Kagi et al. 1996) In this study, unprimed wild type mice were able to mediate anti-
tumour effects, suggesting a role for NK cells, which lack the requirement for priming to
effectively mediate cytolysis. Finally, antibody-targeted depletion of NK cells in mouse studies
have supported the results from perforin knockout mice, showing NK cell-deficient mice are
more susceptible to 3-methylcholanthrene-induced sarcomas than wild-type mice(Smyth, Swann
et al. 2005) thus supporting NK cells as relevant in tumour immune surveillance.
28
Perforin deficiency caused by either a homozygous nonsense or missense mutation leads to
familial hemophagocytic lymphohistiocytosis (type 2) in humans, accounting for 60% of
cases.(Stepp, Dufourcq-Lagelouse et al. 1999) Granule exocytosis is dependent in part on
Munc13-4, a granule trafficking protein. Mutation in Munc13-4, has been shown to present in at
least 20% of familial hemophagocytic lymphohistiocytosis patients designated as type
3.(Feldmann, Callebaut et al. 2003)
Six granzymes have been identified in humans; A, B, H, K, M and tryptase-
2/granzyme3.(Hameed, Lowrey et al. 1988) Granzymes have one of four substrate specificities
cleaving after particular amino acids: tryptase (Arg or Lys), Asp-ase (Asp), Met-ase (Met or
Leu), and chymase (Phe, Tyr, or Trp).(Kam, Hudig et al. 2000) Granzyme B tends to cleave
proteins following aspartic acid residues in the substrate P1 position. Granzyme A, is a tryptase,
which cleaves substrates with basic residues in the P1 position (e.g arginine). The other human
granzymes have other enzymatic functions: H (chymase), K (tryptase) and M (metase).(Kam,
Hudig et al. 2000).
Granzyme B induces DNA damage through activation of caspase activity and has been shown to
partially process procaspase 3, which requires release of other proapoptotic factors from the
mitochondria to complete apoptosis.(Sutton, Wowk et al. 2003) By contrast, granzyme A is
unable to activate caspases, but instead targets nuclear proteins directly to induce DNA single-
stranded DNA breaks and fragmentation by a caspase-independent pathway.(Beresford, Xia et al.
1999) These nuclear proteins include histones, lamins, and DNA damage repair proteins, Ku70
and PARP-1.(Lieberman 2010) Granzyme A has also been shown to target mitochondria and
cause non-apoptotic death by cleaving the complex I protein NDUFS3, and ultimately generating
superoxide anions(Martinvalet, Dykxhoorn et al. 2008). Granzyme knockout mice do not have
significant immunodeficiency or decreased cytotoxicity of T- and NK cells in vitro, indicating
redundancy, and supporting a primary role of perforin in cytolysis of lymphocyte effectors.
29
1.4.2.2 Tumour necrosis factor family mediated cytotoxicity
The tumour necrosis factor family in humans has been well studied, with 27 members as of
2013(Aggarwal 2003; Bremer 2013). All are capable of binding ligands present on target cells to
induce apoptosis (extrinsic pathway) and are expressed primarily by NK and CTL. TNF-α and
TNF-β were the first discovered members of this family. Most TNF ligands are type II
transmembrane proteins containing an extracellular domain that can be cleaved by
metalloproteinases to generate soluble cytokines, which can mediate cytotoxic effects. The most
relevant effectors of cytotoxicity are Fas ligand and TNF-related apoptosis inducing ligand
(TRAIL). Major roles for FasL and TRAIL have been shown for tumour immunity. Fas ligand
is a type II transmembrane protein, which also has a secreted soluble form (Tanaka et al., 1996)
expressed on both cytotoxic CD8 T-cells and NK cells. Binding by Fas ligand of target Fas
allows for calcium-independent cytotoxicity against a range of tumour cells. This is typically
demonstrated by using EGTA, a calcium chelator, when conducting cytotoxicity assays.
Residual killing in calcium-free conditions is attributed to ligand-mediated killing.
While granule exocytosis models of cell death have focused primarily on perforin and
granzymes, FasL has been found to be localized to the outer membrane of cytoplasmic granules,
implicating it in granule mediated cytotoxicity.(Kojima, Kawasaki-Koyanagi et al. 2002) This
finding might also provide a mechanism of control over ligand mediated killing which must be
regulated, otherwise normal cells expressing Fas could be killed by casual contact with immune
effectors bearing FasL. However, conventional approaches to inhibit granule exocytosis have
demonstrated that ligand mediated killing occurs in the absence of granule exocytosis,
questioning the proposed model of Kojima et al.
Fas (CD95), the binding partner for Fas Ligand, is present on a wide range of normal tissues
such as liver as well as on malignant cells. Cytotoxic T-lymphocytes (CTL), in the absence of
perforin, granule exocytosis and calcium, were shown to be cytotoxic against L1210 cells
transfected with Fas, providing early evidence for a ligand mediated killing pathway.(Rouvier,
Luciani et al. 1993) Fas Ligand binding of Fas leads to trimerization of Fas, which causes
aggregation of death domains in the cytoplasmic region of the receptor, and subsequent
30
signalling via the adaptor molecule FADD (Fas associated death domain), ultimately leading to
apoptosis.
TRAIL is also expressed by NK and CTL, and depends on FADD-dependent signalling(Kuang,
Diehl et al. 2000). In addition to the apoptosis-signalling TRAIL receptors, DR4 and DR5, there
are receptors that lack a functional death domain, thought to serve as a ‘decoy’ mechanism to
regulate TRAIL-mediated apoptosis. TRAIL is also upregulated on NK cells following exposure
to IL-2, IL-15 and IFNs.(Smyth, Cretney et al. 2005; Smyth, Swann et al. 2005)
The physiological function of apoptosis by death ligands and receptors primarily functions as
mechanism of homeostatic regulation of lymphocyte and other hematopoietic cell populations.
The generation of FasL (gld) and Fas (lpr) double-knockout mice has demonstrated the
important role of death-receptor mediated pathways of cytotoxicity (Watanabe-Fukunaga,
Brannan et al. 1992; Takahashi, Tanaka et al. 1994). FasL -/- (gld) and Fas -/- (lpr) mice
develop autoimmune nephritis and other features analogous to human systemic lupus
erythematosus (SLE) (Matiba et al., 1997). The lymphoproliferation and autoantibody
production displayed in the lpr and gld mice confirms their role in the control and depletion of
‘self’ reactive lymphocytes. Fas receptor mutations occur in humans as well, leading to an
autosomal lymphoproliferative disorder(Fisher, Rosenberg et al. 1995; Rieux-Laucat, Le Deist et
al. 1995).
TRAIL-deficient mice were generated and had a more rapid onset of fibrosarcoma formation in
response to exposure to MCA, a carcinogen. Further, infusion of TRAIL-blocking antibodies
into wildtype mice revealed an anti-metastatic role for this receptor in the Renca tumour
metastases.(Cretney, Takeda et al. 2002) Also, TRAIL deficient mice have less ability to
mediate graft-versus-tumour effects in experimental murine models of transplantation.(Schmaltz,
Alpdogan et al. 2002)
TRAIL is constitutively expressed on murine NK cells in the liver and can suppress tumor
metastasis. Administration of anti-TRAIL blocking antibody increased experimental liver
metastases of TRAIL-sensitive tumor cell lines. This anti-metastatic effect of TRAIL was not
observed in NK cell-depleted or IFN-γ-deficient mice, which lack TRAIL on NK cells derived
31
from the liver.(Takeda, Hayakawa et al. 2001) NK cell control of Renca carcinoma hepatic
metastases in the liver was partially TRAIL-dependent as evidenced by administration of IL-12
which upregulated TRAIL expression on liver, spleen, and lung NK cells.(Smyth, Cretney et al.
2001)
1.4.2.3 NK cell recognition receptors
1.4.2.3.1 NK adhesion molecules
Prior to engagement of formal recognition receptors, NK cells must adhere to cells that they are
probing for abnormal cell surface antigens. This is accomplished by several receptors shared
with other lymphocytes including CD2, CD11a, CD18, CD54 and CD58. Resting human NK
cells express, lymphocyte function antigen-1 (LFA-1) (CD11a/CD18), LFA-3 (CD58) and
intercellular adhesion molecule-1 (ICAM-1)(CD54) which increase after incubation with IL-2.
Increases in NK cell adhesion molecule expression was associated with enhanced formation of
E:T cell conjugates and cytotoxicity, which could be partially inhibited by blocking CD2,
CD11a, or CD54 with specific antibodies.(Robertson, Caligiuri et al. 1990)
1.4.2.3.2 NK cell inhibitory receptors
NK cells express a variety of inhibitory receptors that regulate their cytotoxicity against normal
tissue. There are two major families of MHC-specific inhibitory receptors in humans. The first
is the Ig superfamily, which includes the killer immunoglobulin Ig-like receptors (KIRs) and
leukocyte Ig-like inhibitory receptors (LIRs). The second is the C-type lectin-like receptor
superfamily (Carretero et al., 1998; Colonna and Samaridis, 1995; Raulet et al., 2001). Each NK
cell expresses multiple receptors in various combinations, which leads to subpopulations of NK
cells able to detect the loss of MHC class I proteins (Gazit et al., 2004; Moretta et al., 1996). The
major HLA class I-specific NK inhibitory receptors in humans are listed in Table 1.2.
32
Table 1.2: NK cell inhibiting receptors
Name(s) CD marker Ligands
KIR
KIR2DL1 CD158a HLA-C2
KIR2DL2 CD158b HLA-C1
KIR2DL3 CD158b2 HLA-C1
KIR2DL4 CD158d HLA-G
KIR3DL1 CD158e2 HLA-Bw4
KIR3DL2 CD158k HLA-A3, HLA-A11
KIR2DL5 CD158f Unknown
LIR
LIR-1, ILT-2 CD85j HLA-A, -B, -C,-G, hCMV UL18
LIR-2, ILT-4 CD85d HLA-G
C-Type lectin-like
NKG2A/CD94 CD159a/CD94 HLA-E
NKR-P1A CD161 LLT1 (CLEC2D)
1.4.2.3.2.1 Inhibitory killer immunoglobulin like receptors
The KIRs are type I transmembrane glycoproteins that are classified into two groups based on
whether there are two (KIR2D) or three (KIR3D) Ig-like domains in the extracellular region of
the protein(Moretta, Bottino et al. 1996), and possess either inhibitory or activating functions.
Inhibitory KIRs have a long cytoplasmic tail with an immunoreceptor tyrosine-based inhibitory
motif (ITIM) that delivers an inhibitory signal. The consensus sequence for an ITIM is:
V/L/IxYxxL (where V= valine, Y= tyrosine, L= leucine, I= isoleucine, and x= any amino acid).
Following KIR binding to its cognate ligand tyrosine residues in the ITIM become
phosphorylated and recruit Src homology region 2-containing protein tyrosine phosphatase
(SHP)-1and SHP-2(Burshtyn, Scharenberg et al. 1996; Olcese, Lang et al. 1996). Further work
on KIR2DL1 signalling demonstrated that β-Arrestin 2(Yu, Su et al. 2008; Bari, Bell et al. 2009)
first binds to the phosphorylated ITIM and subsequently recruits SHP-1 and SHP-2. These
phosphatases prevent or interfere with activating signals. KIR2DL1 recognizes class I HLA-C2
group and KIR2DL2 and 3 recognized class I HLA-C2 group. KIR3DL1 recognizes HLA-Bw
group and KIR3DL2 recognizes HLA A3 and A11.(Lanier 2005) The specificity of KIR2D
receptors is dependent on the presence of either a K or N residue at position 80 of the HLA-C
molecule.(Mandelboim, Reyburn et al. 1996)
33
1.4.2.3.3 Leukocyte immunoglobulin-like inhibitory receptors
The leukocyte Ig-like inhibitory receptors (LIRs) have 13 family members with homology to
LIR1, an inhibitory receptor expressed on NK cells. LIR1 receptor contains four cytoplasmic
ITIMs that delivers inhibitory signals upon binding to a conserved region in MHC class I
proteins and the human cytomegalovirus class I homolog (UL18). The crystal structure of LIR-1
reveals two immunoglobulin-like domains resembling the basic structure of KIRs, but with a
different structural binding domain. (Chapman, Heikema et al. 2000). LIR-1 is able to recognize
a large range of MHC class I proteins including HLA-A, HLA-B and HLA-C with most efficient
binding to HLA-G(Colonna, Navarro et al. 1997) (Colonna, Nakajima et al. 1999).
1.4.2.3.4 C-type lectin-like receptors
CD94 and NKG2 family proteins (NKG2A, NKG2C, and NKG2E) are all type II integral
membrane glycoproteins containing C-type, carbohydrate recognition domains which covalently
assemble together to form heterodimers and recognize HLA class I allotypes(Lazetic, Chang et
al. 1996). These receptors are expressed on NK cells and a subset of CTLs (Carretero et al.,
1998). CD94 can associate with NKG2A (which has an ITIM), creating an inhibitory receptor
that binds to HLA-E (Lee, Llano et al. 1998) NKR-P1A (CD161) is a C-type lectin-like receptor
initially identified on rat NK cells and then later discovered to be expressed on human NK cells
(Lanier, Chang et al. 1994). This initial report was unable to confirm the definitive function of
NKR-P1A. A subsequent study of NKR-P1A using a rat NK cell line demonstrated activating
function against tumour targets.(Ryan, Niemi et al. 1995) However, a study of human NKR-P1A
demonstrated that lectin-like transcript-1(LLT1) was a natural ligand and LLT1 expressing
targets inhibited NK cell cytotoxicity via NKP-P1A supporting that it was an inhibitory receptor
in humans.(Aldemir, Prod'homme et al. 2005; Rosen, Bettadapura et al. 2005; Rosen, Cao et al.
2008)
1.4.2.4 NK cell activating receptors
NK cells also have a number of activating receptors that recognize ligands on target cells that can
lead to signal transduction, degranulation and target cell death. Direct evidence for the existence
of NK activating receptors was first provided by the development of monoclonal antibodies that
34
could blocked the NK cell-mediated cytotoxicity.(Sivori, Vitale et al. 1997; Pessino, Sivori et al.
1998; Vitale, Bottino et al. 1998; Pende, Parolini et al. 1999)
In humans, the major NK activating receptors include NKG2D(Houchins, Yabe et al. 1991;
Pende, Cantoni et al. 2001), CD16(Mandelboim, Malik et al. 1999) and the natural cytotoxic
receptors, which include NKp46(Sivori, Vitale et al. 1997), NKp44(Vitale, Bottino et al. 1998)
and NKp30(Pende, Parolini et al. 1999). Molecular cloning of the NCRs reveals that they share
no homology with each other or other human proteins.(Moretta et al., 2002). Resting human
NK cell activation has been studied using insect cells transfected with various individual and
combinations of ligands, demonstrating that different receptors are required for adhesion,
polarization, degranulation with some being activating, co-activating or co-
stimulating.(Bryceson, March et al. 2006) Activating receptors signal via an adaptor protein
containing an immunoreceptor tyrosine based activation motif (ITAM), such as CD3ζ, FcεRIγ,
DAP10 and DAP12.(Lanier 2003) The consensus sequence for an ITAM is: YxxL/I(x)6-
8YxxL/I (where Y= tyrosine, L= leucine, I= isoleucine and x= any amino acid) The major NK
activating receptors in humans and their ligands are summarized (Table 1.3).
35
Table 1.3: NK cell activating receptors
Name CD marker Ligand(s)
Activating
NKp30 CD337 heparan sulfate proteoglycans, BAT3 (BAG-6), B7-H6
NKp44 CD336 Influenza hemagluttinin
NKp46 CD335 Influenza hemagluttinin, heparan sulfate proteoglycans
DNAM-1 CD226 CD112, CD155
FcγRIII CD16 IgG (Fc portion)
NKG2C CD159c HLA-E
NKG2D CD314 MICA, MICB, ULB-1, -2, -3, -4, -5, -6, Raet-1
KIR2DS1 CD158h HLA-C2
KIR2DS4 CD158i HLA-Cw4
LEU-9 CD7 SECTM1, Galectin
Hyaluronate receptor CD44 Hyaluronan
BY55 CD160 HLA-C
Lag3 CD223 HLA Class II
2B4 CD244 CD48
CRACC CD319 CRACC
NTB-A CD352 CD352, SH2D1A, SAPPTN6, PTN1
Adhesion and
activating
Protectin CD59 C8, C9
Mac-1 CD11b ICAM-1, Fibrinogen
LFA-1 CD11a ICAM-1,-2,-3,-4,-5
LFA-2 CD2 CD58 (LFA-3)
CLEC2C CD69 Unknown
TACTILE CD96 CD155
1.4.2.4.1 NKG2D
NKG2D is a type II transmembrane glycoprotein expressed on the surface of most human and
murine NK cells with some expression on CD8+ and γδ T-cells (Eagle and Trowsdale 2007).
NKG2D has little homology to the other NKG2 gene family members (ie. A and C) and does not
form a heterodimer with CD94. Initially, NKG2D was shown to recognize the stress inducible
protein MICA, facilitating cytotoxicity by NK and T-cells.(Bauer, Groh et al. 1999)
Engagement of NKG2D activates NK cells independent of other activating stimuli (Jamieson et
al., 2002; Pende et al., 2001), leading to signalling via the adaptor molecules DAP10 (human and
murine) or DAP12 (murine), and subsequent cytotoxic response(Rosen, Araki et al. 2004). One
report has shown some capacity for human NK cells to signal via DAP12.(Karimi, Cao et al.
2005) NKG2D has several ligands identified including MICA, MICB and ULBP1–6.(Eagle and
Trowsdale 2007; Eagle, Traherne et al. 2009) These proteins have structural homology to MHC
36
class I proteins and can be upregulated on cells in stress conditions. NKG2D ligands are also
induced in response to DNA damage, which can arrest cell cycle and activate DNA repair,
including in cancer cells with unstable genomes.(Gasser, Orsulic et al. 2005) NKG2D ligands
have been demonstrated on some tumours(Groh, Rhinehart et al. 1999; Diefenbach, Jamieson et
al. 2000). In particular, NKG2D has been implicated in the recognition of leukemic
blasts.(Diermayr, Himmelreich et al. 2008)
1.4.2.4.2 NKG2C
NKG2C is an activating receptor of the NKG2 family (Lazetic, Chang et al. 1996), which
associates with CD94 to form a functional resceptor, and utilizes DAP12 for signalling.(Lanier,
Corliss et al. 1998) HLA-E is the natural ligand of NKG2C(Braud, Allan et al. 1998). The
contribution of NKG2C to tumour recognition has not been clearly defined.
1.4.2.4.3 Natural cytotoxicity receptors
The natural cytotoxicity receptors (NCRs) are members of the Ig superfamily that associate with
ITAM-bearing adaptor molecules. The NCRs are involved in recognition and killing of tumour
cells and when blocked by monoclonal antibodies, reduce cytotoxicity of NK cells.(Sivori, Vitale
et al. 1997; Pessino, Sivori et al. 1998; Vitale, Bottino et al. 1998; Pende, Parolini et al. 1999)
NKp46 and NKp30 are expressed exclusively on activated and resting NK cells, while NKp44 is
upregulated after activation(Fuchs, Cella et al. 2005).
NCRs have also been linked to clinical outcome in AML, where it has been demonstrated that
many AML patients have an NCRdull
NK cell immunophenotype with lower levels of NKp30,
and NKp46, which can be reversed following successful therapy.(Fauriat, Just-Landi et al. 2007)
Further, this group demonstrated that AML blasts can directly interact with NK cells to reduce
NCR expression, demonstrating a form of immune evasion. Analysis of their cohort of 71
patients showed that patients with NK cell immunophenotypes predicted survival. Patients with
NKp30 dull vs bright NK cell populations had a 33% versus 60% overall survival, while for
NKp46 dull versus bright subgroups it was 28% versus 72%.
37
1.4.2.4.3.1 NKp30
NKp30 was identified by Pende et al. in 1999 through the generation of novel murine anti-
human anti-NK cell antibodies and screening them in a reverse ADCC assay using the FcγRII
expressing P815 cell line.(Pende, Parolini et al. 1999) The gene is located in the class III major
histocompatibility complex.(Gruen and Weissman 2001) NKp30 is a 30 kDa glycoprotein that
contains one V-type Ig-like extracellular domain capable of recognition and cytoxicity of targets
that are relatively resistant to NKp46/44-mediated killing.(Pende, Parolini et al. 1999) NKp30
and NKp46 are typically co-expressed on NK cells. NKp30 has 13% identity and 15% similarity
to NKp46.(Pende, Parolini et al. 1999)
NKp30 is selectively expressed on all human NK cells (Pende et al., 1999) except those present
in the lymph nodes and in the endometrium during the menstrual cycle(Manaster, Mizrahi et al.
2008). The transmembrane portion of NKp30 contains an arginine residue, which is probably
involved in the association with CD3ζ chains for the transduction of the downstream activating
signals (Pende et al., 1999). NKp30 recognizes heparan sulfate proteoglycans (HSP) similar to
NKp46.(Bloushtain, Qimron et al. 2004) Subsequently, The nuclear factor HLA-B-associated
transcript 3 (BAT3) was demonstrated to be a ligand of NKp30 required for tumor rejection in a
multiple myeloma model.(Pogge von Strandmann, Simhadri et al. 2007) A fragment of BAT3
(amino acids 686-936) was subsequently shown to be constitute a subdomain that was essential
and sufficient to inhibit NKp30-mediated NK cell cytotoxicity.(Binici, Hartmann et al. 2013)
Another ligand for NKp30 was subsequently identified as a B7-H6, a B7 family
member.(Brandt, Baratin et al. 2009) Shedding of B7-H6 has been shown to serve as mechanism
of immune tumour evasion from NK cell-mediated killing.(Schlecker, Fiegler et al. 2014)
1.4.2.4.3.2 NKp44
NKp44 is the second NCR identified on human NK cells encoding a 44Dka surface glycoprotein
involved in cytotoxicity against MHC class I-deficient targets.(Vitale, Bottino et al. 1998;
Cantoni, Bottino et al. 1999) The simultaneous blocking of both NKp44 and NKp46 led to a
significantly increased inhibition of NK cell cytotoxicity.(Vitale, Bottino et al. 1998)
Engagement of NCRs with mAbs demonstrated NKp46 and NKp30 signalling via CD3ζ and
NKp44 signalling via DAP12(Vitale, Bottino et al. 1998; Cantoni, Bottino et al. 1999) converged
on a common signalling pathway that was distinct from that of CD16 or KIR2DS4, and was
38
inhibited by engagement of CD94/NKG2A.(Augugliaro, Parolini et al. 2003) NKp44 is not
expressed on resting NK cells, but requires activation for its expression.(Vitale, Bottino et al.
1998) NKp44 recognizes viral hemagglutinnins(Arnon, Lev et al. 2001; Mandelboim, Lieberman
et al. 2001; Mandelboim and Porgador 2001) such as influenza hemagglutinin which directly
binds and activates NK cell cytotoxicity(Arnon, Lev et al. 2001; Arnon, Achdout et al. 2004)
1.4.2.4.3.3 NKp46
NKp46 is a 46 kDa glycoprotein with two C2 immunoglobulin-like domains expressed on
resting and activated human NK cells only, and when crosslinked leads to calcium mobilization,
cytotoxicity and cytokine release.(Sivori, Vitale et al. 1997; Pessino, Sivori et al. 1998) The
crystal structure of NKp46 reveals similarity to the LIR1 and KIR2D receptors.(Foster, Colonna
et al. 2003) Signalling of NKp46 is mediated via the association with the ITAM containing
adaptor molecules CD3ζ and FcεRγ.(Lanier 2003) NKp46 recognizes hemagglutinin molecules
of different influenza strains(Arnon, Lev et al. 2001; Mandelboim, Lieberman et al. 2001; Arnon,
Achdout et al. 2004). NKp46 has further been shown to recognize heparan sulfate proteoglycans
(HSP).(Bloushtain, Qimron et al. 2004)
1.4.2.4.4 NKp80
NKp80 is an 80 kDa activating homodimeric C-type lectin-like type II transmembrane protein
(similar to NKG2D) shown to stimulate NK cell cytotoxicity and induces calcium influx in
human NK cells after triggering by specific antibodies (Vitale, Falco et al. 2001). NKp80 signals
via a hemi-ITAM-like sequence with an essential tyrosine at position 7, resulting in Syk
phosphorylation required for cytotoxic responses.(Dennehy, Klimosch et al. 2011) Activation-
induced C-type lectin (AICL) was identified as a ligand of NKp80 and is expressed by
monocytes, macrophages and granulocytes.(Hamann, Montgomery et al. 1997) NKp80 mediates
NK cell cytolysis of malignant myeloid cells expressing AICL.
1.4.2.4.5 CD16
CD16 (FcγRIIIa) is a type I transmembrane receptor containing two extracellular Ig-like
domains. Most human NK cells express CD16(Trinchieri and Valiante 1993), which can
facilitate antibody-dependent cellular cytotoxicity (ADCC) by NK cells. CD16 signals via
association with the adaptor molecules CD3ζ and FcεRIγ, which contain activation ITAM
39
motifs(Vivier, Rochet et al. 1991). The primary function of CD16 is to bind to the Fc portion of
IgG molecules that have opsonised cellular targets (Figure 1.4). NK cell cytotoxicity can be
specifically activated upon binding to Fcγ of IgG. This molecule therefore serves to bridge both
humoral and innate immune systems, allowing NK cells to co-ordinate with an antibody response
to a foreign antigen. CD16 was reported to be the most potent activating receptor on freshly iso-
lated human NK cells, able to elicit strong cytotoxicity and cytokine production signalling via
phospholipase-C-gamma and phosphatidylinositol-3-kinase, leading to degranulation.(Bryceson,
March et al. 2006) CD16 has also been shown to exert cytotoxicity against virally infected cells
and tumor cells, independent of its antibody binding capacity. In addition to IgG Fc specificity,
a cell surface CD16-specific ligand has been demonstrated through use of a CD16-Ig fusion
protein assay system, but this molecule is yet to be isolated and identified.(Mandelboim, Malik et
al. 1999)
Figure 1.4: NK cell antibody dependent cell mediated cytotoxicity
Engagement of the Fc portion of a target bound antibody by Fcγ receptor IIIA (CD16) on NK
cells leads to signal transduction and granule exocytosis.
40
1.4.2.4.6 Activating killer immunoglobulin like receptors (KIRs)
There are six genes encoding for the KIRs clustered on chromosome 19(Lanier 2005) that lead to
an activating signal when bound to class I HLA(Moretta, Sivori et al. 1995) Activating KIRs
are type I transmembrane glycoproteins that consist of either two (KIR2D) or three (KIR3D)
extracellular C2-type Ig-like domains. However, they possess a charged amino acid in their
transmembrane domain and contain a short cytoplasmic tail, without any known signalling motif.
(Biassoni, Cantoni et al. 1996) Instead, they are associated (via the charged amino acid) with the
ITAM-containing signalling protein, DAP12. KIR2DS1 recognizes HLA-C2 while KIR2DS2
recognizes HLA-C1. KIR2DS4 recognizes HLA-C and a non-MHC class I protein expressed on
melanomas, (Katz, Gazit et al. 2004) resulting in enhanced NK killing. KIR3DS1 recognizes
HLA-B Bw4 while KIR2DS3 and KIR2DS5 have unknown ligands.
1.4.2.4.7 2B4
2B4 is an activating cell surface glycoprotein related to CD2 found on human NK and T-
cells(Lanier 2005), whose ligand was shown to be CD48(Brown, Boles et al. 1998). It contains
two immunoglobulin-like external domains and its cytoplasmic tail contains four
immunoreceptor tyrosine-based switch motifs (ITSM). The ITSM consensus sequence is:
TxYxxV/I (where T= threonine Y= tyrosine, V= valine, I= isoleucine and x= any amino acid).
The ITSM motif defines a family of receptors sharing a common signalling pathway and
includes the NTB-A and CRACC receptors that are also expressed by NK cells and are also able
to activate their killing.(Veillette 2006)
Upon phosphorylation of the tyrosine in the ITSM motif, 2B4 binds to the SAP or EAT-2(Perez-
Quintero, Roncagalli et al. 2014), intracellular adaptor proteins, or to the Src homology 2
domain-containing protein tyrosine phosphatase (SHP)-1 and SHP-2 tyrosine phosphatases. 2B4
is capable of triggering effector cell functions when bound by antibody or its natural ligand
CD48.(Nakajima, Cella et al. 1999; Sivori, Parolini et al. 2000) Further, transfection of CD48
into NK-resistant target cells can render them sensitive to NK cell-mediated cytotoxicity and
lead to secretion of IFN-γ following effector and target cell contact. (Tangye, Cherwinski et al.
2000) SAP mutations in humans leads to dysfunctional 2B4 mediated activation of NK cells and
41
switching the receptor to an inhibitory function(Nakajima, Cella et al. 2000; Parolini, Bottino et
al. 2000), leading to the X-linked lymphoproliferative disorder.(Nakajima and Colonna 2000)
1.4.2.4.8 DNAM-1
DNAM-1 (CD226) is a member of the Ig superfamily, is expressed by human NK cells, T cells, a
subset of B cells, monocytes and platelets,(Lanier 2005) and recognizes CD155 (polio virus
receptor) and CD112 (nectin-2).(Bottino, Castriconi et al. 2003) Interactions between DNAM-1
on NK cells and its ligands on tumour targets facilitate NK cell–mediated cytotoxicity and
cytokine production.(Bottino, Castriconi et al. 2003; Tahara-Hanaoka, Shibuya et al. 2004)
Moreover, it was shown that the interaction of DNAM-1 with CD112 and CD155 contributes to
the NK-mediated lysis of dendritic cells (Pende et al., 2006).
1.4.3 NK cell lines
Eleven true NK cell lines have been established(Drexler 2010): HANK-1 (Nasal-type NK/T-cell
lymphoma)(Kagami, Nakamura et al. 1998), IMC-1, KHYG-1 (Aggressive NK-cell leukemia)
(Yagita, Huang et al. 2000), MEC04, NK-92 (NHL with LGL cells)(Gong, Maki et al. 1994),
NK-Y (Nasal NK-cell lymphoma)(Tsuchiyama, Yoshino et al. 1998) NKL (NK-LGL
leukemia)(Robertson, Cochran et al. 1996), SNK-1, SNK-6 (Nasal NK/T-cell
lymphoma)(Nagata, Konno et al. 2001), SRIK-NKL and YT (ALL + thymoma) 1983(Yodoi,
Teshigawara et al. 1985). Six of these NK cell lines were further characterized by Drexler et al.
in 2000 (HANK1, KHYG-1, NK-92, NKL, NK-YS and YT) (Drexler and Matsuo 2000)
showing that five of these cell lines were IL-2-dependent with a common immunophenotype:
CD1-, CD2+, CD3-, CD4-, CD5-, CD7+, CD8-, CD16-, CD56+, CD57-, T-cell receptor (TCR)-.
It is notable that transformation led to a loss of CD16 expression in all these cell lines which is a
hallmark of endogenously derived NK cells that allows for antibody-dependent cell-mediated
cytotoxicity (ADCC). SNK-6 was later characterized by Matsuo et al. as another bona fida NK
cell line.(Matsuo 2003) Using a calcein cytotoxicity assay, only NK-92, KHYG-1 and SNT-8
showed significant cytotoxicity against K562 targets.(Matsuo 2003) Thirty-five cell surface
markers were assessed on these seven NK cell lines to establish a common profile and variations
between NK cell lines.(Matsuo 2003) KHYG-1 was the only NK cell line notably positive for
CD8 and strongly positive for CD158a (KIR2DL1) and CD158b (KIR2DL2).
42
1.4.3.1 NK-92
NK-92 is an IL-2 dependent cell line derived from a patient with a NK-cell non-Hodgkin
lymphoma with a CD56+CD3-CD16- immunophenotype, which retained cytotoxic antitumour
activity, consistent with activated NK cells(Gong, Maki et al. 1994). Potential use of NK-92 as a
purging agent in preparing autologous transplant products had been conducted using a neomycin-
resistant K562 line to spike normal PBMCs with 10% K562 and then incubate with NK-92 and
measure colony growth in methylcellulose.(Klingemann, Wong et al. 1996). NK-92 was highly
cytotoxic against K562 CFU. It was further shown that irradiation of NK-92 with 1000 cGy
inhibited proliferation, but no significant impact on cytotoxicity.(Klingemann, Wong et al. 1996)
Also, cytotoxicity of these irradiated cells persisted for 2 days after IL-2 deprivation, but then
rapidly decreased. Finally, NK-92 had no impact on CFU-GEMM, BFU-E or CFU-C from
normal hematopoietic cells.
NK-92 has been demonstrated to have enhanced cytotoxicity over endogenously derived NK
cells against a variety of human leukemia cell lines and primary leukemic blasts in vitro (Yan Y
et al. 1998). Further, non-irradiated NK-92 can impact survival in a primary AML xenograft
SCID model, which can be enhanced by the administration of IL-2 to mice. Non-irradiated NK-
92 cells (20x106 x 5 doses) were not able to engraft in SCID mice or cause disease.
1.4.3.2 Gene-modified variants of NK-92
Several gene modified variants of NK-92 have been generated to enhance its function as a
therapeutic agent or for basic studies of NK cell function. The approach to introduction of new
genes has been either from electroporation or lentiviral transduction. These include low and high
affinity CD16+, IL-2, KIRs and chimeric antigen receptors.
1.4.3.3 CD16+NK-92
NK cells typically express CD16 and are able to engage in antibody-dependent cell-mediated
cytotoxicity (ADCC) against antibody-coated targets ,allowing for a bridge between the adaptive
and innate immune responses. NK-92 lost CD16 expression during malignant transformation,
making it incapable of ADCC. Two different major CD16 alleles occur in humans, a low (176F)
and high (176V) affinity, each with a particular amino acid at position 176. The distribution of
43
CD16 alleles in the populations is is 0.56 (176F) and 0.44 (176V) yielding a genotypic
distribution of 61% V/F, 26% F/F and 13% V/V.(Wu, Edberg et al. 1997)
However, gene modified variants of NK-92 were generated using a retroviral system (pBMN-
IRES-EGFP vector), whereby both the low and high affinity Fc gamma receptor (CD16) were
transduced into parent line NK-92 with and without green fluorescence protein (GFP).
Individual clones have different integration sites of the retroviral expression vector. These
CD16+NK-92 cells expressing either low (NK-92.176F and NK-92.176F.GFP) or high (NK-
92.176V and NK-92.176V.GFP) affinity CD16 have demonstrated ADCC in vitro against
CD20+ targets treated with Rituximab.(Binyamin, Alpaugh et al. 2008) However, no evidence
of in vivo efficacy has been demonstrated.
1.4.3.4 KHYG-1
Another NK cell line with therapeutic potential is KHYG-1 which was originally derived from a
patient with an NK cell leukemia with a p53 mutation.(Yagita, Huang et al. 2000) Furthermore,
KHYG-1 has a high degree of cytotoxicity against tumour targets over a range of culture
conditions, mediated in part by constitutively phosphorylated ERK2, granzyme M release(Suck,
Branch et al. 2005), and constitutively polarized granules.(Suck, Branch et al. 2006) Like NK-
92, irradiation of KHYG-1 prevents proliferation, but preserves cytotoxicity(Suck 2006), making
it a potential therapeutic agent for the treatment of cancer. A CD16+ gene modified KHYG-1
has been generated which can mediate ADCC against CD20+ target in combination with
Rituximab as low as 0.1 µg/ml).(Kobayashi, Motoi et al. 2014)
1.5 Antibody therapy for cancer
The discovery of a means to generate murine monoclonal antibodies by George Köhler and
César Milstein garnered the 1984 Nobel Prize in Medicine and paved the way for a new class of
therapeutics.(Kohler and Milstein 1975) Monoclonal antibody therapy has transformed therapy
for numerous diseases, including cancer. Initial antibodies were murine (suffix -omab) and of
limited therapeutic value, because they were rejected as foreign antigens. Chimeric
murine/human antibodies reduced the immunogenicity (suffix -ximab), but still had issues with
rejection due to residual murine elements in the antibody variable regions. Humanized
antibodies (suffix -zumab) further modified the antibodies to have a greater percentage of human
sequences, excepting the complementary determining regions. Fully human antibodies (suffix -
44
umab) of a given specificity were subsequently developed using transgenic mice expressing
human immunoglobulin genes and phage display technology. Rituximab (anti-CD20 chimeric
antibody) was the first monoclonal antibody approved for use in cancer and tested
experimentally in a clinical trial for lymphoma in 1998.(McLaughlin, Grillo-Lopez et al. 1998)
NK cells play a major role in the therapeutic activity of humanized monoclonal antibodies, such
as rituximab (anti-CD20) or alemtuzumab (anti-CD52), because ADCC is the principal
mechanism of action of these mAbs (Bowles, Wang et al. 2006) There are now 30 FDA
approved therapeutic monoclonal antibodies, twelve specific to cancer therapy and four with
ADCC as a major mechanisms of action (Table 1.1).(Scott, Allison et al. 2012)
45
Table 1.4: FDA approved therapeutic monoclonal antibodies for cancer*
Antibody name Target Disease Mechanism
Generic Trade
Trastuzumab Herceptin ERBB2 Breast cancer Inhibition of ERBB2 signalling and
ADCC
Bevacizumab Avastin VEGF Colon cancer Inhibition of VEGF
Cetuximab Erbitux EGFR Squamous cell
carcinoma
Inhibition of EGFR signalling and
ADCC
Panitumumab Vectibix EGFR Colon cancer Inhibition of EGFR signalling
Ipilimumab Yervoy CTLA4 Inhibition of CTLA4 signalling
Rituximab Mabthera CD20 Lymphoma ADCC, direct induction of apoptosis
and complement dependent
cytotoxicity
Alemtuzumab Campath CD52 B-cell ALL Direct induction of apoptosis and
complement dependent cytotoxicity
Ofatumumab Arzerra CD20 CLL ADCC and complement dependent
cytotoxicity
Gemtuzamab
ozogamicin
Mylotarg CD33 AML Toxic conjugate calicheamicin
Brentuximab
vedotin
Adcetris CD30 Hodgkin’s
lymphoma
Toxic conjugate auristatin
90Y-labelled
ibritumomab
Zevalin CD20 Follicular B-cell
lymphoma
Radioconjugate 90
Y
131I-labelled
tositumomab
Bexxar CD20 Follicular
lymphoma/NHL
Radioconjugate 131
I
*Adapted from Table 3, Scott et al. Nat Rev Cancer 12:284 (Scott, Allison et al. 2012)
Other non-FDA approved chimeric, humanized or fully human antibodies are at various stages of
preclinical or clinical development. The murine anti-human CD123 monoclonal antibody 7G3
has been modified into two humanized versions CSL360 and CSL362. CSL360 has the variable
region of 7G3. CSL362 has been Fc optimized to bind CD16A on NK cells with better affinity,
as well as affinity matured to better bind to CD123 by its variable region. CSL360 was tested in
a phase I clinical trial with 40 relapsed and refractory AML patients, with no major toxicities and
minimal therapeutic benefit in most patients, except two responses, one being a durable
remission (He, Busfield et al. 2014). CSL362 testing is ongoing in another phase I clinical.
Other antibodies in the development pipeline include Daratumomab (fully human anti-CD38
antibody) for multiple myeloma, Galiximab (chimeric anti-CD80 antibody) for B-cell lymphoma
and Lucatumumab (human anti-CD40 antibody) for treatment of multiple myeloma, non-
46
Hodgkin’s lymphoma and Hodgkin’s lymphoma. Given that monoclonal antibody therapy is
dependent on the innate cellular immune system in many cases, it is logical to consider means to
augment the efficacy of therapeutic monoclonal antibodies in combination with an ADCC-
capable cell therapy treatment.
1.6 Cell therapy for cancer
1.6.1 Allogeneic hematopoietic stem cell transplantation
Bone marrow transplantation or allogeneic hematopoietic stem cell transplantation (HSCT) was
pioneered by E. Donnell Thomas, garnering him the 1990 Nobel prize in medicine. In 1956, he
performed the first successful HSCT for leukemia from a twin donor. Given the syngeneic
nature of the transplant, there were no issues of graft rejection.(Thomas, Lochte et al. 1957)
Subsequently, six patients were treated with unmatched HSCT, after receiving radiation and
chemotherapy. These patients ultimately died, as there was no knowledge of the issue posed by
HLA incompatibility of randomly selected donors. Further work was done on allografting bone
marrow using dogs as a model system.(Ferrebee, Lochte et al. 1958) When HLA typing became
a possibility, the first treatments of leukemia using matched sibling donors was
conducted(Buckner, Epstein et al. 1970), followed by the first matched unrelated HSCT(Clift,
Hansen et al. 1979). This work paved the way for a novel immunotherapeutic approach that
remains today the definitive therapy for many advanced and refractory leukemias.
The benefit of allogeneic hematopoietic stem cell transplantation (HSCT) is greatest for chronic
myeloid leukemia(Arora, Weisdorf et al. 2009), with some benefit for AML(Reiffers, Gaspard et
al. 1989; Woods 2001; Litzow, Tarima et al. 2010) and slight benefit in ALL(Burke, Cao et al.
2009). However, HSCT remains the only effective immunotherapy for AML that can improve
survival and cure some relapsed and refractory AML patients. Further, patients who are deemed
high risk and some intermediate risk patients are typically treated with a HSCT after completion
of chemotherapy, provided that there is a suitable matched related (optimal) or unrelated donor
(less optimal). However, Only 40% of patients will have a matched sibling donor, limiting
consistent application of HSCT (Ruggeri et al 2005). Therefore, a significant number of high
risk AML patients requiring a bone marrow transplant will not have a suitable donor. These high
47
risk patients over 60 years of age without a matched donor for transplantation have a five-year
survival of approximately 5% (Frohling S et al., 2006).
The primary means by which an allogeneic HSCT treats leukemia is by application of high dose
chemotherapy and subsequent immunotherapeutic effect mediated by T-cells. This graft-versus-
leukemia effect (GVL) facilitates elimination of MRD due to alloreactivity of donor T-cells. One
other type of HSCT transplant involves a half-matched relative donor (haplotype transplant).
Without T-cell reduction, such a half-matched transplant would lead to fatal graft versus host
disease. However, by T-cell depleting and stem cell enriching the haplotype transplant product,
GVL can be minimized at the cost of increased infections post-transplant and less optimal
engraftment compared to a conventional HSCT. In this context, NK cells are able to mediate a
powerful GVL, even in the absence of T-cells. While GVL was initially thought to be
exclusively mediated by T cells recognizing tumour cells via major and minor histocompatibility
elements(Pierce, Field et al. 2001), there is growing evidence supporting an important role of NK
cells in mediating GVL(Ruggeri, Mancusi et al. 2005).
1.6.2 Adoptive immunotherapy
Adoptive immunotherapy involves the removal of cells from a patient, typically ex vivo
expansion in a sterile culture system and reinfusion into the patient. Typically, lymphocytes
derived from leukopheresis have been used and then cultured with various cytokines to enhance
the cytotoxicity of T and/or NK cells in the peripheral blood white cell populations. More
advanced protocols may enrich cell populations with magnetic cell sorting or gene modify the
cells. Several representative approaches to adoptive immunotherapy are described below.
1.6.2.1 Lymphokine activated killer (LAK) cells
The field of adoptive immunotherapy began with attempts to infuse peripheral blood derived
lymphocytes termed lymphokine activated killer (LAK) cells by Steven Rosenberg in
1985.(Rosenberg, Lotze et al. 1985) This involved development of ex vivo lymphocyte
expansion protocols using IL-2 containing growth medium to expand T and NK cell mixed
populations into a population of MHC-unrestricted LAK cells. The initial patients treated with
this approach had metastatic melanoma and some responses were seen, demonstrating proof-of-
principle. A larger randomized study of IL-2 with or without LAK cells showed a trend toward
48
increased survival in the IL-2 + LAK therapy group in metastatic melanoma patients
only.(Rosenberg, Lotze et al. 1993) LAK cells were tested in patients with AML post
autologous transplant, with or without IL-2, and extended remissions were obtained in small
numbers of patients.(Fefer, Benyunes et al. 1993) To improve on this approach, tumour
infiltrating lymphocytes (TILs) were harvested and expanded in a similar fashion ex vivo with
IL-2.
Figure 1.5: Schematic of adoptive immunotherapy using LAK cells
1.6.2.2 Tumour infiltrating lymphocytes (TIL)
Following the failure of LAK cell therapy to successfully treat metastatic melanoma patients,
TILs were explored as a potentially therapeutic cell population, initially in animal models of
cancer by Rosenberg et al.(Rosenberg, Spiess et al. 1986) This required the development of
techniques to homogenize solid tissue and extract lymphocyte populations and expand them ex
vivo with IL-2 in culture. The harvest approach was refined using patient-derived renal tumours,
and the subsequently generated TILs exhibited promising cytotoxicity.(Belldegrun, Muul et al.
1988) In a subsequent trial of TIL therapy in six patients metastatic melanoma, indium-111
labelled TILs were infused and demonstrated to home back to sites of tumour using serial whole
body gamma camera imaging (Fisher, Packard et al. 1989) Use of IL-2 alone and in combination
with LAK and TIL was evaluated, with some evidence of improvement in responses with IL-2
49
and TIL therapy but not with with LAK cell therapy.(Rosenberg, Lotze et al. 1989) More
recently, patients with refractory metastatic melanoma treated with a cytoreductive regimen
followed by TIL therapy had objective response rates 50-70% of cases. (Dudley, Yang et al.
2008) While not curative, this approach demonstrated the potential for cellular immunotherapy
to a impact metastatic solid malignancy.
Figure 1.6: Schematic of adoptive immunotherapy using TILs
1.6.2.3 Autologous NK cells
Adoptive immunotherapy with autologous NK cells derived from the patient has been dependent
on expansion protocols and purification techniques enabling immunotherapy with a relatively
pure NK cell population, distinguishing this approach from LAK cell therapy, where the
population originates from both NK and T cell populations in the peripheral blood. The
expansion process is similar to that for LAK cells (Figure 1.5) requiring IL-2 or IL-15, except
there is a T-cell depletion step. Autologous NK cell therapy was tested in a phase I trial with 11
metastatic colorectal cancer patients treated with 0.001-0.3x109 cells/dose iv for 1-4 doses per
cycle and up to 6 cycles with no toxicities noted.(Krause, Gastpar et al. 2004) In another trial,
seven metastatic melanoma and one metastatic renal cell carcinoma patients treated with NK cell
therapy consisting of 4.7x1010
cells in combination with chemotherapy had no toxicities noted,
50
but also no therapeutic effect.(Parkhurst, Riley et al. 2011) Three other trials of autologous NK
cell therapy for a variety of malignancies are in process [reviewed in(Cheng, Chen et al. 2013)].
1.6.2.4 Allogeneic NK cells
NK cells are able to recognize and destroy cancer cells, which have downregulated HLA class I
molecules on their surface, via lack of signaling from inhibitory KIRs, provided at least one
activating receptor is engaged by the target. Ruggeri et al. were the first to demonstrate that, in
the context of a haplotype HSCT, when there is a KIR ligand mismatch between donor NK cells
and recipient HLA, a potent GVL can occur leading to improved survival for AML
patients.(Ruggeri, Capanni et al. 2002) This prompted attempts to use haploidentical allogeneic
NK cells from related donors. The first trial of haploidentical NK cells was conducted by Jeffrey
Miller’s group at the University of Minnesota in 2005 for patients with AML.(Miller, Soignier et
al. 2005) They utilized two low-dose immunosuppressive regimens to facilitate in vivo
expansion of the transferred NK cells in the outpatient setting: low dose cyclophosphamide with
methylprednisolone and fludarabine. Poor prognosis AML patients received more intense
cyclophosphamide and fludarabine regimens prior to NK cell transfer. They were able to detect
expansion of NK cells, a rise in IL-15 levels, and induction of a complete response in 5/19 poor
prognosis patients. A phase I trial of haploidentical relative donor NK cell therapy for advanced
non-small cell carcinoma (16 patients) of the lung revealed no toxicity, two partial responses and
stabilization of disease in five patients.(Iliopoulou, Kountourakis et al. 2010) A trial at St. Jude
in pediatric patients with favourable or intermediate risk AML in remission was conducted using
lower doses of cyclophosphamide and fludarabine than the Minnesota group prior to
administration of haploidentical NK cells and lower doses of IL-2 to facilitate in vivo
expansion.(Rubnitz, Inaba et al. 2010) All patients were in remission at two years post therapy.
A phase II study of haploidentical NK cells for intermediate risk AML was initiated at St. Jude
Children’s research hospital and is ongoing. Another phase I trial of haploidentical relative
donor NK cell therapy for 14 ovarian and 6 breast cancer patients was undertaken, achieving
transient donor chimerism, but limited efficacy, possibly due to Treg stimulation by IL-2
administration.(Geller, Cooley et al. 2011)
51
1.6.2.5 Chimeric antigen receptor T-Cells
An evolutionary step in autologous adoptive immunotherapy has been the advent of chimeric
antigen receptor (CAR) expressing T-cells. CARs are antigen-specific receptors constructed
from the single chain variable fragment (scFv) of antibodies fused to the signalling domains of
the T-cell receptor ζ chain or FcRγ (termed first generation). The first functional CAR was
developed in 1989 (Gross, Gorochov et al. 1989) Subsequent CAR designs have included an
additional one (second generation), or two (third generation), co-stimulatory signalling domains
derived from CD28, 4-1BB or other similar molecule. CARs are then transduced into a patient’s
T-cells, typically by gamma retroviral(Scholler, Brady et al. 2012) or lentiviral(Biffi, Bartolomae
et al. 2011) transduction. Carl June’s research group developed a CD19 second generation CAR
with CD3ζ and 41BB signalling domains (Figure 1.7) used to generated CD19 CAR T-cells to
treat a CLL patient with refractory disease.(Porter, Levine et al. 2011) The patient went into
complete remission and developed a delayed tumour lysis syndrome and had a persistent B-cell
aplasia after the treatment. A follow-up study including this patient and two others confirmed
the efficacy of this approach with two achieving a complete remission with establishment of
memory CAR T-cells.(Kalos, Levine et al. 2011) A subsequent trial of CD19 CAR T-cell
therapy in five adults with refractory, MRD+ or MRD- ALL led to maintenance or conversion to
MRD- in all cases.(Brentjens, Davila et al. 2013) Four patients were subsequently treated with a
HSCT and one relapsed at 90 days. Another trial of CD19 CAR T-cells for eight patients with
follicular lymphoma, CLL and splenic marginal zone lymphoma demonstrated six partial
remissions, one complete remission and one patient with stable disease.(Kochenderfer, Dudley et
al. 2013) Four of these patients also developed B-cell aplasia.
52
Figure 1.7: Design of CD19 CAR utilized in a clinical trial for CLL
Plasmid map of the CD19 CAR construct incorporating a single-chain variable fragement (ScFv)
comprised of a variable heavy (VH) and light chain (VL) from an anti-CD19 mAb, with signaling
domains (4-1BB and CD3ζ) and hinge (human CD8α) region. The plasmid is transduced into
patients T-cells using a self-inactivating lentiviral vector creating antigen specific T-cells
targeting CD19 expressing cells. (Porter, Levine et al. 2011)
53
T-cell
Zeta chain + 41BB
CD 19
VL
VH
Linker
CLL cell
Figure 1.8: Schematic of CD19+ CAR T-cell recognition of CD19+ CLL cells
T-cells transduced with a CD19 specific chimeric antigen receptor can recognize and target
CD19+ targets such as CLL cells and normal B-cells.
Once a CAR T-cell product has been infused the cells can multiply to an unknown degree
limiting the significance of the starting dose and preventing modulation of cell proliferation and
activity once in circulation. Complications from CAR T-cells include a cytokine release
syndrome which can be fatal as reported for ERb2 second-generation CAR T-cell
therapy(Morgan, Yang et al. 2010) However, the cytokine release syndrome can be successfully
treated with anti-IL-6 antibody therapy, which can dampen the excess release of cytokines by
CAR T-cells. In addition, CD19 CAR T-cell therapy is not specific to leukemia cells and leads
to a permanent B-cell aplasia and state of hypogammaglobulinemia. This side effect can be
managed with life-long IV immunoglobulin treatments, but is a significant and undesired side
effect of targeting a tumour-associated antigen present on normal B-cells.
54
1.6.2.6 Cell lines
The ability to create cell lines from primary tumour samples has been critical to facilitate
experimental work leading to our current understanding of tumour biology and in the
development of effective cancer therapies. Malignant transformation can occur in the cells of the
immune system such as T or NK cells, with occasional retention of the ability to recognize and
lyse other tumour cells, making these rare cell lines potential therapeutic agents. There have
been two cell lines tested in clinical trials; T-ALL104(Visonneau, Cesano et al. 2000) and NK-
92(Arai, Meagher et al. 2008; Tonn, Schwabe et al. 2013). The current approach to use of cell
line therapy requires the maintenance of a master frozen cell bank, from which doses can be
expanded in a GMP facility and infused into the patient (Figure 1.9).
Figure 1.9: Schematic of adoptive immunotherapy using cell lines
55
The advantages of using a cell line versus other form of cellular immunotherapy is that they are
more standardized, expand easily with predictable kinetics and any gene modifications need only
be performed once. This is particularly important given that autologous cells from patients have
intrinsic variability and often come from patients who are immunosuppressed from
chemotherapy. Inter-patient variability in cell number, cytotoxicity and growth potential limit
current cellular immunotherapy strategies. Disadvantages include the potential for rejection of
allogeneic tissue, although there have been only occasional immune responses detectable against
NK-92. The potential for engraftment and inducing a malignancy is also present, but can be
abrogated by irradiated of cells. In the future, suicide gene systems can be incorporated into cell
lines to allow for in vivo expansion of the cells and subsequent induction of cytotoxicity. The
advantages and disadvantages of cell line therapy are listed in Table 1.5.
Table 1.5: Comparison of autologous cell therapy and cell line therapy
Issue Cell therapy approach
Autologous Cell line
Cell acquisition Variable, requires leukopheresis Expanded from frozen stock
Cell expansion Variable, expansion within ~3-6
weeks
Expansion within 2-3 weeks
Cytotoxicity Variable Less variable
Gene modification Labour intensive- done for each
patient
Done once
Rejection Low potential High potential, but not observed
Malignancy risk Low potential. Transduction of
CAR T-cells has low risk of
transformation
Some potential- obviated by
irradiation in current protocols and in
the future potentially by suicide gene
insertion
In vivo expansion Yes, limited by homeostatic
regulation of lymphocytes, but
can be overcome by
preconditioning with
chemotherapy.
No, current protocols irradiated cells
preventing in vivo expansion. Could
be done with cells that are either
cytokine dependent or transduced
with suicide genes.
Memory Yes, CAR T-cell therapy can
lead to immunologic memory.
Unclear with other forms of
autologous cell therapy such as
LAK and TIL or NK.
No, cell lines cannot form memory
cells.
56
1.6.2.6.1 T-ALL104
The first attempt to utilize cell lines as therapeutic agents was with cell line, TALL-104 derived
from a patient with T-cell acute lymphoblastic leukemia which had the ability to kill a wide
range of tumour targets in an MHC unrestricted manner(Cesano and Santoli 1992). TALL-104
was tested in a phase I study of fifteen patients with metastatic breast cancer (infiltrating ductal,
lobular or medullary carcinoma) (Visonneau, Cesano et al. 2000). In this study, lethally
irradiated cells were utilized at doses ranging from 106 to 10
8 cells/kg daily for five days
(induction). Nine patients had progression and were taken off study, while the remainder with
stable disease received monthly two day doses (maintenance) for up to six months. Most
toxicities were limited to grade I/II, with one patient experiencing grade IV liver toxicity from
hepatic tumor necrosis (108 cells/kg dose level), which occurred three weeks after induction.
Responses in some patients were noted, including improvement in liver metastases and reduction
of bony pain. Anti-HLA antibodies were detectable in only one patient.
1.6.2.6.2 NK-92
NK-92 has been evaluated in four completed or ongoing phase I clinical trials. The first
published trial of NK-92 therapy was a phase I study for renal cell carcinoma and melanoma
with 12 patients. Minimal toxicities were noted including one grade III fever and grade IV
hypoglycemia.(Arai, Meagher et al. 2008) The second published trial of NK-92 enrolled thirteen
patients (age, 9-71 years) with advanced solid tumors/sarcomas, and two with
leukemia/lymphoma, who received two infusions of NK-92 cells, given 48 h apart.(Tonn,
Schwabe et al. 2013) Dose escalation was done with 1x109, 3x10
9 and 1x10
10 cells/m
2 with
minimal side effects including one patient with fever and another with back pain that responded
to morphine. A third phase I is ongoing at Princess Margaret Hospital for relapsed and refractory
hematologic malignancies. Eight patients with lymphoma who relapsed after autologous
transplantation and three patients with refractory multiple myeloma have been treated in this
trial, none of whom developed serious side effects. Two of the lymphoma patients survived long
term, indicating a potential cure for patients who were otherwise incurable by intensive standard
of care therapy. A fourth trial of NK-92 for relapsed refractory AML patients has been opened at
the University of Pittsburg and is recruiting patients.
57
2 Chapter 2: Hypotheses and experimental approach
2.1 Thesis aims
AML is a cancer that has been shown to be hierarchically structured with rare leukemic stem
cells at the base. These LSCs are resistant to chemotherapy and lead to relapse after
conventional therapy. Hematopoietic stem cell transplantation is an effective therapy for AML
demonstrating that a cellular immunotherapy can be effective at curing patients and thus
eliminating LSCs. Further, NK cells have been implicated in improved survival in haplotype
stem cell transplants with KIR receptor ligand mismatches. However, there is a paucity of
studies on the interaction of cellular immune effectors with cancer stem cells, leaving
unanswered questions about the sensitivity of these cells relative to bulk tumour and the
relationship between elimination of leukemic stem cells and survival. The purpose of this thesis
was to apply and develop approaches to study the interaction of natural killer cell lines with
leukemic stem cells in vitro and in vivo. Ultimately, this was to be applied to optimize a
therapeutic strategy to treat AML patients with minimal residual disease after conventional
chemotherapy who lack a suitable hematopoietic stem cell donor. There are three separate
studies in this thesis, each with hypotheses outlined below.
2.2 Hypotheses
2.2.1 Leukemic stem cells are present in cell line KG1 and are sensitive to
NK-92 mediated cytotoxicity
We hypothesized that the CD34+CD38+ cell line KG1 cell line would have a rare population of
CD34+CD38- cells that would have exclusive stem cell capacity, as determined by the ability to
self renew and differentiate into the CD34+CD38+ immunophenotype. This was based upon the
prior work demonstrating that LSCs were present in the CD34+CD38- fraction of most primary
AML samples(Lapidot, Sirard et al. 1994). Based upon one paper showing that LAK cells were
equally effective at killing primary AML LSCs and bulk leukemia, we also hypothesized that the
LSCs would be similarly sensitive to killing by NK-92. We tested this by determining the
frequency of LSCs in KG1 using several techniques, and sorted a rare CD34+CD38- fraction and
CD34+CD38+ of this cell line, then cultured over them time to determine if there was an
58
exclusive stem cell compartment. We then compared the cytotoxicity of NK-92 against KG1
using the chromium release assay, flow cytometric cytotoxicity assay, proliferation assays and
clonogenic cytotoxicity assays, conducted under standardized incubation conditions to determine
impact on bulk leukemia and LSCs.
2.2.2 Primary AML leukemic stem cells have greater sensitivity to NK92
than bulk leukemia and can be targeted by CD16+NK-92 and anti-
CD123 mAb mediated ADCC in vivo
Based upon our work with cell line KG1, we hypothesized that NK-92 would preferentially kill
primary AML leukemic stem cells relative to bulk tumour. To evaluate this we utilized a
standardized incubation and simultaneous application of the chromium release assay and
methylcellulose cytotoxicity assay. We also conducted a more classical approach by cell sorting
primary AML into bulk and LSC fractions, followed by a standard chromium release assay.
Given that NK-92 is irradiated prior to administration to patients, we then addressed, for the first
time, the issue of in vivo efficacy of irradiated NK-92 in a primary AML xenograft model,
predicting that irradiated cells would have less efficacy due to lack of capacity for in vivo
expansion. We also designed an approach to target LSCs with anti-CD123 antibodies in
combination with gene modified CD16+NK-92 cells. We hypothesized that CD16+NK-92
combined with infusions of an anti-CD123 antibody would enhance cytotoxicity against LSCs in
vivo and improve survival in a primary AML xenograft model.
2.2.3 KHYG-1 has less cytotoxicity than NK-92 against leukemic targets
which can be modulated by antibody pretreatment of targets and
effectors
We then sought to compare the cytotoxicity of another NK cell line, KHYG-1, against NK-92.
We hypothesized that KHYG-1 would be less effective against leukemia than NK-92 because of
additional inhibitory KIRs expressed by KHYG-1. Previous work from our lab using RT-PCR
demonstrated more inhibitory KIR expression in KHYG-1 than NK-92. However, this mRNA
data was never confirmed by flow cytometry. We, therefore used high throughput screening
(HTS) flow cytometry to characterize a large number of markers, including activating and
inhibiting receptors on both NK-92 and KHYG-1. Subsequently, cytotoxicity against a panel of
leukemia cell line and primary AML blasts was determined, and blockade of receptors on targets
and NK cell lines conducted to elucidate mechanism of cytotoxicity of each line. Blockade of
59
class I HLA on targets was conducted to determine if inhibitory KIR ligands were involved in
recognition. Unexpectedly, pretreatment of NK cell lines with putative blocking antibodies to
common NK activating receptors dramatically enhanced cytotoxicity of NK cell lines, prompting
an evaluation of the mechanism and generation of an additional hypothesis: NK cell line killing
of leukemia cells is enhanced by reverse antibody dependent cell mediated cytotoxicity via
NKp30 and NKp44 and target cell Fcγ receptor II. This hypothesis was tested using targets with
variable Fcγ receptor II expression and conducting regression analysis on target CD32
expression versus delta cytotoxicity.
60
3 Chapter 3: Clonogenic assays measure leukemia stem cell
killing not detectable by chromium release and flow cytometric
cytotoxicity assays
This chapter has been published: Reprinted with minor modifications from B.A.
Williams X.-H. Wang and A. Keating. Clonogenic assays measure leukemia stem cell
killing not detectable by chromium release and flow cytometric cytotoxicity assays.
Cytotherapy 2010: 12(7);951-60. License #: 3564810548348
Contributions:
X.-H. Wang: Assisted in experimental design and execution of animal experimentation.
A. Keating: Supervised the overall research project, contributed to experimental design,
and data analysis.
61
3.1 Abstract
NK-92, a permanent NK cell line, shows cytotoxicity against a variety of tumors and has been
tested in a phase I trial. We tested the toxicity of NK-92 and chemotherapy drugs against against
the stem cell capacity of the acute leukemia cell line, KG1. While the chromium release assay is
the most common method to assess cytotoxicity of immune effectors, and flow cytometry is
increasingly used, the relationship of either assay to clonogenic readouts remains unknown.
KG1 was assessed for stem cell frequency by serial dilution, single cell sorting and colony
growth in methylcellulose. KG1 was sorted into CD34+CD38+ and CD34+CD38- populations
and recultured in liquid medium or methylcellulose to determine proliferative capacity of each
fraction. Cytotoxicity of NK-92, daunorubicin and cytarabine against KG1 was measured using
the chromium release assay, flow cytometry and clonogenic assays. The culture-initiating cell
frequency of whole KG1 was between 1 in 100 to 1000 by serial dilution and single cell sorting.
Although a rare (1-3%) CD34+CD38- population could be demonstrated in KG1, both fractions
had equivalent proliferative capacity. The cumulative flow cytotoxicity assay was more
sensitive than the chromium release assay in detecting target cell killing. At a 10:1 ratio, NK-92
eliminated clonogenic capacity of KG1 that was not predicted by the chromium release assay.
Clonogenic assays provide a more sensitive means to assess the effect of a cytotoxic agent
against putative cancer stem cells within cell lines, provided that they grow well in liquid culture
medium or methylcellulose.
62
3.2 Introduction
The utility of standard methodologies to assess cytotoxic agents against tumor targets have been
questioned by a growing body of literature supporting rare cancer stem cells in a number of
cancers(Lapidot, Sirard et al. 1994; Bonnet and Dick 1997; Al-Hajj, Wicha et al. 2003; Singh,
Clarke et al. 2003; Singh, Clarke et al. 2004; O'Brien, Pollett et al. 2007; Ricci-Vitiani, Lombardi
et al. 2007) The cancer stem cell hypothesis holds that only a fraction of cells within a tumour
are capable of unlimited proliferation. If true, this has profound implications for studies of
cytotoxicity against bulk tumour samples.
Acute myeloid leukemia was the first malignancy to have evidence of a stem cell hierarchy
demonstrated(Lapidot, Sirard et al. 1994; Bonnet and Dick 1997) and cancer stem cells have
subsequently been demonstrated in a variety of cancers including brain tumors(Singh, Clarke et
al. 2003; Singh, Hawkins et al. 2004), breast cancer(Al-Hajj, Wicha et al. 2003), multiple
myeloma(Matsui, Huff et al. 2004) and colon cancer(O'Brien, Pollett et al. 2007; Ricci-Vitiani,
Lombardi et al. 2007). Other work has demonstrated cancer stem cells within cell lines(Kondo,
Setoguchi et al. 2004; Matsui, Huff et al. 2004), expanding the hypothesis beyond primary
human tumours. Controversy remains, however, as to which other malignancies may be
structured hierarchically in a stem cell model, the cell of origin, as well as the frequency of
cancer stem cells and their precise immunophenotype.
KG1 is a commonly used leukemia cell line derived from a patient with erythroleukemia in
myeloblastic relapse reported as having an immunophenotype of CD34+CD38+ and to date no
cancer stem cell has been isolated or demonstrated from it. NK-92 is a cell line that has been
derived from a patient with non-Hodgkin’s lymphoma with an NK cell immunophenotype(Gong,
Maki et al. 1994) and has been demonstrated to have enhanced cytotoxicity over endogenously
derived NK cells against a variety of human leukemia cell lines and primary leukemic
blasts(Yan, Steinherz et al. 1998). The ability of these novel NK cell effectors to proliferate can
be abrogated with radiation at doses that do not affect their cytotoxicity(Yan, Steinherz et al.
1998), thereby allowing their use as clinical cellular immunotherapeutic agents which has been
evaluated in a phase I clinical trial for advanced renal cell carcinoma or melanoma(Arai,
Meagher et al. 2008).
63
The implication of rare cancer stem cells potentially driving a given tumour system have
profound implications for the interpretation of assays that make an assumption of homogeneity
within a given tumour. This issue is particularly problematic for cytotoxicity assays used to
determine the effect of therapeutic agents against cancer cells. Typically, these assays focus on a
snap shot assessment of bulk tumour cells to determine whether a tumour has signs of apoptosis
(e.g. annexin V positivity), mitochondrial damage (e.g. MTT assessment of mitochondrial
activity), cell permeability (e.g. Cr release assay and PI staining in flow cytometry), or some
other readout deemed indicative of viability or cell death. Given that leukemic stem cells have
been demonstrated to have differential sensitivity to chemotherapeutic agents relative to non-
stem cells(Costello, Mallet et al. 2000), it is important to consider this as a possibility in other
systems. Where a cancer stem cell has been identified with specific cell surface markers and
validated with appropriate in vitro and in vivo assays, cell sorting can be used in conjunction
with one of the previously mentioned approaches to address toxicity against the cancer stem cell
population. However, most primary cancers and cell lines do not have a clearly identifiable
cancer stem cell population, making this approach problematic. Furthermore, even in the few
cases with well-defined cancer stem cell populations, fractionating them with cell sorting and
testing toxicity of an agent using a conventional assay such as PI and annexin V staining does
not address whether proliferative capacity has been abrogated. Although clonogenic readouts
have been used in the past to assess chemotherapydrugs(Curtis, Minden et al. 1995; Curtis,
Metcalf et al. 2000; Jacobs and Wood 2005), this approach has not been applied to the study of
immune effectors. Here, we characterize the stem cell capacity of KG1 and demonstrate
immunophenotypic heterogeneity of a putative stem cell population, which can be ablated by
NK-92 and chemotherapy drugs, as demonstrated by clonogenic assays when other conventional
cytotoxicity assays demonstrate only fractional cell kill.
64
3.3 Materials and methods
3.3.1 Cell lines
KG1 and K562 was obtained from the ATCC and maintained in IMDM +20% FBS and RPMI
(RPMI, Invitrogen, Grand Island, NY, USA) +10% FBS respectively. NK-92 was originally
provided by Dr. Hans Klingemann (at the time, Rush University Medical Center, Chicago, IL,
USA), expanded and stored in liquid nitrogen and retrieved as required and maintained in GM1
medium supplemented with IL-2 (450 U/mL) (Chiron, QC, Canada). Cellular cytotoxicity
assays were done in GM1 medium and drug cytotoxicity assays were done in IMDM +20% FBS.
3.3.2 Antibodies and reagents
Anti-CD34 PE antibody (BD biosciences) and anti-CD38 APC (Becton Dickson) were used to
immunophenotype KG1 and cell sort fractions. Propidium iodide (Sigma) and Annexin V-FITC
(BD Pharmingen) were used at to stain apoptotic cells in Annexin V buffer (BD Pharmingen) for
10 minutes on ice. IMDM and RPMI (Sigma) were supplemented with 10% and 20% fetal
bovine serum (FBS) (Gibco), respectively. Cytarabine (Mayne Pharma (Canada) Montreal)
came as a premised solution and daunorubicin powder (Novopharma Toronto) was reconstituted
in 4 ml of distilled water prior to dilution in Iscoves Modified Dulbecco’s Medium to appropriate
concentrations. FACS buffer was made with PBS +2mM EGTA + 2% FBS.
3.3.3 Chromium release assay
Target cells were washed in 10 ml of IMDM and 2x10e6 cells were resuspended in 200 ul of
GM1 or IMDM +20% FBS (control) and treated with 100 µCi of Na251
CrO4 for 2 hours prior to
treatment and washed x2 in AIM-V serum free medium prior to treatment with NK-92 or drugs.
5000 radiolabelled KG1 were added to individual wells of a 96 well U bottom plate. NK-92 at
various concentrations were added to KG1 to yield 10:1 and 1:1 E:T ratios and plates were
centrifuged at 500 rpm and incubated at 37°C 5% CO2. Plates were incubated for 24 hr at 37°C
in a humidified atmosphere containing 5% CO2 and centrifuged at 400 g for 5 min, and 100 μl of
supernatant were collected from each well and transferred into collection tubes. The plates were
then centrifuged, supernatants collected and assayed in a gamma counter. The amount of 51
CrO4
present in supernatants was determined using a gamma counter. Percent lysis was calculated
65
using the formula:% lysis = E-S/M-S x100% where E is the 51
Cr-release from an experimental
sample, S is the spontaneous release in the presence of complete IMDM medium and M is the
maximum release upon cell lysis with Triton X 100 10%. Data are presented as the mean
percent lysis of triplicate samples (+/-SD) from a representative experiment repeated 3 times.
3.3.4 Flow cytometry and cell sorting
Immunophenotyping was done using a BD LSR II (Becton Dickson) flow cytometer and cell
sorting using FacsAria (Becton Dickinson). 1 ul CD34 PE and 3 ul anti-CD38 APC alone and in
combination were used to stain 1x106 cells for 30 minutes prior to assessment by flow cytometry.
For cell sorting 5x106 KG1 cells were resuspended in 5 ml of FACS buffer and stained with
anti-CD38 APC (3 µl/million). Gates were set to define CD38- yielding ~0.01%. KG1 was
99.9% positive for CD34+ so was not sorted on this parameter, effectively yielding
CD34+CD38+ and CD34+CD38- fractions following sorting with the anti-CD38 APC antibody.
Cells were then resuspended in IDMD +20% FBS + antibiotics for long-term culture in T25
flasks or infused into methylcellulose (5000 per plate).
3.3.5 Flow cytometric cytotoxicity assay
The flow cytometric cytotoxicity assay was done with a BD LSR II flow cytometer and Coulter
FC500 using PI and Annexin V staining. Co-incubations of NK-92 and KG1 at a 1:1 ratio were
done in an identical manner to the chromium release assay (5000 targets per well) with
additional wells per treatment group pooled together for flow cytometric analysis. NK-92 and
KG1 were discriminated by expression of CD34 (KG1 positive, NK-92 negative) using an anti-
CD34 PE antibody. Cumulative cytotoxicity assays were done using flow count beads (Coulter)
to account for cell loss. Drug treatments were done in 10 ml of IMDM +20% in T25 flasks. All
co-incubations were at 37°C, 5% CO2 and 100% humidity. Cell counts and viability were
determined by trypan blue prior to assessment by flow cytometry using PI, Annexin V to
ascertain viability. Flow count bead were used to enumerate the number of cells to account for
cell loss relative to the untreated control. Cells were spun down in microfuge tubes at 2000 RPM
and resuspended in 1 ml of PBS, spun down, supernatant discarded and pellets resuspended in
Annexin V 1x buffer (800 ul total volume including PI and AV FITC). PI and AV-FITC were
added. Samples were split and run on two different flow cytometers. Flow cytometry was set up
with a doublet discriminator to exclude doublets or larger cell associations. A ‘vertical gate’ was
66
used to exclude fragments on FS axis using operator judgement. 20 000 single whole cell events
assayed for viability. Because KG1 is CD34+ and NK-92 is CD34- a gate was established to
delineate them using a best fit quadrant strategy. A histogram was set up to assess PI and
Annexin V. Viability gates were based on cytarabine 10 µg/ml which yielded partial cell killing
with early apoptotic and dead cells used to define the midpoint between viable and early
apoptotic/dead cells. Beads were added to a second stained control and treatment sample
(CD34PE, PI and Annexiv V) to determine absolute cell counts relative to control. Calculations
for percent cytotoxicity (PC) used the formula: (% viable treated- % viable untreated)/ (% viable
untreated), as previously published (Suck G. et al. 2005). For corrected cumulative counts (CC)
using beads the formula was: (Corrected % viable treated)- (corrected viable
untreated)/(corrected % viable untreated %). Data are presented as the mean percent lysis of
triplicate samples (+/-SD) from a representative experiment repeated 3 times.
3.3.6 Methylcellulose and liquid reculturing cytotoxicity assays
5000 KG1 cells were added to wells of a 96 well U bottom in either 200 µl of IMDM + 10%
FBS or GM1 medium (for NK-92 treatments) and co-incubated with daunorubicin (1 µM),
cytarabine (0.1, 1 and 10 µg/ml) or NK-92 (1:1 or 10:1 ratio) for 24 hours. Cell co-cultures (200
µl) were suspended in 10 ml of liquid culture medium or methylcellulose base medium (R&D
Systems, Minneapolis). KG1 cell suspension +/- treatments were transferred into 10 ml of
IMDM +10% FBS + streptomycin in T25 culture flask and evaluated one month later by cell
counting using the trypan blue exclusion assay. Three 35 mm petri dishs per treatment were
plated with 1 ml of methylcellulose containing ~5000 KG1 cells +/- treatments and the three
plates were stored in a 135 mm petri dish and maintained in 5% C02, 100% humidity and 37C.
Controls for both methylcellulose and liquid re-culturing assays included 24 hour incubations of
KG1 in 96 well U-bottom plates under the following conditions: 1) no drug or cell treatment of
KG1 in IMDM +20% FBS (baseline growth) or GM1 medium (impact of GM1 on KG1 growth)
2) NK-92 alone in GM1 for 24 hours followed by infusion into methycellulose or IMDM +20%
FBS liquid culture (growth potential of NK-92 in methylcellulose or IMDM +20% FBS) 3) NK-
92 alone in GM1 for 24 hours followed by addition to KG1 in methylcellulose or IMDM +20%
liquid culture (impact of NK-92 on KG1 growth in methylcellulose or low density liquid
culture). Colonies (more than 50 cells) growing in methylcellulose were enumerated at one
67
month and colony numbers were averaged for each treatment group and standard deviation
calculated.
3.3.7 Animals
NOD.CB17-prkdcscid
(NOD/SCID) mice were maintained in the Ontario Cancer Institute animal
facility according to protocols approved by the Animal Care Committee. Mice were fed
irradiated food and water ad libitum. Mice were irradiated with 325 cGy of irradiation prior to
injection with KG1 leukemia cells or PBS via tail vein (200 µl volume). Four mice were used
per treatment cohort to receive 104, 10
5 or 10
6 KG1. Mice were evaluated for signs and
symptoms of leukemia and sacrificed at appropriate humane endpoints.
68
3.4 Results
3.4.1 Clonogenic capacity of KG1 in vitro and in vivo
To determine the stem cell frequency in KG1, several in vitro methods were used, assessing
growth in both liquid culture medium and semi-solid methylcellulose followed by an in vivo
assessment in NOD/SCID mice. KG1 was seeded using ten-fold dilutions into liquid medium
and assessed for recovery at 2 weeks and ability to perpetuate beyond one month yielding a stem
cell frequency between 1 in 100 to 1000 (Table 3.1). Subsequently, serial two-fold dilutions of
5000 KG1 (12 samples) in rows of 96 well flat bottom plates yielded complete confluence at two
weeks when 625 or more cells were plated, but not with 312 or less (Table 3.2). Further, cell
sorting was used to replicate serial dilutions with more numerical precision providing evidence
for a stem cell frequency between 1 in 250 and 1 in 1000 (Table 3.3).
Table 3.1: Frequency of KG1 stem cell frequency using liquid culture repopulation
# cells added Culture regrowth at 2 weeks and ability to perpetuate a
liquid culture >1 month
105 +
104 +
103 +
102 -
10 -
Table 3.2: Frequency of KG1 stem cell frequency using two fold serial dilutions in 96 well
confluence assay (5000 to 0.3 per well)
Cells/well Average % confluence @ 2 weeks
5000 100
2500 100
1250 100
625 100
312 70
156 20
78 10
<=39 0
69
Table 3.3: Frequency of KG1 stem cell frequency using cell sorting and 96 well confluence
assay (1000 to 1 per well)
Cells/well Average % confluence @ 2 weeks
1000 100
500 100
250 70
100 60
75 60
50 60
20 50
1 0
Single cell sorting of KG1 into wells of ten 96 well flat bottom plates yielded a stem cell
frequency of 3 in 1000, in which only a small fraction led to confluence of a single well from one
cell─the majority of wells had no significant growth (data not shown). The methylcellulose
colony-forming assay was more variable, yielding colony-forming units ranging from 1 in 69 to
1 in 343. KG1 was infused into NOD/SCID mice at doses of 104, 10
5, 10
6 cells via tail vein to
determine the leukemia-initiating cell frequency within KG1. Mice infused with 105 and 10
6
KG1 developed symptoms of leukemia (weight loss, hindlimb tumors and paresis) by 3 months
and did not survive past 6 months. At a dose of 104 KG1 some mice appeared disease free for
over six months and survived as long as eight months after the initial infusion, but ultimately all
succumbed to progressive leukemia. Therefore, based on these observations, the leukemia-
initiating frequency of KG1 in NOD/SCID mice was less than 1 in 10 000 (Figure 3.1).
70
Figure 1
Group10e410e510e6
Censor
80 100 120 140 160 180 200 220 240
100
80
60
40
20
0
Time
Surv
ival p
robabili
ty (
%)
Figure 3.1: NOD/SCID leukemia initiating frequency of KG1
Four NOD/SCID mice irradiated with 325 cGy were injected via tail vein with one of three dose
of KG1 (104, 10
5, 10
6) and monitored for signs of leukemia and survival with results being
plotted in a Kaplan Meier survival curve.
71
3.4.2 Immunophenotyping and fractionation studies of KG1
To determine if KG1 had an immunophenotypically identifiable stem cell exclusively in the
CD34+CD38- fraction cell sorting combined with clonogenic assessments were performed. The
immunophenotype of KG1 was confirmed by two color flow cytometry to be predominantly
CD34+CD38+ with 99.9% CD34 positivity, but a rare (1-3%), previously unreported,
CD34+CD38- fraction was identified after multiple assessments (Figure 3.2).
10 100 1000 10000 1x105
<PE-A>: CD34/FAS
10
100
1000
10000
1x105
<A
PC
-A>
: CD
38
0.024 97.3
2.590.062
CD34 PE
CD
38
AP
C
Figure 3.2: Immunophenotype of KG1
1x106 KG1 were stained with anti-CD34 PE and anti-CD38 APC and assessed by two color flow
cytometry using a Facscalibur flow cytometer.
72
Both the CD34+CD38+ and CD34+CD38- sorted fractions, however, regrew the culture from as
few as 1700 cells and propagated a suspension culture beyond 2 months. The expression of
CD38 from the CD34+CD38- sorted fraction was 30% CD38+ at 7 weeks and 98% positive at
ten weeks, reconstituting the original immunophenotypic distribution of CD38. At 7 weeks, the
CD34+CD38+ sorted fraction was 73% CD38+ and 27% CD38-, while at 10 weeks became 93%
CD38+ approximating the original culture immunophenotype which remained stable after being
passed through the cell sorter without sorting into fractions (Figure 3.3).
73
CD34 PE CD34 PE
CD34 PECD34 PE
CD34 PE CD34 PE
Figure 3.3: Reconstitution of CD38 distribution following cell sorting KG1
5x106
KG1 cells were sorted into CD34+CD38- and CD34+CD38+ fractions using a FACsAria
cell sorter. As a control, KG1 cells were passed through the cell sorter without sorting
(unsorted). 1700 cells from the unsorted (A), sorted CD34+CD38- (B) and sorted CD34+CD38+
(C) fractions were added to T25 flask with IMDM +20% FBS. All samples reconstituted a
continuous growing culture after one month and cells were assessed by flow cytometry for CD34
and CD38 expression at 7 and 10 weeks.
74
Further, seeding of sorted of CD34+CD38+ and CD34+CD38- KG1 cells into methylcellulose
yielded comparable colony formation (data not shown). Attempts to sort using other known
stem cell markers expressed on KG1 such as CD133 and CD123 were unsuccessful in
significantly enriching the stem cell capacity of KG1 (data not shown).
3.4.3 Chromium release assay (CRA) and flow cytometric cytotoxicity
assay of NK-92 and chemotherapy drugs versus KG1
Toxicity of NK-92 and chemotherapeutic agents were assessed using both the chromium release
assay and flow cytometric cytotoxicity assay, calculating both percent toxicity (cross-sectional)
and cumulative toxicity. Using the CRA, NK-92 was highly cytotoxic to KG1 at a 1:1
effector:target (E:T) ratio relative to K562 (% lysis: 53% versus 29%), but at 10:1 killing of each
target was comparable (Figure 3.4).
0
10
20
30
40
50
60
70
80
90
100
1:1 10:1
% L
ys
is
Effector Target ratio
KG1
K562
Figure 3.4: Chromium release assay of NK-92 against KG1 and K562
KG1 or K562 (5000 targets) were plated in 96 well plates and treated with NK-92 in ratios of
10:1 and 1:1 for 24 hours. % lysis results are the average of triplicate wells +/- SD. Results are
representative of three separate experiments.
75
To compare with the CRA for cellular cytotoxicity, the flow cytometric cytotoxic assays were
conducted on the same day with KG1 in a 1:1 E:T ratio under identical co-incubation conditions,
and the percent cytotoxicity (PC) and cumulative cytotoxicity (CC) was determined to be 48%
and 85%, respectively (Figure 3.5).
Figure 3.5: Chromium release assay versus flow cytometric cytotoxicity assay
KG1 was treated with NK-92 in a 1:1 E:T ratio for 24 hours in 96 well U bottom plates in two
identical simultaneous experiments. The first sample was used in a chromium release assay and
% lysis is the average of triplicate wells +/- SD. The second sample set was used in a flow
cytometric cytotoxicity assay using CD34 to identify KG1 targets (CD34+) from effector cells
(CD34-) and staining with PI and Annexin V to determine viability of target cells. Percent
cytotoxicity (PC) was calculated from the viable cell counts of treated and untreated KG1
samples as outlined in the methods and cumulative cytotoxicity (CC) was determined by
adjusting the PC value using flow count beads to account for cell loss relative to control over the
24 hour incubation. Results of PC and CC are the average of two simultaneous independent
measurements of the same sample on different flow cytometers based on 20 000 events.
76
The three methods were used to assess the toxicity of daunorubicin and cytarabine at several
dose levels. Daunorubicin 1 µM treatment of KG1 for 24 hours yielded cyotoxicity values of
23+/-4% (CRA), 36+/-3% (PC) and 64+/-2% (CC) and for cytarabine 1 µg/ml was 19+/-1%
(CRA), 19+/-3% (PC) 49+/-5% (CC) (Figure 3.6).
Figure 3.6: Chromium release assay versus flow cytometric drug assay
KG1 was treated with daunorubicin 1 µM and cytarabine 0.1, 1 and 10 µg/ml for 24 hours in
either 96 well U bottom plates (CRA) or T25 flasks (flow cytometry assays). The first sample
was used in a chromium release assay and % Lysis is the average of triplicate wells +/- SD.
Control and drug treated samples were prepared for flow cytometry and stained with PI and
Annexin V to determine viability of target cells. Percent cytotoxicity (PC) was calculated from
the viable cell counts of treated and untreated KG1 samples as outlined in the methods and
cumulative cytotoxicity (CC) was determined by adjusting the PC value using flow count beads
to account for cell loss relative to control over the 24 hour incubation. Results of PC and CC are
the average of two simultaneous independent measurements of the same sample on different
flow cytometers based on 20 000 events.
77
3.4.4 Clonogenic and proliferation assays of NK-92 and chemotherapy
drugs versus KG1
Clonogenic assessment of KG1 following treatment with NK-92 or chemotherapy drugs were
performed using identical incubation conditions for the chromium release and flow cytometric
assays to facilitate a comparison of cytotoxic readouts. Duplicate samples of 5000 KG1 were
treated in 96 well plates with either NK-92 in 1:1 or 10:1 ratios, cytarabine (0.1, 1 and 10 µg/ml)
or daunorubicin (1 µM). After 24 hours, cell samples were resuspended in liquid culture or
plated in methylcellulose and assessed at one month. NK-92 was not able to proliferate
significantly in long term culture medium as it lacked IL-2. Cells in re-suspension cultures were
enumerated after one month (Figure 3.7): control 1: KG1 in IMDM medium (34.8x106),
daunorubicin 1 µM (0), cytarabine 0.1 µg/ml (0.9x106), cytarabine 1 µg/ml (0), control 2: KG1
in GM1 medium (38.5x106), NK-92 1:1 (27.7x10
6), NK-92 10:1 (0).
Figure 3.7: Liquid reculturing cytotoxicity assay
KG1 was incubated with or without NK-92 (10:1 and 1:1 E:T ratio), daunorubicin (1 µM) or
cytarabine (0.1, 1, 10 µg/ml) and cytotoxic drugs for 24 hours in 96 well U bottom plates
comparable to a chromium release assay setup. Cells were enumerated after three weeks using a
hemacytometer and trypan blue dye. Controls included KG1 cultured for 24 hours in IMDM
+10% FBS (IMDM) or Ex Vivo + 10% human AB serum + 450 U/ml IL-2 (GM1) , NK-92
alone. Results are representative of two separate experiments.
78
Addition of NK-92 (10:1 ratio) to 5000 KG1 cells in long term suspension culture without prior
24 hour co-incubation in 96 well U-bottom plates did not significantly affect proliferation at 3
weeks, yielding 26.9x106 cells in the control vs 26.0x10
6 cells in the NK-92 treatment group.
The methylcellulose cytotoxicity assay for NK-92 vs KG1 at a 1:1 E:T ratio reduced colony
formation from 54.7 +/- 4.0 to 39.3 +/- 13.1 while at 10:1, colony formation was eliminated
(Figure 3.8). A comparable chromium release assay of NK-92 vs KG-1 at a 10:1 E:T ratio
yielded 78% lysis. All doses of cytarabine and daunorubicin tested eliminated colony formation
of KG1 in methylcellose (data not shown).
79
Figure 3.8: Methylcellulose cytotoxicity assay KG1 was incubated alone (A) or with NK-92 at 1:1 (B) and 10:1 (C) ratio for 24 hours in 96 well
U bottom plates comparable to a chromium release assay setup. Subsequently cells were
transferred into methylcellulose and 5000 KG1 were transferred to each of three 35 mm plates.
Controls included KG1 alone, NK-92 alone, NK-92 infused at 10:1 ratio into methylcellulose
without prior 24 hour co-incubation in 96 well U bottom plates. Methylcellulose plates were
examined for colonies at one month and enumerated (colony definition >=50 cells).
80
3.5 Discussion
The cancer stem cell hypothesis postulates that only a subpopulation of cells within a tumour
have unlimited proliferative capacity while the remainder become more differentiated with
limited proliferative capacity. We attempted to determine if KG1 had a stem cell-driven
hierarchy similar to the original findings for acute myeloid leukemia primary blasts(Lapidot,
Sirard et al. 1994; Bonnet and Dick 1997) and if killing of the putative cancer stem cell in the
line could be detected.
As an initial step we determined the stem cell frequency with several methods, including liquid
reculturing in a whole flask, 96 well plate liquid confluence assay using serial dilution and single
cell sorting, and the methylcellulose colony-forming assay, all of which yielded a similar stem
cell frequency of between 1 in 100 to 1 in 1000. The NOD/SCID repopulating capacity was <1
in 10,000 although it took 4-7 months for mice injected with 10,000 KG1 to develop symptoms
and ultimately a fatal disease. This frequency analysis established that only a rare sub-
population of KG1 has the potential to perpetutate the culture and grow in NOD/SCID mice.
Previous work has demonstrated cancer stem cells in brain tumour cells lines(Kondo, Setoguchi
et al. 2004) and multiple myeloma cell lines(Matsui, Huff et al. 2004). We therefore attempted
to determine if the stem cell capacity in KG1 is a stochastic process or is driven by a rare
population of immunophenotypically identifiable stem cells.
Although KG1 did contain rare CD34+CD38- cells (1-3%) similar to that reported for the
majority of primary human AML blast samples, both the CD34+CD38- and CD34+CD38+
fractions had unlimited proliferation capacity and the ability to recapitulate a culture with a
distribution of CD38+ cells similar to those of the original culture. Moreover, growth of the
CD34+CD38+ and CD34+CD38- fractions in methylcellulose was equivalent. A recent report
demonstrated that primary human AML blasts from some patient samples have stem cell
capacity in the CD34+CD38+ fraction, as shown by Lapidot(Taussig, Miraki-Moud et al. 2008),
which questioned the earliest report identifying the leukemic stem cell as being restricted to the
CD34+CD38- fraction(Lapidot, Sirard et al. 1994). The more recent study demonstrated that
the anti-CD38 antibody used in the original publication facilitated clearance of CD38+ stem cells
by the reticuloendothelial system and this effect could be blocked using IVIg or anti-CD122 (IL-
81
2Rβ) treated immunodeficient mice(Taussig, Miraki-Moud et al. 2008). Specifically, they
showed that in seven AML patient samples, leukemic stem cells could be detected in the
CD34+CD38+ fraction, as determined by engraftment in IVIG or anti-CD122 treated
immunodeficient mice. Although, this has reopened debate about the validity of the cancer stem
cell hypothesis in AML, significant evidence remains that in primary AML, stem cells can be
enriched with anti-CD34, anti-CD123 or other antibodies that are not susceptible to the same
form of immune-mediated clearance. Other tumour systems can be enriched for cancer stem
cell populations based on the presence, rather than absence, of cell surface markers, indicating
that this clearance mechanism does not apply in all systems. However, in our studies of KG1 it
seems that the putative stem cell is contained in both the CD38+ and CD38- fractions, an
observation consistent with the recent findings of Taussig et al.(Taussig, Miraki-Moud et al.
2008). Attempts to sort other sub-populations within KG1 using anti-CD133 and anti-CD123
markers did not significantly enhance proliferative capacity.
Only one study to date has looked at the in vitro sensitivity of CD34+CD38- AML cancer stem
cells to immune effector cell killing. In that study, lymphokine activated killer cells and
allogeneic lymphocytes exerted a modest cytotoxic effect on AML cancer stem cells that were
intrinsically resistant to the chemotherapeutic agent, daunorubicin.(Costello, Mallet et al. 2000)
Given that no cell surface marker could significantly enrich stem cell capacity in KG1, to address
the effect on the cancer stem cell fraction, we turned to functional readouts of cellular
proliferation such as methylcellulose colony formation and liquid culture reconstitution, not
typically used in assessing immune effector cell cytotoxic function.
The study of immune effector cytotoxicity has predominantly focused on the chromium release
assay by measuring cell permeabilization since its initial use in the 1960s.(Brunner, Mauel et al.
1968) Previous work showed that the cross sectional assay approximates the chromium release
assay and the cumulative assay is more sensitive at detecting cell kill(Ozdemir, Ravindranath et
al. 2003). Our comparison of the chromium release assay to flow cytometric methods further
demonstrated that the cumulative cytotoxicity readout was significantly greater than both the
chromium release assay and percent cytotoxicity methods for all drugs used as well as for NK-
92. Neither of these assays however, addresses the issue of proliferative capacity, the most
82
important aspect of assessing whether a cytotoxic intervention has had a true impact on the cell
population of interest.
There was partial killing of KG1 by NK-92 in 24 hr co-incubations at a 10:1 ratio as measured
by the chromium release assay, but complete ablation of the ability of 5000 cells to regrow a
liquid suspension culture or grow colonies in methylcellulose when assessed after one month.
This methodologic discrepancy is important as it demonstrates that the chromium release assay
significantly underestimated the cytotoxicity of NK-92 against the stem cell fraction in KG1.
The toxicity against the stem cell capacity of KG1 was 100% by the clonogenic and liquid
reculturing cytotoxicity assay at an E:T of 10:1 over 24 hrs while toxicity against the bulk
tumour was 78% as determined by the chromium release assay. Given that other studies of
cancer stem cells show them to be resistant to chemotherapy, this finding is important in
demonstrating that an immune effector can preferentially kill a leukemic stem cell. The
clonogenic readouts for daunurubicin 1 µM and cytarabine 0.1 µg/ml also ablated the growth of
colonies or liquid reculturing capacity. These doses are therapeutically achievable and indicate
that NK-92 can exert a powerful impact relative to agents used in standard-of-care protocols that
are potentially curative.
Here, we show that that conventional chromium release assay can be approximated with the flow
cytometric cytotoxicity assay to evaluate immune effector cell killing and drug cytotoxicity. The
sensitivity is increased when Annexin V is used in addition to propidium iodide readouts to
determine viability as early apoptotic cells can be detected as well. When counting beads are
used to convert a cross sectional assay to a cumulative one, a significantly greater amount of
killing is detected as this method accounts also for disintegrated cells that would otherwise not
be detected. Over longer co-incubations at 24 hours, the cumulative assay also measures
differential proliferation, as underscored by the low percent cytotoxicity, but high cumulative
cytotoxicity readouts observed with the cytostatic agent, cytarabine.
Although the cumulative flow cytotoxicity assay detects greater cytotoxicity compared with the
chromium release assay for both drugs and NK-92, neither method predicts the elimination of
leukemia stem cells as determined by the lack of regrowth in liquid culture or colony formation
83
in methylcellulose. Thus, KG1 stem cell capacity appears to be more sensitive to immune
effector cell killing and drugs than are the majority of cells in the culture.
Although we were unable to isolate a cancer stem cell by immunophenotypic profile, we provide
evidence that clonogenic cell readouts enable the proliferative capacity of target cells to be
assessed regardless of whether or not an immunophenotypically identifiable stem cell can be
isolated. We conclude that colony-forming assays and liquid culture methods provide a more
important parameter of cytotoxic readout than the chromium release assay or flow cytometric
cytotoxicity assays.
84
4 Chapter 4: Irradiated CD16+NK-92 prolongs survival
in an AML xenograft model in combination with anti-
CD123 monoclonal antibody therapy by targeting
leukemic stem cells through antibody dependent cell
mediated cytotoxicity (ADCC)
The results of this chapter are unpublished.
Contributions:
X.-H. Wang: Assisted in experimental design and execution of animal experimentation.
J. V. Leyton: Assisted in experimental design and flow cytometry of leukemic stem cell
fractions in mouse bone marrow for secondary transplant experiments.
S. Maghera: Assisted in clonogenic assays.
B. Dief: Assisted in clonogenic assays.
85
4.1 Abstract
Patients with acute myeloid leukemia (AML) often relapse after initial therapy, because of the
presence of minimal residual disease containing leukemic stem cells (LSCs) that express the IL-3
receptor alpha chain CD123. We evaluated the use of NK-92, an infusible CD16- NK cell line,
against leukemic cell lines and primary AML, using bulk and clonogenic assays followed by
testing in a murine AML xenograft model. NK-92 could preferentially target LSCs over bulk
leukemia. Further, both irradiated and non-irradiated NK-92 infusions improved survival in a
primary AML murine xenograft model. To enhance therapeutic efficacy, a gene modified
CD16+NK-92 cell line expressing the high affinity Fc gamma receptor was utilized in
combination with an anti-CD123 monoclonal antibody used to coat AML targets. Cytotoxicity
of CD16+NK-92 against CD123+ AML targets by could be enhanced with the anti-CD123 mAb
7G3 in vitro. Further, combination therapy with irradiated CD16+NK-92 and 7G3 resulted in
improved survival in an AML xenograft model of leukemia relative to controls. We demonstrate
for the first time that NK-92 targets LSCs and that irradiated NK-92 treatment can improve
survival in an AML xenograft model. Finally, we demonstrate that an irradiated CD16+NK-92
cell line can be redirected to kill LSCs more effectively by ADCC both in vitro and in vivo,
leading to prolonged survival of AML xenografted mice.
86
4.2 Introduction
AML accounts for a significant proportion of acute leukemias in both adults (90%) and children
(15-20%)(Hurwitz, Mounce et al. 1995; Lowenberg, Downing et al. 1999). While 70-85% of
AML patients treated with current chemotherapy protocols are able to achieve a morphologic
remission(Hurwitz, Mounce et al. 1995; Ribeiro, Razzouk et al. 2005), many relapse because of
recurrence from minimal residual disease (MRD) containing leukemic stem cells (LSCs) leading
to an overall five year survival of approximately 40%(Lowenberg, Downing et al. 1999). Acute
myeloid leukemia (AML) was the first malignancy to have evidence of a stem cell hierarchy
with enrichment of LSCs demonstrated in the CD34+CD38- fraction(Lapidot, Sirard et al. 1994;
Bonnet and Dick 1997). These LSCs express the IL-3 receptor alpha chain (CD123), a marker
not found on normal hematopoietic stem cells(Jordan, Upchurch et al. 2000). When standard
chemotherapy fails, AML can be treated successfully with bone marrow transplantation due to
the administration of higher doses of myeloablative chemotherapy and the graft-versus-leukemia
effect (GVL) derived from the donor immune system. GVL has been shown to be mediated
primarily by T-cells(Horowitz, Gale et al. 1990), but there is evidence to show that natural killer
(NK) cell mediated GVL can prolong survival in AML patients treated with haplotype bone
marrow transplantation(Ruggeri, Capanni et al. 2002; Ruggeri, Mancusi et al. 2005). Several
permanent NK cell lines have been generated with therapeutic potential and the most notable of
these is NK-92.
NK-92 is a CD16- cell line that has been derived from a patient with non-Hodgkin’s lymphoma
with an NK cell immunophenotype(Gong, Maki et al. 1994) and has been demonstrated to have
enhanced cytotoxicity over endogenously-derived NK cells against a variety of human leukemia
cell lines and primary leukemic blasts(Yan, Steinherz et al. 1998). The ability of these novel NK
cell effectors to proliferate can be abrogated with radiation at doses that do not affect their in
vitro cytotoxicity significantly(Yan, Steinherz et al. 1998). NK-92 has been tested in a two
published phase I clinical trials (Arai, Meagher et al. 2008; Tonn, Schwabe et al. 2013) and in an
ongoing clinical trial by our group for relapsed and refractory hematologic cancers (lymphoma
and multiple myeloma) with minimal toxicities. However, to prevent potential engraftment of
NK-92 in patients it is irradiated with 1000 cGy to prevent proliferation. While this dose of
irradiation does not significantly decrease in vitro cytotoxicity (Klingemann, Wong et al. 1996;
87
Tam, Miyagawa et al. 1999; Tonn, Becker et al. 2001) it renders the cells incapable of in vivo
expansion. The original preclinical animal study utilizing NK-92 to treat cell line and primary
cell-induced leukemia have shown efficacy using non-irradiated NK-92 cells, which would be
capable of in vivo expansion.(Yan, Steinherz et al. 1998) Therefore, understanding the efficacy
of irradiated NK-92 cells in vivo is an open question highly relevant to clinical translation of this
approach, given that these cells are irradiated in all ongoing and completed clinical trials
utilizing NK-92 (Arai, Meagher et al. 2008).
NK cells typically express CD16 and are able to mediate antibody-dependent cell-mediated
cytotoxicity (ADCC) against antibody-coated targets, allowing for a bridge between the adaptive
and innate immune response. As NK-92 lost CD16 expression during malignant transformation,
a gene-modified variant of NK-92 was generated whereby the high-affinity allelic variant (valine
at position 176 instead of phenylalanine) of the CD16A Fc gamma receptor has been transduced
into parent line NK-92. These CD16-NK-92 cells (NK-92.176V and NK-92.176V.GFP) allow
for antibody-dependent cell-mediated cytotoxicity (ADCC) with proven efficacy in
vitro(Binyamin, Alpaugh et al. 2008), but with no testing of this gene modified cell line in vivo.
In this report, we show that NK-92 preferentially kills leukemic stem cells relative to bulk
leukemia and can prolong survival with and without radiation pre-treatment. Gene modified NK-
92 expressing the high affinity CD16 receptor (NK-92.176V.GFP) more effectively killed
CD123+ targets in vitro demonstrating a means to enhance the innate ability of NK-92 to target
LSCs. Finally, irradiated CD16+NK-92 therapy combined with 7G3 antibody therapy enhanced
survival in a primary AML xenograft model.
88
4.3 Methods
4.3.1 Cell lines and primary samples
K562 was obtained from the ATCC and maintained in IMDM + 10% FBS respectively. NK-92
was originally provided by Dr. Hans Klingemann, expanded and stored in liquid nitrogen and
retrieved as required and maintained in Ex Vivo medium supplemented with 450 U/ml of IL-2
and 10% human AB serum (GM1). Five primary AML samples were obtained from the
Princess Margaret Hospital Leukemia Tissue Bank as per institutional protocol. For in vivo
studies, primary AML from a single patient derived from one of two separate visits
(080179/080315) was obtained. The AML sample was M4 with cytogenetic abnormalities
(45XY inv3 -7) with an immunophenotype positive for CD4, CD7, CD11, CD13, CD15, CD34,
HLA-DR obtained at diagnosis (080719) or first relapse (080315). NK-92 and NK-92.176V
GFP (expressing high affinity Fc gamma receptor) was obtained from Conkwest under a
Material Transfer Agreement and maintained as described for NK-92.
4.3.2 Chromium release assay
Target cells (1x106) were resuspended in 200 ul of MEM-Alpha medium and treated with 100
µCi of Na251
CrO4 for 2 hours prior to treatment and washed x2 in AIM-V serum free medium
prior to treatment with NK-92. Radiolabelled targets (1x104) were added to individual wells of a
96 well U bottom plate. NK-92 was added at various concentrations to yield 25:1, 10:1, 5:1 and
1:1 effector:target (E:T) ratios and plates were centrifuged at 500 rpm and incubated at 37°C 5%
CO2 x24 hrs. Plates were incubated for 4 hr at 37°C in 5% CO2 and centrifuged at 400 g for 5
min, and 100 μl of supernatant were collected from each well and transferred into collection
tubes. The amount of 51
CrO4 present in supernatants was determined using a gamma counter.
Percent lysis was calculated using the formula:% lysis = E-S/M-S x100% where E is the 51
CrO4-
release from an experimental sample, S is the spontaneous release in the presence of target cell
growth medium and M is the maximum release upon cell lysis with Triton X 100 10%. To
inhibit granule exocytosis calcium was chelated from medium during four hour incubations using
ethylene glycol tetraacetic acid (EGTA) and MgCl2 at a final concentration of 4 mM with 3 mM
respectively. For ADCC assays target cells were labelled with 100 µCi of Na251
CrO4 for 2 hours
prior to treatment +/-10 µg/ml of mAb prior to treatment with CD16+NK-92 in 96 well plates.
89
4.3.3 ADCC chromium release assay
Target cells were labelled with 100 µCi of Na251
CrO4 for 2 hours with simultaneous incubation
with +/- 10 µg/ml of mAb (anti-class I HLA A, B, C or 7G3 or isotype control) prior to treatment
with CD16+NK-92 in 96 well plates in a standard chromium release assay as previously
described. Additional spontaneous and maximal release controls were conducted with and
without antibody pretreatment. Plates were centrifuged at 400 g and 100 μl of supernatant were
collected and assayed in a gamma counter.
4.3.4 Flow cytometry
Immunophenotyping of BM was done using an FC500 or Facscalibur flow cytometer. FACS
buffer was made with PBS + 2mM EGTA + 2% FBS. Primary AML and leukemic stem cell
fractions were detected using the following antibodies: anti-CD34 PE (BD biosciences), anti-
CD34 FITC (BD biosciences), anti-CD38 APC (Becton Dickson) and anti-CD123 PE (BD
Biosciences), anti-CD45 APC (BD Biosciences) and anti-class I HLA A, B, C (BD Biosciences)
NK-92 cells lines were assessed for CD16 expression using CD16 PE (BD Biosciences).
Leukemia cell lines were evaluated using anti-CD123 PerCy5.5 (BD Biosciences).
4.3.5 Cell sorting
Cell sorting was done using a FacsAria cell sorter. Primary AML samples were stained with
anti-CD34 PE and anti-CD38 APC (BD Biosciences). 10 million fresh primary AML blasts
were thawed, washed and resuspended in alpha-MEM +20%FBS +10% 5637 conditioned
medium. Viability was assessed using trypan blue exclusion test. Samples were then washed in
10 ml of FACS buffer (PBS, 1% FBS, 2 mM EDTA), supernatant discarded and resuspended in
2 ml FACS buffer (~5x106 cells/ml). Cells were then filtered using 5ml polystyrene round tube
with cell-strainer caps (BD Falcon). Samples were split samples into five groups to use in
setting gates: not sorted control, untreated control (ie no antibody treatment, but run through
sorter), CD34 PE control, CD38 APC control, CD34 PE + CD38 APC control. Following
antibody addition, samples were incubated at room temperature for 20-30 minutes in the dark,
washed with 10 ml of FACS buffer and resuspended in 1-2 ml of FACS buffer to yield 5 million
cells/ml. Following establishment of the CD34+CD38+ and CD34+CD38- gates, remaining
primary AML cells with double staining were sorted into these two fractions for later use a
chromium release assay as outlined previously. Purity checks were conducted to confirm the
90
efficacy of the sorting procedure and viability of cells were again determined with trypan blue
exclusion assay prior prior to use in chromium release assay.
4.3.6 Methylcellulose cytotoxicity assay
Effectors and targets were incubated together in 96 well U bottom plates for four hours and spun
at 500 rpm (high density conditions), and then infused into methylcellulose (low density
conditions) and evaluated at two weeks for cell lines and four weeks for primary AML samples.
Controls included tumour alone and tumour and effector incubated in separate wells for four
hours and co-infused into methylcellulose (low density control) to determine the impact of
effectors on targets in methycellulose under low density conditions. Five primary AML samples
(10 000 cells/plate) were incubated with or without NK-92 at 25:1 E:T ratio for four hours in 96
well U bottom plates comparable to a chromium release assay setup. Subsequently cells were
transferred into methylcellulose and 10 000 whole AML blasts from each treatment group were
transferred to each of three 35 mm plates. Methylcellulose plates were examined for colonies at
2-4 weeks and enumerated (colony definition >=50 cells) and number of colonies on each of the
three plates per group were averaged and presented +/- SD. Percent clonogenic inhibition was
calculated according to the following equation:
% Colony inhibition = [(#ColoniesLDC)- (#ColoniesTreatment)] x100%
[#ColoniesLDC]
#ColoniesLDC=the number of colonies in the low density control.
4.3.7 Animals
NOD/SCID gammanull
(NSG) mice from The Jackson Laboratory were bred and maintained in
the Ontario Cancer Institute animal facility according to protocols approved by the Animal Care
Committee. Mice were fed irradiated food and Baytril containing water ad libitum during
experimental periods. Prior to infusion with AML NSG mice were irradiated with 325 or 225
cGy to facilitate engraftment. We developed a primary AML xenograft model utilizing a patient
derived AML sample outlined in the results.
4.3.8 Statistics
Survival analysis was done with Kaplan Meier survival curves using the log rank rest with
Medcalc software. Comparison of cytotoxicity data was done using two tailed student’s t-test
were done to compare in vitro cytotoxicity and engraftment data using Medcalc software.
91
4.4 Results
4.4.1 NK-92 is cytotoxic against primary AML by granule exocytosis
We initially set out to determine cytotoxicity of NK-92 against primary AML using a standard
chromium release assay as a measure of bulk tumour cell kill. Primary AML was killed in a
dose-dependent manner, and in the presence of the calcium chelator EGTA, killing was
abrogated (Figure 4.1). A larger panel of five primary AML blast samples treated with NK-92
yielded a dose-dependent response and moderate degrees of cytotoxicity against 4 samples at a
25:1 E:T (% lysis): 080179 (42.3+/-3.6%), 080078 (29.8+/-3.6%), 43.9+/- 1.47%, 42.6+/-0.1%
(Figure 4.2).
-10
0
10
20
30
40
50
60
70
80
90
100
1:1 5:1 10:1 25:1
% L
ysis
Effector:Target ratio
AIM-V
EGTA
Figure 4.1: Chromium release assay of NK-92 a against a primary AML sample at a range
of Effector:Target ratios with and without calcium chelation
AML blasts (sample 080078) were labelled with 100 µCi of Na251
CrO4 prior to treatment with
NK-92 at four E:T ratios with and without calcium chelator EGTA 4 mM and MgCl2 3 mM.
Data are presented as the mean percent lysis of triplicate samples (+/-SD) from a representative
experiment done three times.
92
0
10
20
30
40
50
60
70
80
90
100
080179 080078 080008 0909
% L
ys
is
Patient sample ID
1:1
5:1
10:1
25:1
Figure 4.2: Chromium release assay of NK-92 against a panel of primary AML patient
samples at a range of Effector:Target ratios
Freshly thawed primary AML blast samples were labelled with 100 µCi of Na251
CrO4 prior to
treatment with NK-92 at four E:T ratios. Data are presented as the mean percent lysis of
triplicate samples (+/-SD) from a representative experiment done three times.
93
4.4.2 Chromium release assay versus methylcellulose cytotoxicity assay
We tested NK-92 against two AML cell lines in a simultaneous chromium release assay using
OCI/AML2 and OCI/AML3 as targets. As demonstrated previously with KG1 (Chapter 3),
OCI/AML3 clonogenic cells were more sensitive to NK-92 than bulk tumour. However,
OCI/AML2 bulk and clonogenic cells were equivalently sensitive to NK-92 (Figure 4.3). K562
and OCI/AML5 also showed less sensitivity in the CRA than the MCA (data not shown), making
OCI/AML2 the only exception to this phenomenon. This demonstrates that the enhanced
cytotoxicity measured in the MCA relative to the CRA for most targets is not intrinsically related
to the method of data comparison.
0
10
20
30
40
50
60
70
80
90
100
OCI/AML2 OCI/AML3
% ly
sis
or
colo
ny
inh
ibit
ion
Cell line
CRA
MCA
*
Figure 4.3: Clonogenic cytotoxicity assay of NK-92 against OCI/AML2 and OCI/AML3
NK-92 cytotoxicity was determined against cell lines OCI/AML2 and OCI/AML3 using a
methylcellulose cytotoxicity assay (MCA) and chromium release assay (CRA) both with four
hour incubations. For the CRA, OCI/AML2 and OCI/AML3 were labelled with 100 µCi of
Na251
CrO4 for 2 hours prior to treatment with NK-92 at a 25:1 E:T in 96 well U bottom plates.
For the MCA, targets cells, target cell + effector cells incubated separately and target cells and
effector cells incubated together were infused into methylcellulose. Colonies were quantified by
inverted microscopy following 10 days at 37°C incubation. CRA is presented as % lysis and
MCA results are presented as % colony inhibition. Data are presented as the mean of triplicate
samples (+/-SD) from a representative experiment repeated 3 times. *= p<0.05.
94
4.4.3 NK-92 preferentially kills leukemic stem cells relative to bulk
leukemia
To determine the impact of NK-92 on LSCs we sorted a primary AML sample into
CD34+CD38- LSCs and CD34+CD38+ fractions for further testing using a CRA. Primary
AML-derived CD34+CD38- sorted LSCs were more sensitive to killing than CD34+CD38+
blasts by NK-92 in a 4 hour CRA at E:T ratios of 1:1 (58.9+/-11.5%, 20.3+/-1.7%), 5:1 (78.3+/-
9.7%, 43.5+/-11.1%) and 10:1 (72.9+/-5.6%, 38.5+/-2.4%), but this difference was not
significant at a 25:1 E:T ratio (Figure 4.4).
0
10
20
30
40
50
60
70
80
90
100
1:1 5:1 10:1 25:1
% L
ysis
Effector:Target ratio
CD34+CD38+
CD34+CD38-
Figure 4.4: NK-92 cytotoxicity against sorted leukemic stem cells (CD34+CD38-) and bulk
leukemia (CD34+CD38+)
Primary AML samples were stained with anti-CD34PE and anti-CD38APC and sorted into
CD34+CD38- and CD34+CD38+ fractions for subsequent testing in a chromium release assay.
Sorted primary AML cells were labelled with Na251
CrO4 and 10 000 labelled cells were plated
into wells of a 96 well U bottom plate and treated with NK-92 at four E:T ratios. Data are
presented as the mean percent lysis of triplicate samples (+/-SD). Data are representative of two
separate experiments.
95
To test the impact of NK-92 against LSCs relative to bulk tumour, we performed a CRA with a
methylcellulose cytotoxicity assay (MCA) designed to measure the killing during the four-hour
co-incubation. To correct for the effect of NK-92 against targets during the 4 week incubation in
methylcellulose we also utilized a ‘low density control’, whereby NK-92 and targets that were
co-infused into methylcellulose without prior incubation together in the 96 well plate under
‘high density’ conditions (Figure 4.5). There was a minor inhibitory effect of colony formation
from low density incubation with NK-92 on sample 0909. Therefore, all calculations of
cytotoxicity were done relative to the low density control rather than baseline control. The MCA
showed that NK-92 at 25:1 E:T eliminated clonogenic growth of 3/5 primary AML blast samples
with minimal colony growth in 2/5 yielding % colony inhibition values of: 100+/-0%, 86.3+/-
2.3%, 98.4+/-2.8%, 100+/-0% and 100+/-0%, demonstrating much higher cytotoxicity than
obtained with the CRA, which was done on the same day (Figure 4.6).
15 ml conical tube 33 mm petri dish96 well U bottom plate
4 hrs 4 weeks
4 hrs 4 weeks
4 weeks
4 hrs 4 weeks
Tumour cell target NK-92 effector cell
Cancer stem cell target
Dead cell Colony
Control
Treatment
Low density control
Figure 4.5: Schematic of methylcellulose cytotoxicity assay
96
A
Control (i) Low density control (ii) Treatment (iii)
B
0
20
40
60
80
100
120
080179 080078 08008 0909
% L
ysi
s o
r %
co
lon
y in
hib
itio
n
Sample #
CRA
MCA
Figure 4.6: NK-92 against primary AML blasts using the methylcellulose cytotoxicity assay
compared to the chromium release assay
An example of the MCA (A) shows a representative assay for one sample (080179) with a
control (AML only) (i), low density control (AML + NK-92 in methylcellulose only) (ii) and
treatment group (AML + NK-92) (iii). Four primary AML samples were incubated with or
without NK-92 at a 25:1 E:T ratio for 4 hours in 96 well U bottom plates and utilized in a
chromium release assay setup and methylcellulose cytotoxicity assay (MCA) and % lysis values
and % colony inhibition compared (B). Methylcellulose plates were examined for colonies at
two weeks and enumerated (colony definition >=50 cells) and number of colonies on each of the
three plates per group were averaged and presented +/- SD.
97
4.4.4 Primary AML xenograft model
We developed a primary AML xenograft model utilizing a patient derived M4 sample with
aggressive engraftment features infused into irradiated NSG mice. The primary AML sample
was CD34+ and contained a small fraction of CD34+CD38- cells that were predominantly
CD123+ (Figure 4.7). NSG mice were irradiated with 200 or 325 cGy of irradiation followed by
immediate injection with 3x106 of freshly thawed primary AML cells or in vivo passaged
primary AML derived from the spleens of mice with end stage leukemia. This led to a
symptomatic leukemia by week 6. Leukemic progression led to significant weight loss, bone
marrow infiltration with human leukemia, anemia and splenomegaly. Further, potency of
serially transplanted BM or SPL cells from NSG with end stage leukemia was maintained and
could lead to leukemia in mice given secondary, tertiary or quaternary transplants with the same
kinetics of freshly thawed primary AML samples from the leukemia bank (Figure 4.8). Spleen
derived AML was as potent as that derived from BM at inducing leukemia (data not shown).
Figure 4.7: Primary AML immunophenotype
Primary AML 080078 was immunophenotyped using CD34 PE and CD38 APC antibodies and
CD34+CD38+ and CD34+CD38- subfractions were identified.
98
Group1st passage AMLprimary AML
Survival
0 20 40 60
100
80
60
40
20
0
Time
Surv
ival pro
babili
ty (
%)
Figure 4.8: Survival of NSG mice with primary AML versus 1
st passage AML derived from
BM
NSG mice were infused with 3x10e6 freshly thawed primary AML (080791) or spleen derived
first passage AML. Mice were monitored until humane endpoint and sacrificed. Survival was
analyzed using Kaplan Meier curves and log rank test (p= NS).
99
4.4.5 In vitro treatment of primary AML cells by irradiated NK-92 reduce
engraftment potential
In vitro cytotoxicity against LSCs was assessed by treating 1x10e6 first-passage BM-derived
primary AML with or without 5x10e6 iNK-92 and injecting into two cohorts mice (Figure 4.9).
At 6 weeks, mice were sacrificed and bone marrows harvested. Average leukemic engraftment in
the iNK-92 group (79.8, 3.48, 92.1, 81.3, 86.1, Av= 68.6%) was less than untreated AML
inoculated group (95.0, 93.4, 19.4, 95, 97.3, Av=80.0%), but not statistically significant
(p=0.62). Each group had a poor engrafting mouse, possibly related to an interstitial injection of
AML cells rather than directly into the tail vein, which contributed to a very high variance for
each group. Removing one poorly engrafted outlier mouse from each group yielded a higher
engraftment in the control (Average=95.1%) versus the treatment group (Av=84.8%) and was
significant (p=0.011) (Figure 4.10).
Engraftment levels were determined by proportion of cells expressing human class I HLA and
subgated on to assess the CD34+CD38- LSC fraction (Figure 4.10). iNK-92 (HLA+CD34-)
could be differentiated from AML (HLA+CD34+) using this panel and iNK-92 did not
significantly engraft in the BM (<1%) (data not shown).
Day 1
NOD/SCID IL-2Rgc-/- mice
Day X
225 cGy
+/-1000 cGy
iNK-92 Primary AML
+spin 4 hrs
Sacrifice
Inject
Figure 4.9: Schematic of in vitro cytotoxicity assay with in vivo engraftment readout
Primary AML cells (5x106) were incubated with and without iNK-92 (25x10
6) in 15 ml conical
tubes, spun down at 400 g, and incubated a 4 hours at 37° C in 5% CO2. 1x10e6 primary AML
cells +/- 5x106 iNK-92 were injected via tail vein into cohorts of five mice. At six weeks mice
were sacrificed to assess BM engraftment.
100
A B
HLA +
95.0
1 0- 1
1 00
1 01
1 02
1 03
HLA
0
2 0
4 0
6 0
8 0
100
Co
un
t
Q1
0.10
Q2
96.5
Q3
3.23
Q4
0.17
1 0- 1
1 00
1 01
1 02
1 03
CD34
1 0- 1
1 00
1 01
1 02
1 03
CD
38
HLA +
79.8
1 0- 1
1 00
1 01
1 02
1 03
HLA
0
2 0
4 0
6 0
8 0
100
Co
un
t
Q1
0.19
Q2
96.6
Q3
3.02
Q4
0.19
1 0- 1
1 00
1 01
1 02
1 03
CD34
1 00
1 01
1 02
1 03
CD
38
C p=0.62
Engraftment of AML
Control Treatment0
50
100
150
% E
ngra
ftm
ent
Figure 4.10: In vitro cytotoxicity assay with in vivo engraftment readout NSG mice were sacrificed 6 weeks after injection of primary AML +/- iNK-92 BM assayed for
leukemic engraftment using anti-class I HLA antibody followed by subgating of CD34+CD38+
and CD34+CD38- subfractions. Plots are of a typical control mouse with 95.0% engraftment
(top) and therapy mouse with 79.8% engraftment (bottom) (A). Spleen sizes were enlarged in all
mice except one mouse in control and treatment groups with very low BM engraftment (B).
Engraftment of mouse bone marrow in controls versus treatment is shown (C).
101
4.4.6 Irradiated NK-92 reduce leukemic stem cell fraction in secondary
transplantation assay
To assess the cytotoxic effect of irradiated NK-92 (iNK-92) on LSCs in the in vivo setting,
secondary transplantation experiments were conducted to evaluate total engraftment and fraction
of LSCs in secondary recipients. Primary AML cells (3x10e6) were also infused into two
cohorts of four mice and treated with or without iNK-92 from day 2 and given 15x10e6 cells
twice weekly to a total dose of 75x10e6. At six weeks mice were sacrificed and bone marrow
(1x10e6 cells) from each of 4 primary recipients in control or treatment were serially
transplanted 1:1 into four new NSG mice. Evaluation of BM from secondary recipients
inoculated with AML only revealed a high proportion of human CD45+ cells (80.8, 93.3, 80.4,
96.4 Av=87.7%), while one mouse from iNK-92 group was leukemia free with engraftment at
background levels of non-injected mice (96.4, 94.7, 1.8, 95.7 Av=72.2%). The difference
between average leukemic secondary engraftment between groups was not significant, because
of high sample variance in the treatment group. However, the proportion of CD34+CD38-
CD123+ cells in secondary transplanted mice for the AML group was 7.85% (8.01, 9.48, 8.66,
5.25) and for the AML + iNK-92 group was 3.66% (7.13, 3.46, 0.03, 4.00), which was
significantly lower (p=0.05) (Figure 4.11).
102
A
Day 1 2
NOD/SCID IL-2Rgc-/- mice
Week 6
AML 3x106 i.v.
Sacrifice
5
225 cGy
NK-92
15x106
i.v.
12 16
+/-1000 cGy
9 19
1x106 BM cells infused into two
new cohorts of four iNSG (1:1)
Week 6
BM flow
B C
Control Treatment
0
50
100
150
% E
ng
rag
tme
nt
Control Treatment
0
2
4
6
8
10
% L
eu
ke
mic
Ste
m C
ells *
Figure 4.11: In vivo cytotoxic impact of iNK-92 on secondary BM engraftment of AML
cells and LSCs
3x10e6 AML cells were also infused into two cohorts of four mice and treated with and without
iNK-92 from day 2 and given 15x10e6 cells twice weekly to a total dose of 75x10e6 (A). BM
(1x10e6 cells) from each of 4 primary recipients in control and treatment was serially
transplanted 1:1 into four new NSG mice. These mice were sacrificed at 6 weeks and bone
marrow assayed for overall leukemic engraftment (B =% human CD45+) and LSC engraftment
(C=% human CD34+CD38-CD123+) (* p=0.05).
103
4.4.7 NK-92 prolongs survival in a primary AML xenograft model
We next sought to assess the impact of NK-92 on survival of mice inoculated with primary
human AML. NSG mice injected with 3x106 primary AML cells received 10x10
6 non-irradiated
NK-92 weekly for three doses (Figure 4.12). This treatment increased median survival from 57
to 72 days (log rank test p<0.01) although most succumbed to disease ultimately (Figure 4.13A).
Autopsy revealed enlarged spleens and pale fragile bones relative to controls. Flow cytometry of
bone marrow from NSG mice innoculated with AML only (Figure 4.13B), or AML + NK-92
treatments (Figure 4.13C) that became symptomatic, had human 99% engraftment of human
leukemia detected in bone marrow, while the mouse that survived long term (~9 months) was
healthy at sacrifice and did not have evidence of leukemic infiltration in BM (Figure 4.14D) or
splenomegaly. To determine the impact of irradiation on the in vivo activity of NK-92, the cells
were irradiated with 1000 cGy prior to infusion into NSG mice inoculated 10 days before with
3x10e6 primary AML cells. NSG mice were administered 20x10e6 iNK-92 ip weekly x 5 doses
and monitored for signs of leukemia. Survival was improved in the treatment group (26 to 48
days) to near statistical significance (p=0.0566), but all mice ultimately succumbed to disease
(Figure 4.14).
NK-92 10x106 i.v.
Day 1 Day 8
NOD/SCID IL-2Rgc-/- mice
Day X
AML 3x106 i.v.
Humane endpoint sacrifice
Day 15
325 cGy
Figure 4.12: Schematic of NK-92 therapy for primary AML xenografted NSG mice
104
A
GroupAMLAML + NK-92
Survival
0 20 40 60 80 100 120 140 160 180 200
100
80
60
40
20
0
Time (days)
Surv
ival p
robabili
ty (
%)
B C D
Anti-Class I HLA FITC
Figure 4.13: NK-92 therapy of primary AML xenografted NSG mice
3x106 primary AML cells were injected via tail vein into irradiated NOD/SCID gamma null
mice. 10x106 NK-92 were infused via tail vein weekly for three weeks starting on the day of
AML innoculation. Mice were monitored for signs of leukemia and sacrificed at humane
endpoints. Kaplan Meier survival curves were generated to compare survival in control and
treatment groups (P<0.01) (A). Autopsies were performed on select mice from each cohort,
including flow cytometry of BM to determine leukemic engraftment. Unstained (blue) and anti-
Class I FITC stained (red) specimens are presented from one mouse with symptomatic leukemia
(B), a mouse with AML in the treatment group that developed symptomatic leukemia (C) and a
healthy long term survivor (D).
*
105
Day 0
NOD/SCID IL-2Rgc-/- mice
Day X
AML 3x106 i.v.
Death/sacrifice
10
325 cGy
iNK-92
20x106
i.p.
14 17
+1000 cGy
12 15 19
GroupAMLAML + iNK-92 IP
Survival
0 10 20 30 40 50 60 70 80
100
80
60
40
20
0
Time
Surv
ival p
robabili
ty (
%)
Figure 4.14: iNK-92 therapy of primary AML xenografted NSG mice
3x106 primary AML cells were injected via tail vein into irradiated NOD/SCID gamma null
mice. iNK-92 given ip 20x20e6 weekly x 6 were used to treat of AML xenografted mice starting
10 days after inoculation. Mice were monitored for signs of leukemia and sacrificed at humane
endpoints. Kaplan Meier survival curves were generated to compare survival in control and
treatment groups (p=0.0566)
CD
38
AP
C
106
4.4.8 iCD16+NK-92 can mediate ADCC against bulk and stem cell
antigens in vitro
To develop a strategy to enhance killing of LSCs we utilized a gene-modified CD16+NK-92
transduced with the high affinity CD16 receptor (NK-92.176V GFP), which is capable of
mediating ADCC against antibody coated targets. Expression level of CD16 was 2.3% on NK-
92 and 27.9% on CD16+ NK-92 (Figure 4.15A and B). Three AML cell lines were assessed for
levels of CD123 expression by flow cytometry which demonstrated: OCI/AML2 (69.9%),
OCI/AML3 (11.3%) OCI/AML5 (31.1%) (Figure 4.14C, D and E). We opted to evaluate
OCI/AML5 further because of its considerable degree of CD123 expression, fast cycling time,
excellent colony growth in methylcellulose and its rapid engraftment capability in
immunodeficient mice allowing for in vivo studies in the future.
A B
CD16+
2.26
1 0- 1
1 00
1 01
1 02
1 03
CD16
0
2 0
4 0
6 0
8 0
100
Co
un
t
CD16+
27.9
1 0- 1
1 00
1 01
1 02
1 03
CD16
0
2 0
4 0
6 0
8 0
100
Co
un
t
C D E
CD 123+
31.1
1 0- 1
1 00
1 01
1 02
1 03
CD123
0
2 0
4 0
6 0
8 0
100
Co
un
t
CD123+
11.3
1 0- 1
1 00
1 01
1 02
1 03
CD123
0
2 0
4 0
6 0
8 0
100
Co
un
t
CD123+
69.9
1 0- 1
1 00
1 01
1 02
1 03
CD123
0
2 0
4 0
6 0
8 0
100
Co
un
t
Figure 4.15: Immunophenotyping of CD16+NK-92, NK-92 and OCI/AML 2, 3 and 5
Immunophenotyping of NK-92 (A) and CD16+NK-92 (B) after staining with with CD16 PE and
OCI/AML 2, 3 and 5 (C, D and E respectively) after staining with CD123 PerCy5.5 PE.
Samples were run on an FC500 flow cytometer. Unstained (red) and stained (blue) populations
are presented in the histogram. Gates were set to exclude 99% of unstained events for each cell
line to define positive and negative populations.
107
The chromium release assay was modified to measure ADCC by coating targets with antibodies
during the 2 hour chromium incubation. Isotype control antibody (DNP) and anti-class I HLA
antibodies at 10 µg/ml were utilized to coat primary AML targets known to express high levels
of class I. CD16+NK-92 killed primary AML at E:T ratios of 25:1, 10:1, 5:1 and 1:1 (% lysis
+/-SD: 34.7 +/-4.6, 15.6 +/- 4.7, 11.5 +/- 2.0, 7.7 +/-1.6) which was significantly enhanced (2-
3x) by coating with anti-Class I monoclonal antibodies relative to isotype control (% lysis +/-SD:
64.8 +/-10.4, 31.1 +/- 8.9, 30.2 +/- 9.4, 23.9+/-2.8) (Figure 4.16).
0
10
20
30
40
50
60
70
80
90
100
25:1 10:1 5:1 1:1
None
TNP
HLA I
Figure 4.16: CD16+NK-92 in vitro ADCC assay against primary AML
Primary AML cells were labelled with 100 µCi of Na251
CrO4 for 2 hours +/- 10 µg/ml of mAb
(isotype control-DNP or anti-class I HLA) and washed x2 in AIM-V serum free medium prior to
treatment with CD16+NK-92 in 96 well plates in a standard chromium release assay. Primary
AML pre-treated with anti-Class I HLA antibody or isotype control (clone MG1-45 specific to
TNP) prior to incubation with CD16+NK-92. Data are presented as the mean percent lysis of
triplicate samples (+/-SD) from a representative experiment done two times.
108
Isotype control antibody (DNP) and anti-CD123 (7G3) antibodies at 10 µg/ml were utilized to
coat OCI/AML5 targets shown to express CD123 previously. CD16+NK-92 was cytotoxic
against OCI/AML5 cells at E:T ratios of 25:1, 10:1, 5:1 and 1:1 (% lysis +/-SD: 35.0 +/-4.0,
9.0+/-6.6, -2.0 +/-0.1, 1.7 +/-3.3) and was significantly enhanced (2-6x) when targets were
coated with anti-CD123 mAb (% lysis +/-SD: 64.3 +/-3.1, 48.5 +/-4.1, 20.9 +/-0.1, 10.1+/-3.3)
(Figure 4.17).
-10
0
10
20
30
40
50
60
70
80
90
100
25:1 10:1 5:1 1:1
% L
ysis
Effector:Target ratio
None
7G3
Figure 4.17: CD16+NK-92 in vitro ADCC assay against OCI/AML5
OCI/AML 5 cells were labelled with 100 µCi of Na251
CrO4 for 2 hours +/- 10 µg/ml of mAb and
washed x2 in AIM-V serum free medium prior to treatment with CD16+NK-92 in 96 well plates
in a standard chromium release assay. OCI/AML5 was treated with anti-CD123 antibody or
isotype control prior to incubation with CD16+NK-92. Data are presented as the mean percent
lysis of triplicate samples (+/-SD) representative of three experiments.
109
4.4.9 CD16+NK-92 improves survival in an AML xenograft model with
enhancement by anti-CD123 mAb therapy
To enhance the therapeutic approach of irradiated NK-92 against AML-xenografted NSG mice,
we treated mice with a CD16+NK-92 cell line, in combination with an anti-CD123 mAb (7G3),
given on the same days to facilitate targeting of leukemic stem cells by antibody-dependent cell-
mediated cytotoxicity. Due to known splenic sequestration of 7G3 in the spleen, a blocking dose
of isotype control antibody BM4 (200 µg) was given prior to the administration of 7G3 at a low
dose (8 µg) (Figure 4.18). In this pilot experiment, we demonstrated that 7G3 (8 µg) could
improve therapeutic efficacy of iCD16+NK-92, as determined by improvement of median
survival by 13 days (p=0.0173) (Figure 4.19).
Day 1 4
NOD/SCID IL-2Rgc-/- mice
Day X
AML3x106 i.v.
Death/sacrifice
5
225 cGy
iCD16+NK-92
20x106
i.p.
10 12
+/-1000 cGy
8 15
8 µg 7G3 mAb
200 µg BM4 mAb
2
Figure 4.18: Schematic of iCD16+NK-92 +/- a single anti-CD123 mAb dose for primary
AML xenografted NSG mice
110
Group
CD16+NK-92
CD16+NK-92 + 7G3
Survival
0 10 20 30 40 50 60 70 80
100
80
60
40
20
0
Time (days)
Su
rviv
al p
rob
ab
ility
(%
)
Figure 4.19: iCD16+NK-92 +/- a single anti-CD123 mAb dose for primary AML
xenografted NSG mice
NSG mice were inoculated with 3x106 passage human AML spleen derived cells (day 0) and
treated with iCD16+NK-92 x 5 doses +/- 8 µg 7G3 starting on day 3. Survival was determined
using Kaplan Meier survival analysis with a log rank test (p=0.0173).
111
We conducted a more rigorously controlled experiment of CD16+NK-92 and anti-CD123 mAb
therapy utilizing an isotype control antibody (BM4) in control arms (Figure 4.20). The cohorts
included: no therapy, 7G3, BM4, iCD16+NK-92, iCD16+NK-92 + 7G3, iCD16+NK-92 + BM4.
The dosing schedule was iCD16+NK-92, with or without 7G3 or BM4, given on day 3, 5, 7, 10,
and 12 after AML inoculation (day 0). NSG mice without therapy had a median survival of 32
days. iCD16+NK-92 alone improved survival to a median of 37 days which was significant
(p<0.001). Treatment with BM4 did not enhance survival with a median survival of 32 days
(p=0.619), but 7G3 improved median survival to 35 days which was significantly increased
relative to the AML only control (p<0.001), but not relative to BM4 isotype control (p=0.1509).
Combination of 7G3 and iCD16+NK-92 produced the best survival outcome, with median
survival of 42 days, which was significantly enhanced over AML only (p<0.001), 7G3
(p<0.0025) and iCD16+NK-92 + BM4 (p<0.0025) (Figure 4.21B). An identically repeated
experiment using an alternative source of 7G3 and a different isotype control (MG2a-53) was
conducted (data not shown). MG2a-53 or 7G3 alone did not have any survival benefit.
iCD16+NK-92 combined with 7G3 enhanced median survival by 8 days relative to iCD16+NK-
92 combined with isotype control (p=0.69) which was statistically significant if one long-term
survivor outlier in the control group was removed from analysis (p=0.03).
Day 0
NOD/SCID IL-2Rgc-/- mice
Day X
AML3x106 i.v.
Death/sacrifice
3
225 cGy
iCD16+NK-92
20x106
i.p.
7 10
+1000 cGy
5 12
100 µg 7G3 mAb i.v. Isotype control BM4
Figure 4.20: Schematic of CD16+NK-92 +/- five doses of anti-CD123 mAb therapy for
primary AML xenografted NSG mice
112
A
Group
iCD16+NK-92
iCD16+NK-92 +7G3
iCD16+NK-92 +BM4
7G3
BM4
No Therapy
Survival
0 5 10 15 20 25 30 35 40 45 50
100
80
60
40
20
0
Time (days)
Surv
ival pro
babili
ty (
%)
B
Group
iCD16+NK-92 +7G3
iCD16+NK-92 +BM4
Survival
0 5 10 15 20 25 30 35 40 45 50
100
80
60
40
20
0
Time (days)
Surv
ival pro
babili
ty (
%)
Figure 4.21: iCD16+NK-92 with and without 7G3 or isotype control treatment for primary
AML xenograft model NSG mice were inoculated with 3x10
6 passage human AML spleen derived cells (day 0) and
treated with iCD16+NK-92 +/- 7G3 or BM4 x 5 doses (3x/week) starting on day 3. Controls
included no therapy and antibodies alone. Survival was determined using Kaplan Meier survival
analysis with a log rank test. All curves are presented (A) with a subgroup comparison (B) of
iCD16+NK-92 +7G3 versus iCD16+NK-92 +BM4 (p=0.0015).
113
4.5 Discussion
While the overall long-term survival for AML is around 40% in adults (Lowenberg, Downing et
al. 1999), subgroups of AML continue to fare very poorly, including patients with the recurrent
cytogenetic abnormalities inv(3)/(t3;3), -5/del(5q) and 7/del(7q), where the 10 year survival is 3,
6 and 10% respectively(Grimwade, Hills et al. 2010). Therefore, there is a need for novel
therapeutic approaches in treating AML, particularly those who have completed therapy, but
have detectable minimal residual disease. We have evaluated the mechanism and efficacy of
NK-92 against primary AML samples, with a focus on using methods that evaluate the impact on
leukemic stem cells and overall survival in an AML xenograft model. We confirmed initial
reports that NK-92 could mediate cytotoxicity in vitro against primary AML(Yan, Steinherz et
al. 1998), and were further able to demonstrate killing was mediated primarily by granule
exocytosis rather than ligand-mediated cytotoxicity (e.g. via Fas ligand). This was evidenced by
minimal killing in the presence of the calcium chelator, EGTA, which prevents calcium influx
required for degranulation of NK and T cells(Ostergaard, Kane et al. 1987; Trenn, Takayama et
al. 1987).
We noted that classically defined sorted CD34+CD38- LSCs(Lapidot, Sirard et al. 1994) were
more sensitive to NK-92 killing than bulk leukemia at low E:T ratios utilizing a standard
chromium release assay. Given the conflicting reports in the literature of the definitive
immunophenotype of the leukemic stem cell in AML(Lapidot, Sirard et al. 1994; Bonnet and
Dick 1997; Taussig, Miraki-Moud et al. 2008; Goardon, Marchi et al. 2011), we opted to use a
clonogenic assay to assess the impact of immune effectors against leukemic stem cells in a larger
set of samples. Primary AML grows well in methylcellulose, allowing for measurement of
colony forming units, which is a measure of stem and progenitor leukemic cells capable of
sufficient divisions to form a colony. Specifically, we used a methylcellulose cytotoxicity assay
(MCA) established previously by our lab(Williams, Wang et al. 2010) that is designed to have
comparable four-hour co-incubation conditions and a baseline control for effects of immune
effectors over the two week incubation in methylcellulose (termed low density control). This
allows for a comparison of the degree of bulk killing versus colony inhibition in a four-hour
period, effectively providing another means to assay differential cytotoxicity against bulk and
LSC populations. An additional benefit of this approach is that it does not require the
114
confirmation of the cell surface markers defining the LSC population in each patient sample,
which requires significant cost and time to assay in immunodeficient mice. The MCA showed
that NK-92 at a 25:1 E:T eliminated clonogenic growth of 2/4 primary AML blast samples with
minimal colony growth in the remaining two, demonstrating a 2-3 fold higher % colony
inhibition than the % lysis measured by the CRA. These results support our initial finding that
NK-92 can preferentially recognize and kill LSCs over bulk leukemia.
Only two studies to date have looked at the in vitro sensitivity of CD34+CD38- LSCs to immune
effector cell killing. In the first, lymphokine-activated killer (LAK) cells and allogeneic
lymphocytes exerted a modest cytotoxic effect on AML LSCs comparable to the effect on the
non-stem cell fraction(Costello, Mallet et al. 2000). In a more recent study, endogenous single
killer immunoglobulin-like receptor (KIR)-expressing NK cells, mismatched for the HLA of
primary AML targets, showed killing against LSCs using both the chromium release assay and a
methylcellulose-based cytotoxicity assay, demonstrating equivalent killing of LSCs and
blasts(Langenkamp, Siegler et al. 2009).
Here, we demonstrate preferential killing of LSCs by NK-92 relative to bulk leukemia, not
shown by these other studies, and utilize a better controlled methylcellulose cytotoxicity assay
than Lankencamp et al. to demonstrate differential cytotoxicity relative to the CRA. This is
consistent with our work on NK-92 treatment of multiple myeloma (MM) cell lines. Using the
MCA, NK-92 showed preferential killing of clonogenic MM cells over bulk tumour as
determined by both a flow cytometric cytotoxicity assay and the CRA (Swift, Williams et al.
2012).
To pursue more in depth studies of the effectiveness of NK-92 in killing LSCs, we developed an
animal model of primary human AML by using NSG mice infused with a primary AML sample
containing a small fraction of CD34+CD38- cells. Primary AML, or in vivo passaged primary
AML cells induced leukemia in NSG mice and maintained comparable potency beyond
quaternary transplantations with a stable immunophenotype. There was no difference in potency
of BM or spleen derived cells or delivery by ip or iv injection sites. In using this model we
demonstrate the ability to test a single-patient primary AML sample over multiple in vivo
experiments independent of the quantity of original samples obtained, effectively generating an
‘in vivo’ primary cell line.
115
Secondary transplantation assays are the current gold standard assay to determine the impact of
small molecule therapies against LSCs(Skrtic, Sriskanthadevan et al. 2011), and have not been
used to evaluate cellular therapies for leukemia. We attempted this by treating mice and
transplanting bone marrow from control and treated mice into new mice in a one-to-one manner,
so as to assess the impact on individual mice rather than a pooled outcome. BM engraftment
occurred in all AML-only cohort secondary mice, while one mouse from iNK-92 group was
leukemia free, with engraftment at background levels of non-injected mice, suggesting that the
primary animal receiving treatment was cured. While the average BM engraftment of secondary
transplant mice in the therapy groups was less than the control, this was not statistically
significant. However, the LSC fraction was significantly decreased in secondary recipients,
providing some evidence of NK-92 cytotoxicity against LSCs in the secondary transplant assay.
AML-xenografted NSG mice were effectively treated with NK-92 infusions, leading to
improvement in survival relative to controls, confirming previous work (Yan, Steinherz et al.
1998). We accomplished this with lower doses of NK-92 on a less compressed schedule
(10x10e6 weekly for three doses) than the original study (20x10e6 every other day for five
doses), and without the use of IL-2 in the regimen. Irradiated NK-92 could prolong survival in
mice, but was less effective than the non-irradiated cells. We postulate that the reason for this
reduction in therapeutic efficacy is the lack of ability to expand in vivo.
We have demonstrated for the first time that irradiated NK-92 improves survival in an AML
xenograft model, which has translational relevance given that this is the cellular preparation
given to patients in the phase I setting. However, given the modest effects of iNK-92 on
improving survival in vivo, we recognized the need to enhance its efficacy to achieve better
therapeutic outcomes. We therefore attempted to use a gene modified CD16+NK-92 in
combination with monoclonal antibodies to enhance killing of leukemia by ADCC. CD16+NK-
92 have been utilized in combination with Rituximab to enhance killing of CD20+ malignant
targets, showing their potential to enhance killing of cells expressing a tumour-associated
antigen(Binyamin, Alpaugh et al. 2008). CD16+NK-92 cytotoxicity against primary AML was
enhanced when target were coated with anti-class I antibodies via ADCC, demonstrating that
CD16+NK-92 can be redirected against bulk primary leukemia using a highly expressed cell
surface marker. More importantly, coating of OCI/AML5 with a murine anti-human CD123
116
mAb (7G3) was able to facilitate ADCC indicating the ability to redirect CD16+NK-92 against a
leukemia stem cell-specific antigen.
We then sought to combine iCD16+NK-92 and 7G3 therapy in our AML xenograft model.
Systemic treatment with 7G3 alone has been tested in an AML NOD/SCID xenograft model with
evidence of impact on primary bone marrow engraftment(Jin, Lee et al. 2009). The dosage
regimen used in one experiment in this study was 300 µg IV give three times weekly for 4 doses,
which reduced primary engraftment of 1/3 primary AML samples relative to isotype control. In
this study, survival could only be improved by ex vivo coating of AML cells prior to
administration, or passive immunization of mice with 7G3 prior to injection with AML, which is
not a clinically relevant model.
We initially used a very small quantity of 7G3 in a single dose, preceded by blocking with a non-
specific isotype-matched antibody to block Fc receptors, and improve circulation and binding of
7G3 to CD123 as established by Leyton et al. (Leyton, Hu et al. 2011). This approach worked in
increasing the efficacy of the iCD16+NK-92 cells and improving survival. In a follow-up
experiment, we did not use an Fc blocking pre-dose strategy, but used 100 µg of 7G3 or isotype
control antibody BM4 for five doses given with or without the iCD16+NK-92 cells. This was at
a dose level comparable to that used by initial studies by Jin et al. that would likely have
minimal impact on survival, but lead to circulating levels of antibody capable of facilitating
ADCC from infused cells. Doses of 7G3 were given just after administration of cells to optimize
ADCC, given the 7 hour half-life of 7G3(Leyton, Hu et al. 2011).
In this experiment, iCD16+NK-92 alone prolonged survival over control. The BM4 antibody
had no therapeutic effect and did not enhance iCD16+NK-92, while 7G3 alone had a modest
survival benefit above control, which was not quite statistically significant above BM4 isotype
control group. Of note, the best outcome was in the iCD16+NK-92 + 7G3 treated group which
had a ten day improvement in median survival relative to the iCD16+NK-92 + BM4 treatment
group. Therefore, this demonstrates that the combination of iCD16+NK-92 with 7G3 can
improve survival by antibody-dependent cell-mediated cytotoxicity. Further, this represents the
first demonstration of in vivo efficacy of the CD16+NK-92 cell line alone, and in combination
with antibody which has only previously been tested in vitro(Binyamin, Alpaugh et al. 2008).
Recently, an Fc optimized anti-CD123 humanized monoclonal antibody CSL362 (derived from
117
7G3) was shown to facilitate ADCC from peripheral blood-derived allogeneic NK cells against
primary AML and CD123-expressing cell line targets.(Busfield, Biondo et al. 2014) Further,
CSL362 is in clinical trials and would make an ideal combination therapy with CD16-NK-92.
In summary, we demonstrate that NK-92 can preferentially target LSCs over bulk leukemia in
vitro and irradiated NK-92 can impact survival in an AML xenograft model, which can be
enhanced using CD16+NK-92 in combination with anti-CD123 monoclonal antibodies. This
provides the first proof-of-principle for the targeting of leukemic stem cells by combining an
antibody and a standardized cellular therapy. A humanized version of 7G3 (CDSL360 and
CLS362) has been developed which would allow for therapeutic translation of this approach into
a clinical trial in the future. The approach we demonstrate can be readily applied to enhance
targeting of any antigen and is of particular novelty here, because we are targeting a cancer stem
cell marker and improving survival.
118
5 Chapter 5: NK cell line killing of leukemia cells is
enhanced by reverse antibody dependent cell mediated
cytotoxicity (R-ADCC) via NKp30 and NKp44 and
target cell Fcγ receptor II (CD32)
Contributions:
X.-H. Wang: Assisted in experimental design, chromium release assays and animal
experimentation.
S. Maghera: Assisted in clonogenic assays, flow cytometery and data analysis.
R. Cheng: Assisted in clonogenic assays.
J. Patterson: Conducted HTS flow cytometry on NK-92, KHYG-1, OCI/AML3 and
OCI/AML5.
B. Routy: Assisted in assays involving esophageal cancer cell lines and data analysis.
119
5.1 Abstract
NK-92 and KHYG-1 are natural killer cell lines with the potential to treat cancer. Phase I NK-92
trials show minimal toxicity while KHYG-1 has not been tested in humans. We evaluated their
cytotoxicity against leukemia and esophageal cell lines and primary acute myeloid leukemia
(AML) and modulated efficacy with monoclonal antibodies against NK activating receptors.
Pretreatment of NK-92 and KHYG-1 with antibodies (0.01-10 µg/ml) against the activating
receptors NKp30 and NKp44 led to enhancement of cytotoxicity against a panel of leukemic cell
lines and primary AML samples, but not esophageal cancer cell lines. Immunophenotyping
cancer cell lines showed high expression of Fcγ receptor II (CD32) on leukemia cell lines, which
was absent on esophageal cancer cell lines. There was a significant correlation with CD32 target
expression and anti-NKp30 or anti-NKp44-mediated cytotoxic enhancement for KHYG-1
(p<0.01), implicating reverse antibody-dependent cell-mediated cytotoxicity (R-ADCC) as the
mechanism of enhancement. Clonogenic OCI/AML5 were more sensitive to NK-92 than
KHYG-1. However, anti-NKp30 pretreatment of KHYG-1 enhanced percent colony inhibition
by three-fold with minimal enhancing effect on NK-92. NOD-SCID gamma null mice injected
ip with OCI/AML5 co-incubated in vitro with anti-NKp30-pretreated, irradiated KHYG-1
(iKHYG-1) showed improved survival over control and iKHYG-1 treatments (p<0.05).
Treatment of OCI/AML5-xenografted mice with iKHYG-1 infusions without or with anti-
NKp30 pretreatment improved median survival 35 or 37 days over control, respectively. In
summary, we demonstrate that KHYG-1 cytotoxicity can be enhanced by R-ADCC, leading to
enhanced killing of leukemic targets in both bulk and clonogenic in vitro cytotoxicity assays.
Finally, we demonstrate for the first time, the efficacy of KHYG-1 in improving survival using
an in vivo model of cancer.
120
5.2 Introduction
Acute myeloid leukemia (AML) is a hematopoietic malignancy involving precursor cells
committed to myeloid development, and accounts for a significant proportion of acute leukemias
in both adults (90%) and children (15-20%).(Hurwitz, Mounce et al. 1995; Lowenberg, Downing
et al. 1999) Despite 80% of patients achieving remission with standard chemotherapy (Hurwitz,
Mounce et al. 1995; Ribeiro, Razzouk et al. 2005), survival remains unsatisfactory because of
high relapse rates from minimal residual disease (MRD). The five-year survival is age-
dependent; 60% in children(Rubnitz 2012), 40% in adults under 65(Lowenberg, Downing et al.
1999) and 10% in adults over 65 (Ferrara and Schiffer 2013). These outcomes can be improved
if patients have a matched hematopoietic cell donor, but most do not, highlighting the need for an
alternative approach to consolidation treatment, such as NK cell therapy.
In haplotype transplantation, the graft-versus-leukemia effect is mediated by NK cells when
there is a KIR receptor-ligand mismatch, which can lead to improved survival in the treatment of
AML (Ruggeri, Capanni et al. 2002; Ruggeri, Mancusi et al. 2005). Further, rapid NK recovery
is associated with better outcome and a stronger GVL effect in patients undergoing haplotype T-
depleted hematopoietic cell transplantation (HCT) in AML.(Savani, Mielke et al. 2007) Other
trials have used haploidentical NK cells expanded ex vivo to treat AML in adults(Miller, Soignier
et al. 2005) and children(Rubnitz, Inaba et al. 2010), supporting a therapeutic role for NK cells in
AML therapy. However, all current adoptive immunotherapy protocols are affected by donor
variability in the quantity and quality of effector cells, variables that could be eliminated if
effective cell lines were available to provide more standardized therapy.
Several permanent NK cell lines have been established, and the most notable is NK-92, derived
from a patient with non-Hodgkin’s lymphoma expressing typical NK cell markers except for
CD16 (Fc gamma receptor), making it incapable of antibody-dependent cell-mediated
cytotoxicity (ADCC). NK-92 has undergone extensive preclinical testing and exhibits superior
lysis against a broad range of tumours compared with activated NK cells and lymphokine-
activated killer (LAK) cells.(Gong, Maki et al. 1994) Further, NK-92 has been evaluated in
three phase I clinical trials for: renal cell carcinoma and melanoma (Arai, Meagher et al. 2008),
solid tumours and lymphoid malignancies(Tonn, Schwabe et al. 2013), and lymphoma and
121
multiple myeloma (in progress at our center). There have been minimal cytotoxicities reported
in these trials.
Another NK cell line with therapeutic potential is KHYG-1, derived from a patient with an NK
cell leukemia with a p53 mutation.(Yagita, Huang et al. 2000), with the unique features of
constitutively phosphorylated ERK2 (Suck, Branch et al. 2005) and polarized granules(Suck,
Branch et al. 2006). Like NK-92, irradiation of KHYG-1 prevents proliferation, but preserves
cytotoxicity in vitro (Suck, Branch et al. 2006), thereby making it safe for human administration.
Here, we demonstrate that pretreatment of NK cell lines with monoclonal antibodies to activating
receptors causes several-fold enhancement of cytotoxicity against leukemic cell lines and
primary AML blasts. This effect is most prominent with anti-NKp30 and anti-NKp44
pretreatment of KHYG-1 against CD32-expressing targets implicating reverse antibody-
dependent cell-mediated cytotoxicity (R-ADCC) as the mechanism of enhancement. We further
demonstrate an impact of NKp30 pretreated KHYG-1 in an in vivo model.
122
5.3 Methods
5.3.1 Cell lines and primary samples
K562 was obtained from the ATCC and maintained in IMDM + 20% FBS and 10% fetal bovine
serum (FBS), respectively. KG1 and KG1a was obtained from the ATCC and maintained in
IMDM + 20% FBS and 10% FBS, respectively. OCI/AML 2, 3 and 5 were derived at the
Ontario Cancer Institute (OCI). OCI/AML 2 and 3 were cultured in MEM alpha + 10% FBS and
OCI/AML5 was cultured in MEM alpha + 10% FBS and 10% 5637 bladder carcinoma condition
medium. KHGY-1 was purchased from The Human Science Research Resources Bank
(JCRB0156; Tokyo, Japan) and cultured in GM1 (Ex Vivo medium with 450 U/ml and human
A/B serum). NK-92 was obtained from Dr. Hans Klingemann and also cultured in Ex Vivo with
human A/B serum and 450 U/ml of IL-2 (GM1). KHYG-1 was irradiated (iKHYG-1) with 1000
cGy prior to use in in vivo experiments. Four primary AML samples were obtained from the
Princess Margaret Hospital Leukemia Tissue Bank as per institutional protocol (5890, 080179,
080078, 080008, 0909). The University Health Network HLA database was linked with the
leukemia bank with REB approval to identify the HLA class I type of leukemia specimens.
5.3.2 Chromium release assay
We utilized a standard chromium release assay as previously described by our group (Williams,
Wang et al. 2010) and detailed in the Chapter 2 methods section. Briefly, 1x106 target cells were
labelled with 100 µCi of Na251
CrO4 for 2 hours prior to plating 10 000 cells per wells followed
by treatment with NK-92 at various concentrations. The amount of 51
Cr present in supernatants
was determined using a gamma counter and percent lysis calculated.
5.3.3 Antibody pretreatment of NK cell effectors
All antibodies used were from Biolegend. For NK pretreatment experiments, antibodies against
the following NK receptors were utilized (clone; product #): NKp30 (clone P30-15; 325204),
NKp44 (clone P44-8; 325104), NKp46 (clone 9E2; 331904), DNAM-1 (clone DX-11; 316802),
NKG2D (clone 1D11; 320810), CD7 (CD7-6B7; 343102). Isotype controls specific to trinitrol
phenol + KLH were utilized: MG1-45 (clone MG1-45; 401404) and MG2a-52 (clone MG2a-53,
123
401502). Briefly, 1.5x106 NK cells (NK-92 or KHYG-1) were treated with in 1 ml of AIM-V
serum free medium for 1 hour, washed in 10 ml of AIM-V medium and resuspended in 1.5 ml of
AIM-V medium (1x106/ml). Concentration of antibodies ranged from 10 µg/ml to 0.01 µg/ml.
0.1 ul (105 cells) of NK cell suspension were added to 10 000 tumour targets also in AIM-V
medium in 96 well U bottom plates to yield a 10:1 E:T ratio.
5.3.4 Flow cytometry
Immunophenotyping of BM was done using an FC500. FACS buffer was made with PBS
+2mM EGTA + 2% FBS. For routine flow cytometry of leukemia and esophageal cancer cell
lines the following antibodies to Fcγ receptors were utilized: CD16 APC (clone 3G8, 302011),
CD32 PE (clone FUN-2, 303205), CD64 FITC (clone 10.1; 305005). Antibody concentrations
were utilized at ~1 µg/ml in a 50 µl reaction volume with 200 000 to 1,000 000.
5.3.5 High throughput sampling flow cytometry
Commercially validated FITC, PE or APC conjugated antibodies (374) to cell surface markers
(BD Pharmingen, eBioscience, Abcam, AbD Serotech, BioLegend, Lifespan Biosciences,
Miltenyi, R&D Systems, Beckman-Coulter, and Imgenex) were aliquotted into individual wells
of 96-well plates in Hanks Balanced Salt Solution supplemented with 1% bovine serum albumin
and 2 mM EDTA (FACS buffer) at a dilution of 1:25 (Supplemental Table 1, 2). NK-92 or
KHYG-1 cells (30x10e6) were prepared in 10 ml of PBS, spun down and resuspended in HBSS
+ 1% BSA, 2mM EDTA and volume adjusted to 1x106/ml. 50 ul of cell suspension (50 000
cells) suspension was added to each well to yield a final antibody dilution of 1:50. Cells were
stained for 30 minutes on ice at a concentration of 0.25-1.0x106/mL, washed once with cold
FACS buffer, and resuspended in FACS buffer with 0.1 µg/mL DAPI to allow for dead cell
exclusion. Flow cytometry was performed using a High Throughput Sampler-equipped Becton-
Dickinson LSRII flow cytometer. Plates were placed into an automated flow cytometry plate
reader. Data was acquired and analyzed on FlowJo 9. Gating strategy utilized both a FS and SS
plot and subsequent DAPI staining to exclude non-viable cells, followed by FSC-H and FSC-W
to exclude doublets. Final gate was contoured around viable, unstained cells. Percentage positive
cells and mean fluorescence intensity were quantitated for each marker
124
5.3.6 Animals
NOD/SCID gammanull
(NSG) mice from The Jackson Laboratory were bred and maintained in
the Ontario Cancer Institute animal facility according to protocols approved by the Animal Care
Committee. Mice were fed irradiated food and Baytril containing water ad libitum during
experimental periods. Prior to infusion with AML NSG mice were irradiated with 200 cGy to
facilitate engraftment. We developed ip and iv injection route OCI/AML5 NSG xenograft
models utilizing a dose of 2x106 cells. To determine the impact of in vitro incubation with
iKHYG-1 on proliferative capacity of OCI/AML5, the ip route of injection was utilized, with
sacrifice at humane endpoints. Briefly, OCI/AML5 cells were incubated in 15 ml conical tubes
with or without iKHYG-1 (+/- 1 µg/ml anti-NKp30 pretreatment x 1 hour), at a 10:1 E:T ratio,
spun at 1200 rpm to pellet, and incubated for four hours at 37°C. Cell mixtures were then
washed and resuspended in PBS and 2x10e6 OCI/AML5 cells with or without 20x106 iKHYG-1
or NKp30 iKHYG-1 cells in 200 µl of PBS were injected ip into three cohorts of five NSG mice.
To determine in vivo effect of NK cell line therapy OCI/AML5 or primary AML were injected iv
on day 0 with and without iKHYG-1 or anti-NKp30 pretreated iKHYG-1 treatment started on
day 3 (10x106 x 6 doses; days 3, 5, 7, 10, 12, 14). The primary AML sample (080179) was
derived from an M4 leukemia with aggressive engraftment features and passaged through NSG
mice prior to use in these experiments.
5.3.7 Statistics
Survival analysis was done with Kaplan Meier survival curves using the log rank rest with
Medcalc software. Comparison of cytotoxicity and engraftment data was done using a two tailed
student’s t-test performed on Medcalc software. Linear regression analysis was done using
Medcalc software and used to generate scatter plots with best fit line, coefficients of
determination (R2), F test and degree of significance.
125
5.4 Results
5.4.1 NK-92 and KHYG-1 cytotoxicity against leukemia cell lines
NK-92 and KHYG-1 were tested against a panel of leukemic cell lines (K562, KG1, OCI/AML2,
3 and 5) at a 10:1 E:T ratio using the chromium release assay. Both cell lines demonstrated
cytotoxicity against these targets, with NK-92 showing overall better cytotoxicity than KHYG-1
(Figure 5.1). OCI/AML5 was particularly sensitive to NK-92 killing with percentage lysis of
68%, exceeding that for K562. OCI/AML2 was relatively resistant to killing by both cell lines
with mimimal cytotoxicity demonstrated.
0
10
20
30
40
50
60
70
80
90
100
K562 KG1 OCI/AML2 OCI/AML3 OCI/AML5
% L
ysis
Cell line targets
NK-92
KHYG-1
Figure 5.1: NK-92 and KHYG-1 cytotoxicity against a panel of leukemia cell lines K562, KG1, OCI/AML2, 3, 5 cells were labelled with 100 µCi of Na2
51CrO4 prior to treatment
with NK-92 (A) and KHYG-1 (B) at a 10:1 E:T ratios. Data are presented as the mean percent
lysis of triplicate samples (+/-SD) from a representative experiment. Data are presented as the
mean percent lysis of triplicate samples (+/-SD) from a representative experiment repeated 3
times.
126
NK-92 cytotoxicity against K562 was completely abrogated by calcium chelation at all effector
targets ratios, indicating that granule exocytosis was the primary means of cytotoxicity (Figure
2A). However, there was a small amount of residual killing of K562 by KHYG-1 particularly at
effector:target ratios of 1:1 and 5:1 (Figure 5.2).
A
B
0
10
20
30
40
50
60
70
80
90
100
1:1 5:1 10:1 25:1
% L
ys
is
Effector:Target ratio
Medium
EGTA
Figure 5.2: NK-92 and KHYG-1 cytotoxicity against K562 with and without calcium
chelation K562 cells were labelled with 100 µCi of Na2
51CrO4 prior to treatment with NK-92 (A) and
KHYG-1 (B) at four E:T ratios in a standard four hour chromium release assay. Data are
presented as the mean percent lysis of triplicate samples (+/-SD) from a representative
experiment. Data are presented as the mean percent lysis of triplicate samples (+/-SD) from a
representative experiment repeated 3 times.
127
5.4.2 High throughput screening flow cytometry of NK-92 and KHYG-1
surface receptors
To better understand the differences in cytotoxicity of NK-92 and KHYG-1, we conducted high
throughput screening flow cytometry and identified the expression level of 374 cell surface
markers on NK-92 and KHYG-1 (Appendix I and II). Relevant subsets of activating, inhibiting
and apoptosis inducing cell surface proteins were more closely evaluated, as these are relevant to
recognition of malignant targets and cytotoxic effector function (Table 5.1). Cell surface
expression was categorized into quartiles and labelled tables with a colored heat map as follows:
Minimal-no color (0-1%), low-green (1-25%), intermediate-yellow (25-50%), high-orange (50-
75%) and very high-red (75-100%).
On KHYG-1 we identified the presence of very high levels of NKp30, NKp44, NKG2D and
DNAM-1 and high levels of NKp46. Further, other receptors known to mediate adhesion and
also transmit an activating signal to NK cells were also detected on KHYG-1 to variable degrees:
CD2 (LFA-2), CD7 (LEU-9), CD11a (LFA-1 component), CD11b (Mac-1), CD27 (TNFRSF7),
CD44 (Hyaluronate receptor), CD59 (Protectin), CD96 (TACTILE), CD160 (BY55), CD158i
(KIR2S4), CD161 (NKR-P1), CD223 (Lag3), and CD319 (CRACC). KHYG-1 expressed low
levels of two inhibitory KIRs, CD158b2 (KIR2DL3) and CD158f (KIR2DL5), high levels of
CD158d (KIR2DL4), and very high levels of CD85j (LIR-1). KHYG-1 had some degree of
expression of all ten tumour necrosis family members assayed, with intermediate and high levels
of expression of Fas Ligand CD178 (Fas Ligand) and CD253 (TRAIL), respectively.
NK-92 had a lesser degree of expression of natural cytotoxicity receptors: NKp44 (low), NKp46
(intermediate) and NKp30 (intermediate). DNAM-1 and NKG2D had very high expression.
NK-92 expressed low levels of KIR2DL4 and intermediate levels of KIR2DL5. However, LIR-1
was highly expressed by NK-92. NK-92 expressed nine of ten tumour necrosis family members
to variable degrees. Most notably, CD120b (TNF-β/lymphotoxin-α) was very highly expressed
and CD178 (Fas Ligand) and CD253 (TRAIL) had intermediate expression.
128
Table 5.1: Differential expression of cell surface activating, inhibiting and apoptosis
inducing molecules on NK-92 and KHYG-1
CD
Marker
Name Ligand % expression*
NK-92 KHYG-1 Activating
CD2 LFA-2 CD58 (LFA-3) 100 100
CD7 LEU-9 SECTM1, Galectin 99.9 100
CD11a LFA-1 component ICAM-1,-2,-3,-4,-5 100 100
CD11b Mac-1 ICAM-1, Fibrinogen 5.67 52.7
CD16 FcγRIII IgG 1.78 16.5
CD44 Hyaluronate receptor Hyaluronan 99 100
CD59 Protectin C8, C9 13.7 85.2
CD69 CLEC2C Unknown 0.946 93.7
CD96 TACTILE CD155 99.5 98.6
CD158i KIR2DS4 HLA-C 6.01 18.8
CD159c NKG2C HLA-E 0.971 47.9
CD160 BY55 HLA-C 73.2 28.6
CD223 Lag3 HLA Class II 10.4 99
CD226 DNAM-1 CD112, CD155 75.1 82.6
CD244 2B4 CD48 6.79 3.64
CD314 NKG2D MICA, MICB, ULB-1, -2, -3, -4, -5, -6 78.9 94.7
CD319 CRACC CRACC 92.8 82.2
CD335 NKp46 Influenza hemaglutinins, HSPs 36.9 50.8
CD336 NKp44 Influenza hemaglutinins 4.55 99.5
CD337 NKp30 BAT3, B7-H6 41.6 99.3
CD352 NTB-A CD352 (NTB-A) 100 100
Inhibiting
CD85d LIR-2, ILT-4 HLA-G 12.2 38.3
CD85j LIR-1, ILT-2 HLA-A, -B, -G 95.7 81.8
CD158a KIR2DL1 HLA-C2 0.243 0.175
CD158b KIR2DL2 HLA-C1 0.263 0.193
CD158b2 KIR2DL3 HLA-C1 0.395 14.5
CD158d KIR2DL4 HLA-G 37.3 73.5
CD158e2 KIR3DL1 HLA-Bw4 0.225 0.162
CD158f KIR2DL5 Unknown 6.73 13.5
CD159a NKG2A HLA-E 1.82 17
CD161 NKR-P1 LLT1 8.76 30.5
Apoptosis
inducing
CD120a TNF-alpha 1.99 19.2
CD120b TNF-β/lympotoxin-α 98.7 99.4
CD137L 4-1BB ligand 4-1BB ligand 29.5 76.6
CD153 CD30 ligand CD30 4.88 29.3
CD154 CD40 ligand CD40 0.428 4.4
CD178 Fas Ligand CD95 (Fas) 30.4 31.9
CD252 OX40 Ligand CD134 (OX40) 15.8 17.2
CD253 TRAIL TRAIL R1, TRAIL R2 36.3 59.9
CD256 APRIL TACI 25.3 18
CD257 BAFF BAFF-R, TACI and BCMA 49.5 73.4
CD258 LIGHT HVEM 4.51 36.0
*Percent positivity of cell surface expression was categorized into quartiles and tables labelled
with a colored heat map as follows: Minimal-no color (0-1%), Low-green (1-25%), Intermediate-
yellow (25-50%), High-orange (50-75%) and Very High-red (75-100%).
129
5.4.3 Anti-Class I HLA blockade of AML targets
To explore the role of inhibitory killer immunoglobulin-like receptors (KIRs), we blocked the
class I HLA molecules on target cells, which serve as the ligand for this receptor group. The
antibody used was a pan-HLA blocking antibody with capability to block HLA- A, B and C
groups by binding to generic epitopes on each of these molecules. NK-92 was incubated at a
25:1 E:T ratio with four primary AML blast samples (080179, 080078, 080008 and 0909),
yielding moderate degrees of cytotoxicity by the CRA: 42.3, 29.8, 43.9, 42.6 % lysis. Blockade
of class I HLA on primary AML targets with antibody did not affect cytotoxicity of NK-92 (data
not shown). KHYG-1 at a 25:1 E:T had lower cytotoxicity against this panel: 9.8, 5.1, 17.1 and
8.5 % lysis. However, following class I HLA blockade, sample 080008 was rendered more
sensitive to killing by KHYG-1 increasing from 17.1 +/-2.8% to 35.8 +/-1.2 % lysis (p<0.001)
(Figure 5.3). We obtained the HLA typing on the four primary AML specimens to identify
potential HLA class I ligands that could interact with inhibitory KIRs and noted the following
HLA-C groups: 080179 (C1/C1), 080078 (C2/C2), 080008 (C2/C2), 0909 (C1/C1) (Table 5.2)
No relation between KIR ligand expression and cytotoxicity could be made. Further, predictions
of cytotoxicity enhancement based upon the KIR-ligand receptor mismatch hypothesis were not
confirmed. Finally, blockade of KIR2DL3 (specific for HLA-C1) on KHYG-1 did not enhance
cytotoxicity against primary AML targets 08008 and 0909 (data not shown).
130
0
10
20
30
40
50
60
70
80
90
100
080179 080078 080008 0909
% L
ys
is
Sample ID
Control
1 ug/ml α MHC I
10 ug/ml α MHC I
*
Figure 5.3: KHYG-1 cytotoxicity against 4 primary AML samples +/- class I HLA blockade
AML blasts were labelled with 100 µCi of Na251
CrO4 for 2 hours prior to treatment with KHYG-
1 at a 25:1 E:T in 96 well U bottom plates. Some samples were also incubated with 1 or 10
µg/ml of anti-Class I A, B, C blocking mAb for 1 hour prior to effector cell addition. Plates
were centrifuged at 200 g and incubated at 37°C 5% CO2 x4 hours. KHYG-1 against primary
AML blasts +/- 1 or 10 µg/ml class I blockade. Data are presented as the mean percent lysis of
triplicate samples (+/-SD) representative of two separate experiments (*= p<0.001).
Table 5.2: HLA type of primary AML panel and sensitivity to NK-92 and KHYG-1 +/-
HLA blockade
Sample
ID #
HLA C type
(C1 or C2)
Sensitivity to cytolysis
Effect of HLA class I
blockade on
cytotoxicity
Consistent with KIR
ligand mismatch
hypothesis?
NK-92 KHYG-1 NK-92 KHYG-1 NK-92 KHYG-1
080179 C1 (C*07:01)
C1 (C*07:02)
Intermediate Low None None Yes No
080078 C2 (C*05:01)
C2 (C*15:02)
Intermediate Low None None Yes Yes
080008 C2 (C*02:xx)
C2 (C*06:02)
Intermediate Low None Increase Yes No
0909 C1 (C*07:01)
C1 (C*16:01)
Intermediate Low None None Yes No
131
5.4.4 Effect of pretreating NK-92 and KHYG-1 with activating receptor
specific antibodies
We attempted to address the role of common activating receptors in NK cell line-mediated
recognition of leukemic targets by pretreating NK-92 and KHYG-1 with of antibodies specific
to NKp30, NKp44, NKp46, DNAM-1, NKG2D, and CD7 (10 µg/ml), prior to co-incubation
with the target cells K562, KG1a and OCI/AML5. An off-target antibody was used as an isotype
control. Pretreatment of NK-92 with antibodies to NKp30, NKp44 and NKp46 unexpectedly
increased killing of K562 above isotype control [(1.3X (p<0.0001), 1.2X (p<0.05), 1.2X
(p=0.11)], while anti-NKp30 treatment enhanced killing of KG1a [+1.8X (p<0.0001)] and
OCI/AML5 [1.2X (p<0.01)] (Figure 5.4A). Treatment of KHYG-1 with antibodies to NKp30,
NKp44 and NKp46 increased killing of K562 above isotype control [1.4X (p<0.001), 1.5X
(p<0.01), 1.2X (p<0.05)], while anti-NKp30 treatment increased killing of KG1a [2.6X
(p<0.01)] and anti-NKp30, anti-NKp44 and anti-NKp46 treatment increased killing of
OCI/AML5 [3.4X (p<0.00001), 3.2X (p<0.0001), 1.8X (p<0.0001)] (Figure 5.4B). Pretreating
NK cell lines with antibodies to DNAM-1 and NKG2D had minimal effects on cytotoxicity.
A
0
10
20
30
40
50
60
70
80
90
100
K562 KG1a OCI/AML5
% L
ysis
Cell line
None
MG1-45
NKp30
NKp44
NKp46
NKG2D
DNAM-1
* *
*
*
*= <0.05
*
132
B
0
20
40
60
80
100
120
140
K562 KG1a OCI/AML5
% L
ysis
Cell line
None
MG1-45
NKp30
NKp44
NKp46
NKG2D
DNAM-1
* *
*
*
**
*
Figure 5.4: Effect of antibody pre-treatment of activating receptors on NK-92 and KHYG-
1 cytotoxicity against leukemia cell lines NK-92 (A) and KHYG-1 (B) were pretreated with a panel of antibodies (10 µg/ml) against
receptors with activating function for NK cells including an untreated control and isotype control
as follows: none, MG1-35 (isotype control), NKp30, NKp44, NKp46, DNAM-1, NKG2D, CD7.
NK-92 and KHYG-1 were incubated for one hour with antibodies and then washed in AIM-V
medium to remove residual antibody. Effector cells were utilized in a standard chromium
release assay against K562, KG1a and OCI/AML5 labelled with 100 µCi of Na251
CrO4 at an E:T
ratio of 10:1. Statistical comparisons were done using a student’s test between isotype control
(MG1-45) and antibody pretreated groups (* =p<0.05).
We then attempted a similar experiment with K562 and two primary AML cell lines. NK-92 and
KHYG-1 were pre-treated with antibodies specific to NKp30, NKp44, NKp46, DNAM-1,
NKG2D and CD7 (10 µg/ml) prior to co-incubation with the leukemic target cells K562, and the
primary AML specimens 080078 and 0909. NK-92 cytotoxicity against primary AML samples
demonstrated prominent increases above isotype control when pretreated with anti-NKp30 [7.1X
(p<0.001) and 3.0X (p<0.0001)] (Figure 5.5A).
Pretreatment of KHYG-1 with either anti-NKp30 or anti-NKp44 led to dramatic increases of
cytotoxicity relative to isotype control against primary AML samples 080078 [16.9X (p<0.0001)
and 17.6X (p<0.001)] and 0909 [2.8X (p<0.0001) and 2.9X (p<0.001)], (Figure 5.5B). The dose
dependence of anti-NKp30 and anti-NKp44 mediated enhancement of killing was then explored
by testing several dose ranges.
133
A
0
10
20
30
40
50
60
70
80
90
100
K562 080078 0909
% L
ysis
Cell line or primary AML sample
None
MG1-45
NKp30
NKp44
NKp46
NKG2D
DNAM-1
CD7
*
*
*
*
*
*
B
0
10
20
30
40
50
60
70
80
90
100
K562 080078 0909
% L
ysis
Cell line or primary AML sample
None
MG1-45
NKp30
NKp44
NKp46
NKG2D
DNAM-1
CD7
**
*
**
**
Figure 5.5: Effect of antibody pre-treatment of activating receptors on NK-92 and KHYG-
1 cytotoxicity against primary AML samples
NK-92 (A) and KHYG-1 (B) were pretreated with a panel of antibodies (10 µg/ml) against
receptors with activating function for NK cells (NKp30, NKp44, NKp46, DNAM-1, NKG2D,
CD7), including an untreated control, and isotype control (MG1-45). NK-92 and KHYG-1 were
incubated for one hour, and then washed in AIM-V medium to remove residual antibody.
Effector cells were utilized in a standard chromium release assay against K562 and two primary
AML samples labelled with 100 µCi of Na251
CrO4 at an E:T ratio of 10:1. Data are presented as
the mean percent lysis of triplicate samples (+/-SD) representative of two similar experiments.
Statistical comparisons were done using a student’s test between isotype control (MG1-45) and
antibody pretreated groups (* =p<0.05).
134
In an attempt to determine the linear portion of the dose response curve and approximate the
EC50%, pretreatment of NK cell lines was done with a range of doses of anti-NKp30 and anti-
NKp44 (1, 5 and 10 µg/ml) against K562, OCI/AML3 and OCI/AML5 and primary AML
080078. A dose response was seen with anti-NKp30 pretreatment of NK-92 against OCI/AML3
and primary AML sample 080078 however the EC50% was less than the lowest dose used (1
µg/ml) (data not shown). KHYG-1 had a similar degree of enhancement seen at all dose ranges
of both anti-NKp30 and anti-NKp44 antibody pretreatments (data not shown).
A lower dose range was then selected utilizing NK cell lines pretreated with isotype control, anti-
NKp30 and anti-NKp44 at 0.1, 0.5 and 1 µg/ml. Isotype-control pretreated NK-92 and KHYG-1
had minimal effects on cytotoxicity against K562, OCI/AML3, OCI/AML5 and KG1, with no
dose response (Figure 5.6). Anti-NKp30 pretreatment enhanced NK-92 cytotoxicity against
OCI/AML3 (EC50% ~0.1) and KG1 (EC50%<0.1 µg/ml) only. Combined anti-NKp30 and anti-
NKp44 pretreatment (0.1 µg/ml) of NK-92 did not have additive or synergistic effects on
cytotoxicity against any targets (Figure 5.7A).
Anti-NKp30 pretreatment enhanced KHYG-1 cytotoxicity against all targets with a dose
response seen for K562 (EC50% ~0.1 µg/ml), OCI/AML3 (EC50% <0.1 µg/ml), and KG1
(EC50% <0.1 µg/ml), while OCI/AML5 showed a plateau from the lowest dose (EC50% <0.1
µg/ml). Anti-NKp44 pretreatment enhanced KHYG-1 cytotoxicity against all targets, with a
dose response seen for K562 only (EC50% ~0.1 µg/ml), while OCI/AML3, KG1 and
OCI/AML5 showed a plateau from the lowest dose (EC50% <0.1 µg/ml) (Figure 5.7B).
135
A
0
10
20
30
40
50
60
70
80
90
100
K562 OCI/AML3 OCI/AML5 KG1
% L
ysis
Cell line
None
MG2a-53 (0.1)
MG2a-53 (0.5)
MG2a-53 (1)
B
0
10
20
30
40
50
60
70
80
90
100
K562 OCI/AML3 OCI/AML5 KG1
% L
ysis
Cell line
None
MG2a-53 (0.1)
MG2a-53 (0.5)
MG2a-53 (1)
Figure 5.6: Effect of antibody pre-treatment with isotype control on NK-92 and KHYG-1
cytotoxicity against leukemia cell lines
NK-92 and KHYG-1 were pretreated with isotype control MG2-53, incubated for one hour and
then washed in AIM-V medium to remove residual antibody. Effector cells were utilized in a
standard chromium release assay against K562, OCI/AML3, 5 and KG1 labelled with 100 µCi of
Na251
CrO4at an E:T ratio of 10:1. Data are presented as the mean percent lysis of triplicate
samples (+/-SD).
136
A
0
10
20
30
40
50
60
70
80
90
100
K562 OCI/AML3 OCI/AML5 KG1
% L
ysis
Cell line
None
NKp30 (0.1)
NKp30 (0.5)
NKp30 (1)
NKp44 (0.1)
NKp44 (0.5)
NKp44 (1)
NKp30/NKp44 (0.1)
B
0
10
20
30
40
50
60
70
80
90
100
K562 OCI/AML3 OCI/AML5 KG1
% L
ysis
Cell line
None
NKp30 (0.1)
NKp30 (0.5)
NKp30 (1)
NKp44 (0.1)
NKp44 (0.5)
NKp44 (1)
NKp30/NKp44 (0.1)
Figure 5.7: Effect of antibody pre-treatment with anti-NKp30 and anti-NKp44 on NK-92
and KHYG-1 cytotoxicity against leukemia cell lines
NK-92 (A) and KHYG-1 (B) were pretreated with anti-NKp30 and anti-NKp44 antibodies at 0.1,
0.5, 1 µg/ml incubated for one hour and then washed in AIM-V medium to remove residual
antibody. Effector cells were utilized in a standard chromium release assay against K562,
OCI/AML3, 5 and KG1 labelled with 100 µCi of Na251
CrO4at an E:T ratio of 10:1. Data are
presented as the mean percent lysis of triplicate samples (+/-SD).
137
Combined anti-NKp30 and anti-NKp44 pretreatment (0.1 µg/ml) of KHYG-1 demonstrated
greater enhancement on cytotoxicity than each antibody alone against K562, OCI/AML3 and
KG1, but not OCI/AML5. Not only did the combined dosing of 0.1 µg/ml anti-NKp30 and anti-
NKp40 exceed the effect of each antibody alone at this dose level, it also matched or exceeded
the effect of a 10-fold higher dose (1 µg/ml) of each antibody alone for K562, OCI/AML3 and
KG1, demonstrating true synergy. The % lysis values for untreated, isotype control, anti-
NKp30, anti-NKp40 and combined anti-NKp30 and anti-NKp44 groups (0.1 µg/ml) and anti-
NKp30 and anti-NKp44 (1 µg/ml) for the cell line targets treated with KHYG-1 from Figure 5.6
and Figure 5.7 are tabulated for comparison (Table 5.3).
Table 5.3: Comparison of antibody pretreatment effects on KHYG-1 cytotoxicity and
synergy assessment at 0.1 µg/ml
Cell line % Lysis (mean +/- SD)
Pretreatment
0.1 µg/ml 1.0 µg/ml
None MG2a-
53
Anti-
NKp30
Anti-
NKp44
Anti-
NKp44/
NKp30
Anti-
NKp30
Anti-
NKp44
K562 26
+/- 3.2
37
+/- 2.9
41
+/-6.0
46
+/-11.1 *61
+/- 5.9
54
+/-16.7
59
+/-2.6
OCI/AML3 0
+/- 0.8
9
+/-7.8
20
+/- 2.0
25
+/- 1.5 *39
+/-1.7
34
+/-1.3
26
+/-4.8
OCI/AML5 11
+/- 0.5
19
+/- 3.1
68
+/- 2.7
62
+/- 3.5
69
+/- 3.8
74
+/-42
67
+/- 7.0
KG1 1
+/- 0.4
9
+/- 1.7
16
+/- 1.7
10
+/- 2.2 *24
+/- 1.1
25
+/-0.5
11
+/- 0.7
*Combined anti-NKp30 and anti-NKp44 regimens that yielded statistically significant (p<0.05)
increases above each antibody alone in separate comparisons are in bold font.
While dose responses could be seen in the range of 0.1 to 1 µg/ml in many cases, the lowest dose
exceeded the EC50%. Therefore, an additional experiment testing a dose range one log lower
was conducted (0.01, 0.1 and 1 µg/ml). There was minimal effect of the isotype control (MG2a-
53) antibody in this range, with no dose response seen (data not shown). Pretreatment of NK-92
with 0.01, 0.1 and 1 µg/ml of anti-NKp30 enhanced cytotoxicity of OCI/AML3 only (ED50%
0.01 to 0.1 µg/ml) and there was no effect of anti-NKp44 pretreatment (Figure 5.8A).
138
Anti-NKp30 and anti-NKp44 pretreatment enhanced KHYG-1 cytotoxicity against all targets
with a dose response seen for K562, OCI/AML3 and OCI/AML5 (EC50% 0.01-0.1
µg/ml)(Figure 5.8B). Combined anti-NKp30 and anti-NKp44 pretreatment of KHYG-1 (0.01
µg/ml) demonstrated synergistic effects on cytotoxicity against OCI/AML3 only at this dose
level. The combined dosing of 0.01 µg/ml anti-NKp30 and anti-NKp40 exceeded the effect of
each antibody alone (2X) and the absolute cytotoxicity was comparable to a 10-fold higher dose
of each antibody (0.1 µg/ml). The % lysis values for untreated, isotype control, anti-NKp30,
anti-NKp40 and combined anti-NKp30 and anti-NKp44 groups (0.01 µg/ml) and anti-NKp30
and anti-NKp44 (1 µg/ml) for the cell line targets treated with KHYG-1 from Figure 5.8 and its
isotype controls are tabulated for comparison (Table 5.4).
Table 5.4: Comparison of antibody pretreatment effects on KHYG-1 cytotoxicity and
synergy assessment at 0.01 µg/ml
Cell line % Lysis (mean +/- SD)
Pretreatment
0.01 µg/ml 0.1 µg/ml
None MG2a-
53
Anti-
NKp30
Anti-
NKp44
Anti-
NKp44/
NKp30
Anti-
NKp30
Anti-
NKp44
K562 21
+/- 1.6
29
+/- 2.4
28
+/- 6.0
26
+/-2.5
32
+/- 2.0
35
+/- 0.5
41
+/- 3.4
OCI/AML3 4
+/- 0.1
7
+/- 1.8
7
+/-0.8
7
+/- 0.6 13*
+/- 3.8
13
+/- 0.5
19
+/- 3.4
OCI/AML5 12
+/- 0.8
16
+/- 5.0
38
+/- 1.9
30
+/- 3.8
36
+/- 7.6
65
+/- 1.5
63
+/- 5.4
KG1 9
+/- 7.4
9
+/- 1.2
6
+/- 1.0
9
+/- 4.7
11
+/- 1.0
9
+/- 1.1
15
+/- 4.5
Combined anti-NKp30 and anti-NKp44 regimens that yielded statistically significant (p<0.05)
increases above each antibody alone in separate comparisons are in bold font.
139
A
0
10
20
30
40
50
60
70
80
90
100
K562 OCI/AML3 OCI/AML5
% L
ysis
Cell line
None
NKp30 (0.01)
NKp30 (0.1)
NKp30 (1)
NKp44 (0.01)
NKp44 (0.1)
NKp44 (1)
NKp30/NKp44 (0.01)
B
0
10
20
30
40
50
60
70
80
90
100
K562 OCI/AML3 OCI/AML5
% L
ysis
Cell line
None
NKp30 (0.01)
NKp30 (0.1)
NKp30 (1)
NKp44 (0.01)
NKp44 (0.1)
NKp44 (1)
NKp30/NKp44 (0.01)
Figure 5.8: Effect of antibody pre-treatment with anti-NKp30 and anti-NKp44 on NK-92
and KHYG-1 cytotoxicity against leukemia cell lines and primary AML
NK-92 (A) and KHYG-1 (B) were pretreated without or with anti-NKp30 and anti-NKp44
antibodies at 0.01, 0.1, 1 µg/ml (C and D), incubated for one hour and then washed in AIM-V
medium to remove residual antibody. Effector cells were utilized in a standard chromium
release assay against K562, OCI/AML3 and OCI/AML5 with 100 µCi of Na251
CrO4at an E:T
ratio of 10:1. Data are presented as the mean percent lysis of triplicate samples (+/-SD).
140
To determine if anti-NKp30 and anti-NKp44 pretreatment of NK cell lines could enhance
cytotoxicity against a solid tumour, we performed the same experiment with esophageal cancer
cell lines (FLO-1, OE-33, SKGT-4, KYAE-1). However, pretreatment of NK-92 and KHYG-1
with 0.1 µg/ml of anti-NKp30 and anti-NKp44 mAb did not enhance cytotoxicity against four
esophageal cancer cells lines relative to the isotype control (Figure 5.9). This suggested the
presence of a unique cell surface marker present on leukemia cells, but not esophageal cancer
cells, that was mediating the enhancing effect of antibody-pretreated NK cell lines.
141
A
0
10
20
30
40
50
60
70
80
90
100
K562 FLO-1 OE-33 SKGT-4 KYAE-1
% ly
sis
Cell line
None
MG1-45 (0.1)
NKp30 (0.1)
NKp44 (0.1)
B
0
10
20
30
40
50
60
70
80
90
100
K562 FLO-1 OE-33 SKGT-4 KYAE-1
% ly
sis
Cell line
None
MG1-45 (0.1)
NKp30 (0.1)
NKp44 (0.1)
Figure 5.9: Impact of antibody pre-treatment with anti-NKp30 and anti-NKp44 on NK-92
and KHYG-1 cytotoxicity against esophageal cancer cell lines
NK-92 (A) and KHYG-1 (B) were pretreated with isotype control MG1-45 or anti-NKp30 and
anti-NKp44 antibodies at 0.1 µg/ml, incubated for one hour and then washed in AIM-V medium
to remove residual antibody. Effector cells were utilized in a standard chromium release assay
against K562 and esophageal cancer cell lines FLO-1, OE-33, SKGT-4, KYAE-1 and labelled
with 100 µCi of Na251
CrO4at an E:T ratio of 10:1. Data are presented as the mean percent lysis of
triplicate samples (+/-SD).
142
5.4.5 Relationship of Fcγ receptor expression and enhancement of
cytotoxicity
We conducted HTS flow cytometry on two representative leukemic cell line targets (OCI/AML3
and OCI/AML5) to assess for potential cell surface markers that might be involved in enhancing
the cytotoxicity of anti-NKp30 or anti-NKp44 coated NK cell lines (Appendix III and IV)I). We
noted a high degree of CD32 (FcγRII) expression on both cell lines. Subsequently, we
conducted routine flow cytometry on all leukemic and esophageal cancer cell lines for
expression all Fcγ receptors (CD16, CD32, CD64). The leukemia cell lines showed relatively
high expression of Fcγ receptor II (CD32), but very low expression of Fcγ receptor I (CD64) or
Fcγ receptor III (CD16) on leukemia lines (K562, KG1, KG1a OCI/AML3, OCI/AML5) (Figure
5.10). The histogram shape for K562 suggested the presence of both intermediate and high
CD32 expressing subpopulations. KG1a appeared to have a dual population of negative and low
CD32 expressing subpopulations. OCI/AML3 had a clear dual peak representing two high CD32
expressing subpopulations. There were clear single populations of CD32 expressing cells in
KG1 (moderate) and OCI/AML5 (high). There was no significant expression of Fcγ receptors
on esophageal cancer lines (OE-33, FLO-1, KYAE-1, SKGT-4) (Figure 5.11).
143
A
B
C
D
E
Figure 5.10: Fc gamma receptor expression on leukemia cell lines (K562, KG1, KG1a,
OCI/AML3, OCI/AML5)
Leukemia cell lines K562 (A), KG1 (B), KG1a (C), OCI/AML3 (D), OCI/AML5 (E) were tested
for CD16, CD32 and CD64 expression by flow cytometry. Unstained (red) and stained (blue)
populations are presented in the histogram.
144
A
B
C
D
Figure 5.11: Fc gamma receptor expression on esophageal cancer cell lines (OE-33, FLO-1,
KYAE-1, SKGT-4)
Esophageal cancer cell lines were tested for CD16, CD32 and CD64 expression by flow
cytometery. Unstained (red) and stained (blue) populations are presented in the histogram.
145
The percent positivity of each leukemic and esophageal cancer cell line was determined from
flow cytometry plots (Figure 5.10 and Figure 5.11). Data from prior cytotoxicity assays
measuring the enhancement of cytotoxicity of NK-92 and KHYG-1 when pretreated with 10
µg/ml of either anti-NKp30 or anti-NKp44 antibody were compared to isotype controls and delta
cytotoxicity calculated. The delta cytotoxicity relative to isotype control was correlated with the
degree of CD32 expression using regression analysis to create best fit lines, calculate co-efficient
of determination (R2) and statistical significance. Regression analysis of the relationship of delta
cytotoxicity of antibody-pretreated NK-92 with CD32 expression of targets did not reveal a
correlation for anti-NKp30 (R2=0.13; p=0.34) and anti-NKp44 (R
2=0.22; p=0.20) pretreatments
(Figure 5.12A and B). However, regression analysis of the relationship of delta cytotoxicity of
antibody pretreated KHYG-1 with CD32 expression of targets revealed a strong correlation for
both anti-NKp30 (R2=0.71; p<0.01) and anti-NKp44 pretreatments (R
2=0.64; p<0.01) (Figure
5.12C and D).
146
Figure 5.12: Regression analysis of CD32 expression and delta cytotoxicity of NKp30 and
NKp44 pretreated NK-92 and KHYG-1
Relation between delta cytotoxicity of isotype control versus NKp30 or NKp44 pretreated NK-
92 (A and B) and KHYG-1 (C and D) against leukemic and esophageal cancer cell lines was
determined by using linear regression analysis. Scatter plots with best fit line are presented with
coefficient of determination (R2) calculated and F test for significance performed. NK-92
pretreatments: anti-NKp30 (A) (R2=0.13; p=0.34) and anti-NKp44 (B) (R
2=0.22; p=0.20).
KHGY-1 pretreatments: anti-NKp30 (C) (R2=0.71; p<0.01) and anti-NKp44 (D) (R
2=0.64;
p<0.01).
147
5.4.6 Effect of anti-NKp30 pretreatment on NK cell line cytotoxicity
against clonogenic OCI/AML5
To determine the effect of anti-NKp30 and anti-NKp44 pretreated NK cell lines against
clonogenic leukemic cells, we utilized our previously established clonogenic cytotoxicity assay
utilizing OCI/AML5 as the target. Comparison of killing was made with untreated, isotype
control (MG1-45) and anti-CD7 pretreated NK cell lines. CD7 is highly expressed on NK-92
and KHYG-1, with no confirmed activating capacity in these cell lines. Therefore, anti-CD7
antibody pretreatment was chosen as an additional control. There was no difference between
isotype control and CD7-pretreated NK-92. Pretreatment of NK-92 with 0.1 µg/ml anti-NKp30
had only a slight impact (+3.7%; p<0.05) on OCI/AML5 clonogenic capacity relative to baseline
and isotype control (Figure 5.13A). However, pretreatment of KHYG-1 with anti-NKp30
enhanced % colony inhibition 3-fold (+63.1%; p<0.0001) relative to baseline and isotype
control. While the baseline % colony inhibition of NK-92 (61.9 %) was greater than KHYG-1
(32.0%), anti-NKp30 pretreated KHYG-1 (90.7%) had the greater inhibition relative to NK-92
(+28.8%; p<0.0001).
A
148
B
*
Figure 5.13: Methylcellulose cytotoxicity assay of NK-92 and KHYG-1 +/- pretreatment
with antibodies against OCI/AML5
NK-92 (A) and KHYG-1 (B) were pretreated without (no pretreatment) and with monoclonal
antibodies (MG1-45, anti-CD7, anti-NKp30) at 0.1 µg/ml for 1 hour prior to use in
methylcellulose cytotoxicity assay (MCA) at a 10:1 E:T ratio. OCI/AML5 cells were incubated
with or without effectors prior to infusion into methylcellulose. Plates were incubated at 37°C
with 5% CO2 for two weeks. Colonies were then enumerated by using an inverted microscope
defining a colony as a cell cluster of >50 cells. Cytotoxicity was measured relative to the low
density control (LDC) using untreated effector cells which yielded similar numbers of colonies
to OCI/AML5 cells incubated alone. % colony inhibition was calculated using the following
formula: [(#ColoniesLDC)- (#ColoniesTreatment)] x100% [#ColoniesLDC]. Data are presented
as the mean percent lysis of triplicate samples (+/-SD) representative of two separate
experiments. Statistical comparison was done using a student’s test between no pretreatment, or
isotype control (MG1-45), and anti-NKp30 pretreated groups (* =p<0.0001).
149
5.4.7 In vitro effect of anti-NKp30 pretreated iKHYG-1 against
OCI/AML5 capacity for leukemic progression in an NSG xenograft
model
We tested the in vitro cytotoxic effect of KHYG-1 on in vivo progression of leukemia in an
OCI/AML5 xenograft model. KHYG-1 proliferation was prevented by irradiation with 1000
cGy prior to use. OCI/AML5 cells were co-incubated with irradiated KHYG-1 (iKHYG-1) +/-1
µg/ml anti-NKp30 pretreatment for one hour prior to a 4 hour co-incubation at a 10:1 E:T with
OCI/AML5 cells. Subsequently, cell mixtures were injected ip into three cohorts of NSG mice
with survival as an endpoint. Individual NSG mice were injected with 2x106 OCI/AML5 cells
+/- 20x106 viable effector cells (iKHYG-1 or anti-NKp30 pretreated iKHYG-1). At 9 weeks,
control mice developed progressive malignant ascites with minimal splenomegaly and imbedded
vascular tumours in the omentum (Figure 5.14). Co-incubation with iKHYG-1 did not improve
survival (p=0.92). However, anti-NKp30 pretreated iKHYG-1 improved survival compared to
the no therapy (p<0.05) or iKHYG-1 (p<0.05) cohorts (Figure 5.15).
150
A B
DC
Figure 5.14: OCI/AML5 induced malignant ascites
2x10e6 OCI/AML5 cells were injected ip into NSG mice and monitored for progression of
leukemia. NSG mice developed massive malignant ascites (A and B) with development of
inflamed omental tumours (C red arrow), but minimal increase in spleen size (D red arrow).
151
Group
OCI/AML5
OCI/AML5 + iKHYG1
OCI/AML5 + NKp30 iKHYG1
Survival
0 20 40 60 80 100
100
80
60
40
20
0
Time (days)
Surv
ival p
robability
(%
)
Figure 5.15: In vitro incubation of OCI/AML5 with iKHYG-1 +/- anti-NKp30 and in vivo
proliferation in NSG mice
OCI/AML5 cells were incubated in 15 ml conical tubes with or without iKHYG-1 (+/- 1 ug/ml
anti-NKp30 pretreatment x 1 hour) at a 10:1 E:T ratio, spun at 1200 rpm to pellet and incubated
for four hours at 37°C. Cell mixtures were then washed and resuspended in PBS and 2x10e6
OCI/AML5 cells with or without 20x106 iKHYG-1 or NKp30 iKHYG-1 cells were injected ip
into three cohorts of five NSG mice. Mice were sacrificed at humane endpoint and Kaplan
Meier curves generated and compared with the log rank test (p<0.05).
152
5.4.8 Effect of anti-NKp30 pretreatment of iKHYG-1 on therapeutic
efficacy for OCI/AML5 or primary AML xenografted mice
We evaluated OCI/AML5 engraftment potential in NSG mice by infusing 5x106 OCI/AML5
cells via tail vein and measured bone marrow engraftment at two weeks. Bone marrow
engraftment of OCI/AML5 was detected by measuring human CD33 expression in bone marrow
samples (Figure 5.16) and revealed relatively rapid, but variable bone marrow engraftment (13.0,
52.3, 29.9, 63.5%). In a subsequent survival endpoint experiment, 2x106 OCI/AML5 cells were
injected iv into cohorts of five mice. OCI/AML5-xenografted mice were then treated without
and with iKHYG-1 or anti-NKp30 pretreated iKHYG-1 (10x106 x 6 doses ip). There was
significant improvement in survival of mice treated with either iKHYG-1 (+35 days median
survival; p<0.05) or anti-NKp30 pretreated iKHYG-1 (+37 day median survival p<0.05) above
control. The two day difference in median survival of mice treated with anti-NKp30 pretreated
iKHYG-1 over iKHYG-1 alone was not significant, although the longest surviving mouse was in
the anti-NKp30 pretreated iKHYG-1 group (Figure 5.17).
Figure 5.16: Bone marrow engraftment of OCI/AML5 injected iv into NSG mice
5x106 OCI/AML5 cells were injected via tail vein into four NSG mice. Mice were sacrificed at 3
weeks and bone marrow harvested, stained with anti-CD33 antibody and measured using flowing
cytometry. Flow cytometry plots represent bone marrow from each mouse showing unstained
(red) and anti-CD33 stained (blue) samples.
CD33+
13.0
1 0- 1
1 00
1 01
1 02
1 03
FL4
0
2 0
4 0
6 0
8 0
100
Co
un
t
CD33+
52.3
1 0- 1
1 00
1 01
1 02
1 03
FL4
0
2 0
4 0
6 0
8 0
100
Co
un
t
CD33+
29.9
1 0- 1
1 00
1 01
1 02
1 03
FL4
0
2 0
4 0
6 0
8 0
100
Co
un
t
CD33+
63.5
1 0- 1
1 00
1 01
1 02
1 03
FL4
0
2 0
4 0
6 0
8 0
100
Co
un
t
153
GroupiKHYG-1iKHYG-1 NKp30OCI/AML5 only
Survival
0 10 20 30 40 50 60 70 80 90
100
80
60
40
20
0
Time
Surv
ival p
robabili
ty (
%)
Figure 5.17: Treatment of OCI/AML5 leukemia in NSG mice with iKHYG-1 +/- NKp30
pretreatment
2x106 OCI/AML5 were injected iv on day 0 into three cohorts of mice that were subsequently
treated as follows: no treatment, iKHYG-1 or anti-NKp30 pretreated iKHYG-1. Treatment
started on day 3 (10x106 x 6 doses ip; days 3, 5, 7, 10, 12, 14). Mice were sacrificed at humane
endpoints and Kaplan Meier curves generated and compared with the log rank test.
154
We utilized a primary AML sample (080179) known to engraft and cause leukemia in NSG mice
as a model to test the efficacy irradiated NK-92 and KHYG-1 pretreated with and without anti-
NKp30 (1 ug/ml) prior to injection into NSG mice inoculated with either primary AML.
iKHYG-1 and iNK-92 did not prolong survival in the primary AML model, although iKHYG-1
pretreated with anti-NKp30 showed some longer term survivors (3-4 weeks above control
median) with a trend toward significance (p=0.20) versus iKHYG-1 alone (Figure 5.18).
A
GroupControliKHYG-1iKHYG-1 NKp30
Survival
0 10 20 30 40 50 60 70
100
80
60
40
20
0
Time (days)
Surv
ival p
robabili
ty (
%)
Figure 5.18: Treatment of primary AML xenografted NSG mice with iKHYG-1 +/- NKp30
pretreatment
Primary AML cells (3x106) were injected iv on day 0 with and without iKHYG-1 or NKp30
iKHYG-1 ip treatment started on day 3 (10x106 x 6 doses; days 3, 5, 7, 10, 12, 14). Mice were
sacrificed at humane endpoint and Kaplan Meier curves generated and compared with the log
rank test.
155
5.5 Discussion
Adoptive immunotherapy with NK cells represents a novel emerging treatment modality for
patients with AML with several established protocols for allogeneic NK cell therapy for
AML.(Miller, Soignier et al. 2005; Rubnitz, Inaba et al. 2010) Anti-cancer cell-based
immunotherapy with a standardized, highly cytotoxic NK cell line is an attractive alternative to
autologous or allogeneic NK cells with their attendant variability in cytotoxicity and cell
manufacturing characteristics. The permanent, malignant NK line, NK-92 has been tested in
phase I trials and shown to have minimal toxicities (Arai, Meagher et al. 2008; Tonn, Schwabe et
al. 2013), while KHYG-1 has only been assessed in preclinical studies. Here, we have
conducted all experiments with KHYG-1 using a clinical grade medium (GM1) that lacks fetal
bovine serum and may be suitable for future clinical application. To further characterize both
NK cell lines and provide insight into mechanisms of cytotoxicity, high throughput screening
flow cytometry assaying for 374 distinct cell surface markers was conducted and provides the
most extensive immunophenotypic assessment of these cell lines to date. Prior to this study,
only ~30 receptors were reported for NK-92(Maki, Klingemann et al. 2001) and several for
KHYG-1 (Suck, Branch et al. 2005).
We confirmed previous findings that NK-92 and KHYG-1 are cytotoxic to leukemic cell lines
(Yan, Steinherz et al. 1998; Tonn, Becker et al. 2001; Suck, Branch et al. 2005; Williams, Wang
et al. 2010). We sought initially to determine basic mechanisms of cytotoxicity of these cell
lines and determined that NK-92 and KHYG-1 cytotoxicity was mediated primarily by granule
exocytosis as evidenced by minimal killing of leukemic cell line or primary AML blasts in the
presence of the calcium chelator EGTA. Calcium influx is a key step in the process of granule
exocytosis (Maul-Pavicic, Chiang et al. 2011), which can be specifically blocked by chelation of
calcium from the medium using EGTA without altering other cellular pathways. We observed
however, a small amount of residual killing after EGTA treatment of KHYG-1 at low E:T ratios
implicating ligand-mediated killing not seen with NK-92. Our high throughput flow cytometry
screen of KHYG-1 demonstrated the presence of all ten tumour necrosis factor family members
tested for with high expression of Fas ligand and TRAIL, both major ligands involved in
apoptosis induction by T and NK cells. NK-92 expressed nine tumour necrosis factor receptors
with equivalent Fas Ligand expression to KHYG-1, but lower TRAIL expression.
156
While cytotoxicity of NK-92 against primary AML has been established (Yan, Steinherz et al.
1998), KHYG-1 cytotoxicity against primary AML has not been previously reported. We noted
that KHYG-1 was less effective than NK-92 at killing both leukemia cell lines and primary AML
samples and sought to determine the basis for this finding. KIRs present on NK cells recognize
class I HLA and can be either activating or inhibiting (Velardi 2008). Presence of an inhibitory
KIR ligand, particularly HLA-C alleles can suppress NK cytolytic function. Further, KIR
receptor-ligand mismatch has been a factor associated with improved survival outcomes in
haplotype hematopoietic marrow transplantation for AML in some, indicating that NK cells can
mediate a powerful graft-versus-leukemia effect(Ruggeri, Capanni et al. 2002; Ruggeri, Capanni
et al. 2005).
It was initially reported that NK-92 lacked inhibitory killer immunoglobulin-like receptors
(KIRs)(Tonn, Becker et al. 2001), but we had previously demonstrated that NK-92 expresses
mRNA for KIR- 2DL4, 2DL5, 3DL1 and 3DL3 and KHYG-1 expresses mRNA for KIR- 2DL3,
2DL4, 2DL5, 3DL1, 3DL2, 3DL3(Suck, Branch et al. 2005). Among these receptors, all are
inhibitory, with the exception of KIR2DL4, which has been shown to have both activating and
inhibiting function (Faure and Long 2002). However, RT-PCR measures mRNA levels which
may not be translated to protein or be transported to the cell surface, so we therefore utilized
HTS flow cytometry to analyze the receptor profiles of NK-92 and KHYG-1, to assist in
determining the reason for differential killing against AML. Our HTS flow cytometry did not
detect significant levels of inhibitory KIRs on the surface of NK-92 consistent with previous
work(Tonn, Becker et al. 2001) with the exception of KIR2DL4. HTS flow cytometry of
KHYG-1 revealed expression of low levels of KIR-2DL3, 2DL5, high levels of KIR2DL4, but
no 3DL1, demonstrating that presence of KIR mRNA does not always translate into cell surface
expression of the protein. We did not detect significant levels of KIR2DL1 and KIR2DL2 on
KHYG-1 as previously reported (Matsuo 2003), which may have been related to differing culture
conditions.
The ligands for the inhibitory KIR receptors expressed on KHYG-1 are: KIR2DL3 (HLA-C1),
KIR2DL4 (HLA-G), KIR2DL5 (unknown). We postulated that exclusive KHYG-1 expression
of KIR2DL3 binding to HLA-C1 might explain the poor baseline cytolytic function of KHYG-1
relative to NK-92. We therefore blocked class I HLA on AML cell lines to determine if this or
other undefined receptors involved in HLA recognition might be involved in recognition. Class I
157
HLA blocking was done using antibody from clone W6/32, which results in a pan-HLA class I
blockade including HLA-E(Wooden, Kalb et al. 2005). Blockade of class I HLA of primary
AML target cells did not enhance NK-92 killing, consistent with the minimal inhibitory KIR
expression. However, class I blockade resulted in improved killing by KHYG-1 of one primary
AML sample tested (080008), suggesting blockade of an inhibitory KIR receptor on KHYG-1.
We obtained the HLA typing of these primary AML samples to determine consistency with the
KIR receptor-ligand hypothesis. Sample 080008 was homozygous for HLA-C2, which would
typically engage the inhibitory receptor KIR2DL1, but this receptor is not expressed on KHYG-
1. Therefore, the enhancement following blockade of class I HLA cannot be explained by
interference with an inhibitory signal from KIR2DL1. Sample 080179, 5890 and 0909 were
homozygous for HLA C1 and would be expected to mediate inhibition of KHYG-1 via KIR
2DL3, which could potentially be impacted by HLA class I blockade, but there was no change in
cytotoxicity with target class I blockade. KHYG-1 did have a low expression of KIR2DL3, but
antibody blockade of this receptor did not result in an increase in cytotoxicity against primary
AML sample 0909 which was homozygous for the HLA-C1 group, the natural ligand of this
KIR. KHYG-1 also does not express KIR3DL1 that engages HLA-Bw4 group. However,
KHYG-1 does express mRNA for KIR3DL2, which was not included in the HTS flow cytometry
screen and therefore may be expressed by KHYG-1. However, KIR3DL2 only engages with
HLA-A3 and -A11, making it an unlikely candidate receptor to explain the results with sample
080008.
HLA-G could in theory inhibit KHYG-1 via KIR2DL4, but is predominately expressed on the
placenta and serves to down modulate NK cell cytotoxicity at the materno-fetal interface. HLA-
G was not been detected on acute leukemias in one study (Polakova, Krcova et al. 2003), but was
reported on 68% of primary AML samples in another study.(Locafaro, Amodio et al. 2014)
NKG2A, when engaged by HLA-E can mediate an inhibitory signal.(Lee, Llano et al. 1998)
KHYG-1 does express NKG2A, and blockade of HLA E using W6/32 monoclonal antibody
could impact cytotoxicity if 080008 expresses HLA-E. HL-60 cells (an acute promyelocytic cell
line) express HLA-E (Marin, Ruiz-Cabello et al. 2003), while Majumber et al. demonstrated a
lack of expression of HLA-E on primary leukemic cells (Majumder, Bandyopadhyay et al.
2006). Another study demonstrated that interferon-γ was required to induce primary AML cells
to express HLA-E, leading to inhibition of NK cell-mediated cytolysis.(Nguyen, Beziat et al.
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2009). Therefore, it is conceivable that sample 080008 expresses HLA-E, which could mediate
an inhibitory effect that when blocked by anti-class I HLA monoclonal antibody, could enhance
cytotoxicity.
We then sought to determine the effect of pretreating NK-92 and KHYG-1 with antibodies
against a panel of activating receptors commonly associated with NK cell cytotoxicity (natural
cytotoxicity receptors, NKG2D and DNAM-1). While we anticipated potential blocking of
cytotoxicity at 10 µg/ml against a panel of leukemia cell line targets, we observed stimulation of
cytotoxicity from some of the antibodies and no blocking of cytotoxicity. Treatment of NK-92
with antibodies to NKp30, NKp44, NKp46 increased killing of K562 by approximately 10%,
while only NKp30 treatment enhanced killing of KG1a and OCI/AML5. Further, anti-NKp30,
but not anti-NKp44 pretreated NK-92 had a large degree of enhancement in killing of primary
AML. Previously, we detected expression of NKp30 on NK-92 and found it to mediate
recognition of multiple myeloma cells (Swift, Williams et al. 2012), which was reduced when
NK-92 was blocked by this anti-NKp30 antibody (P30-15 clone). The discrepancy of activity of
this antibody prompted us to consider the mechanism of enhancement against leukemic targets.
HTS flow cytometery demonstrated higher expression of NKp30, NKp44 and NKp46 on
KHYG-1 compared with NK-92. Treatment of KHYG-1 with antibodies to all the natural
cytotoxicity receptors increased killing of K562 and OCI/AML5 to a greater degree than NK-92.
KHYG-1 cytotoxicity against primary AML was enhanced to a greater degree than NK-92 with
pretreatment with anti-NKp30 or anti-NKp44. Pretreatment of NK cell lines with antibodies
against DNAM-1, NKG2D (both commonly involved in NK cell recognition) induced small
statistically significant inhibitory effects against target K562 in some experiments, indicating a
possible role for these molecules in recognition. However, anti-DNAM-1 and anti-NKG2D were
not able to facilitate reverse ADCC despite high expression of DNAM-1 and NKG2D on both
cell lines.
We sought to determine the dose response curve of antibody-mediated cytotoxic enhancement,
which was most prominent with KHYG-1 against OCI/AML5. Increases in KHYG-1
cytotoxicity against OCI/AML5 was seen as low as 0.01 µg/ml, which is near the EC50 of the
stimulatory effect. A plateau in enhancement started to occur at 0.1 µg/ml with slight increase in
efficacy at 1 µg/ml. NK-92 had lesser enhancement than KHYG-1 with this approach, but
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effects could be observed in the 0.01 µg/ml dose range particularly against OCI/AML3, which is
somewhat resistant to NK-92 cytotoxicity.
While the antibodies we used were blocking antibodies, based on studies of endogenous NK cell
cytotoxicity(Markel, Seidman et al. 2009), we postulated that the increased killing was mediated
by reverse-antibody dependent cell-mediated cytotoxicity (R-ADCC). R-ADCC results when an
antibody fixed via the Fab fragment to an effector cell activating receptor can become
crosslinked by engagement with Fc gamma receptors on the target cell. We further tested this
approach with a panel of esophageal lines, but did not observe any significant increase in
cytotoxicity after pretreatment of NK-92 or KHYG-1 with anti-NKp30 or anti-NKp44.
Regression analysis of the delta cytotoxicity with CD32 expression of the cell line targets yielded
highly significant correlation for KHYG-1, but not for NK-92. However, NK-92 had higher
baseline killing against some cell line targets relative to KHYG-1 and had less potential for
enhancement, particularly for OCI/AML5. Also, NK-92 had lower expression of NKp30 than
KHYG-1, and minimal expression of NKp44, which may have affected the degree of
enhancement possible by R-ADCC against leukemia cell lines. However, there was a high
degree of enhancement of NK-92 pretreated with anti-NKp30 against primary AML targets. The
regression analysis supported reverse ADCC via CD32 as the mechanism of the enhancement for
KHYG-1 cytotoxicity against the cancer cell line panel. Given that the enhancing effect of anti-
NKp30 and anti-NKp44 was not generic in nature makes another mechanism such as co-
activation or co-stimulation unlikely. Further, the low concentrations of antibody that could
elicit this effect and the high magnitude of enhancement were consistent with R-ADCC which
can occur <0.1 µg/ml and lead to several- fold increases in cytotoxicity against resistant targets.
NK cells were first implicated in mediating reverse ADCC by Saxena et al.(Saxena, Saxena et al.
1982) The cell lines and primary AML samples we tested were predominantly FAB M4
(myelomonocytic) and M5 (monocytic), which typically express Fc gamma receptors. This
enables them to bind the Fc portions of antibodies, which in turn allows for reverse ADCC to
occur in the context of our assay. Reverse ADCC has been primarily demonstrated using P815
murine mastocytoma cell lines in combination with NK cells pretreated with antibodies against
unknown or known NK cell antigens, to determine novel receptors, and this method allowed for
the discovery of NKp30. (Pende, Parolini et al. 1999). Subsequently, the approach was used to
discover and characterize the NKp44(Vitale, Bottino et al. 1998) and NKp46(Pessino, Sivori et
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al. 1998) receptors. Antibody pretreatment of NK cells with antibodies directed at activating
receptors can allow them to become crosslinked followed binding to an Fcγ receptor positive
target cell. The effect can be very potent in facilitating lysis against resistant P815 cells, with
effects as low as 0.001 µg/ml reported.(Pende, Parolini et al. 1999) While reverse ADCC has
been described in this context, it has never been shown to function in vivo. Ball et al. conducted
a study of the expression of Fcγ receptors on primary AML and noted the following frequencies:
FcγRI (58%); FcγII, (67%); and Fcγ III, (26%). FcγI and II receptor expression was highly
correlated with FAB M4 and M5 morphology. Notter et al. demonstrated that precoating
lymphokine activated killer (LAK) cells with anti-CD3 antibodies could enhance killing of
autologous AML blasts through reverse ADCC via FcγRI (CD64).(Notter, Ludwig et al. 1993)
This was established by showing a 1.5-9.3 fold increase in killing that was dependent on CD64
expression of targets and could be partially reduced by blocking FcγRs with very high doses of
murine IgG2a or human monomeric IgG. In this study, CD34+ hematopoietic stem cells did not
express FcγRs and were not subject to enhanced killing by anti-CD3 coated LAK cells. R-
ADCC was not tested in vivo, but the authors proposed a combination of IL-2, IFN-γ and anti-
CD3 monoclonal antibody as a potential treatment for AML. However, here we demonstrate R-
ADCC as a means of enhancing NK cell line cytotoxicity against leukemic cell lines and, more
importantly, primary AML cells. The combination of antibody and NK cell line together in this
manner provides a potentially novel leukemia therapy.
While this is the first time NK-92 and KHYG-1 have been shown to mediate reverse ADCC,
gene modified CD16+NK-92(Binyamin, Alpaugh et al. 2008) and CD16+KHYG-1(Kobayashi,
Motoi et al. 2014) have been generated and are capable of mediating ADCC against CD20+
targets in combination with Rituximab. However, for CD16+KHYG-1, the lowest effective
concentration of Rituximab was 0.1 µg/ml, while for anti-NKp30 or anti-NKp44 mediated
reverse ADCC the dose was less than 0.01 µg/ml, demonstrating the relative potency of reverse
ADCC.
We then sought to determine if the enhancing effect of pretreating NK cell lines with anti-
NKp30 and anti-NKp44 held against clonogenic cells by using our established methylcellulose
cytotoxicity assay. We noted that NK-92 was relatively effective at inhibiting OCI/AML5
colony formation, but this could not be enhanced by pretreatment with anti-NKp30 over isotype
control. However, KHYG-1 was less effective at inhibiting OCI/AML5 colony formation, but
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this could be enhanced three-fold by pretreatment with anti-NKp30 antibody. The inhibition of
colonies indicates a cytotoxic or cytostatic effect on clonogenic cells within the cell line
populations that represent leukemic stem and progenitor cells. Therefore, this provides indirect
evidence that reverse ADCC can facilitate cytotoxicity against leukemic stem cells.
To evaluate the impact of NK cell line therapy in vivo, we established an OCI/AML5 xenograft
model in NSG mice. OCI/AML5 was derived from a patient with M4 leukemia, and highly
expresses CD33, which is useful for tracking in vivo.(Wang, Koistinen et al. 1991) This cell line
was used previously in a NOD/SCID mouse model to evaluate Indium111
conjugated anti-CD123
antibody therapy.(Leyton, Hu et al. 2011) We confirmed injection of OCI/AML5 iv led to
leukemia with splenomegaly and bone marrow engraftment. However, ip injection tended to
cause progressive malignant ascites over a longer timeframe, rather than classic leukemia.
To determine the effect of in vitro cytotoxicity on in vivo proliferation, we incubated OCI/AML5
with or without iKHYG-1 (+/-anti-NKp30 pretreatment) and injected the cells ip. We utilized
this injection route instead of iv, because of the high cell load (2x106 OCI/AML5 + 20x10
6
viable iKHYG-1 + ~3x106 non-viable iKHYG-1) and relatively larger KHYG-1 cells, which
might have caused pulmonary stress to the mice if injected via tail vein. Co-incubation with
iKHYG-1 had no impact, while anti-NKp30 pretreated iKHYG-1 improved median survival by
10 days.
We subsequently utilized the iv injection OCI/AML5 NSG xenograft model to test therapeutic
efficacy of iKHYG-1 with or without anti-NKp30 pretreatment. Unexpectedly, iKHYG-1 was
able to improve the median survival by 35 days, despite its poor cytotoxicity in the CRA against
bulk OCI/AML5. However, KHYG-1 had three-fold better cytotoxicity against clonogenic
OCI/AML5 than bulk OCI/AML5, as determined by the CRA, providing some basis for this
finding. This is the first demonstration of efficacy by KHYG-1 in an in vivo cancer model and
confirms that the irradiated cells can persist and reduce tumour burden. While it has been shown
that irradiation has minimal effects on cytotoxicity in vitro (Suck, Branch et al. 2006), this is the
first evidence that the irradiated cells can function in vivo. However, pretreatment of iKHYG-1
with anti-NKp30 did not enhance survival over iKHYG-1-treated OCI/AML5-xenografted NSG
mice. We postulate that this is due to antibody dissociating in vivo, given that no iv doses of
antibody were given to the mice. We also used a primary AML xenograft model to test iKHYG-
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1 and anti-NKp30 iKHYG-1, demonstrating lack of efficacy of iKHYG-1, but a trend to
improved survival in the anti-NKp30 iKHYG-1 treated group.
In summary, we have conducted an extensive characterization of the cell surface expression of
the two most potent NK cell lines with therapeutic potential, using high throughput screening
flow cytometry identifying key molecules involved in adhesion, activation and inhibition of
cytotoxicity as well as apoptosis inducing ligands. We demonstrate that NK-92 and KHYG-1
have cytotoxicity against a broad range of leukemic targets that can be enhanced several-fold by
by anti-NKp30 and anti-NKp44 antibodies. For KHYG-1, reverse ADCC has been implicated at
the mechanism of enhancement via interaction of antibody coated effectors with FcγRII (CD32)
on the target cells. Furthermore, NKp30 mediated reverse ADCC can enhance cytotoxicity of
KHYG-1 against clonogenic leukemic cells and affect in vivo proliferation of leukemia. Further
studies of combination of anti-NKp30 antibody pretreatment and NK cell line therapies are
warranted.
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6 Chapter 6: General Discussion
6.1 Overview
Cancer immunology has focused primarily on the interaction of the immune system with bulk
tumour cells. This has related to both a historical conceptual understanding of tumour biology,
and limitations in methodologies to address heterogeneity within tumours, as it relates to immune
cell recognition and cytotoxicity. While tumour heterogeneity was noted in the 19th
century by
Rudolph Virchow,(Brown and Fee 2006) the connection with stem cell biology occurred much
later. However, as early as 1937, leukemia was demonstrated as clonal, driven by rare leukemia-
initiating cells.(Furth, Kahn et al. 1937) The discovery of hematopoietic stem cells(McCulloch
and Till 1960) led to a paradigm shift in thinking about cell biology and initial work supported
that cell fate decisions were stochastic(Till, McCulloch et al. 1964). Around this time, these
concepts were applied to cancer progression, with a cogent leukemia stem cell hypothesis being
postulated as an alternative to a stochastic model(Bruce and Ash 1963). The formal
demonstration of the existence of rare leukemic stem cells(Lapidot, Sirard et al. 1994) opened
the door to questions about how the immune system interacts with these rare cancer stem cells.
This work also called into question the conclusions made from work done using bulk tumour
cells in cancer immunology (Williams, Swift et al. 2013), and other fields of cancer research
such as drug discovery(Williams, Anderson et al. 2013). Here we have investigated the natural
killer cell lines, NK-92 and KHYG-1, against leukemia utilizing techniques to address the impact
on bulk and leukemic stem cells. Both of these cell lines can preferentially target leukemic stem
cells as demonstrated by comparing a novel in vitro clonogenic cytotoxicity assay with the
standard chromium release assay. These findings can be extended in vivo, with NK-92 and
KHYG-1 capable of prolonging survival in AML xenograft models. Using gene-modified NK-
92 expressing the high affinity Fc gamma receptor, LSCs can be targeted by ADCC in vitro
using anti-CD123 monoclonal antibodies, which also can be combined in vivo to improve
survival in a primary AML xenograft model. While KHYG-1 has a lower baseline ability to kill
leukemic targets than NK-92, it can be significantly enhanced when pretreated with anti-NKp30
and NKp44 monoclonal antibodies, via reverse ADCC. These data support the notion that NK
cell lines can target leukemic stem cells, be enhanced by antibodies via ADCC or reverse ADCC,
and lead to enhanced survival in translationally relevant leukemia models. These results are on
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par with recent pre-clinical studies of CD123 chimeric antigen receptor (CAR) T-cell therapy of
AML(Mardiros, Dos Santos et al. 2013; Tettamanti, Marin et al. 2013; Gill, Tasian et al. 2014;
Pizzitola, Anjos-Afonso et al. 2014), a rapidly emerging cellular immunotherapy with
unprecedented clinical success in the context of CD19 CAR T-cell therapy for relapses and
refractory B-cell malignancies(Kalos, Levine et al. 2011; Porter, Levine et al. 2011; Grupp,
Kalos et al. 2013). Therefore, cell line therapy, with its more standardized expansion and
functional characteristics, represents a viable cellular therapeutic platform, with a strong
rationale to test combined cell line and antibody therapy in a clinical trial for AML patients with
minimal residual disease lacking a suitable allogeneic transplant donor.
6.2 Methodologic approaches to measuring the impact of immune
effectors on leukemic stem cell and bulk leukemia
Our initial focus was to develop a cell line model of leukemic stem cells to facilitate a better
understanding of how immune effectors recognize and kill this subpopulation of cells relative to
bulk leukemia. We selected KG1, a CD34+CD38+ cell line, which immunophenotypically
represented a typical AML primary sample, and postulated that it might contain a stem cell-
driven hierarchy, similar to the original findings for acute myeloid leukemia primary
blasts(Lapidot, Sirard et al. 1994; Bonnet and Dick 1997) The stem cell frequency was
determined with liquid reculturing in combination with serial dilution and single cell sorting, and
the methylcellulose colony-forming assay, all of which yielded a similar stem cell frequency
(1:100 to 1:1000). Therefore, we established that KG1 had a relatively rare tumour-initiating
cell, and sought to demonstrate if this was driven by an identifiable cancer stem cell, as had been
shown for some brain tumour(Kondo, Setoguchi et al. 2004), and multiple myeloma cell
lines(Matsui, Huff et al. 2004). KG1 did contain rare CD34+CD38- cells (1-3%), however, both
the CD34+CD38- and CD34+CD38+ fractions had unlimited proliferation capacity and the
ability to recapitulate the original culture immunophenotypic distribution of CD38. While these
findings were at odds with the original report from John Dick’s research group(Lapidot, Sirard et
al. 1994), they were consistent with a follow-up study demonstrating that CD34+CD38+ AML
cells had stem cell capacity. This was masked in the original study because the anti-CD38 mAb
used facilitated clearance by the NOD/SCID reticuloendothelial system, which could be blocked
by pretreating the immunodeficient mice with either IVIg or anti-CD122 (IL-2Rβ).(Taussig,
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Miraki-Moud et al. 2008). This work questioned the earliest report identifying the leukemic
stem cell as being restricted to the CD34+CD38- fraction(Lapidot, Sirard et al. 1994). However,
the experiments supporting CD34 as a LSC marker were not subject to the same artifactual
clearance issue, and it remains a reliable functional LSC marker for the majority of primary
AML samples. In our in vitro studies of sorted KG1, the putative stem cell was contained in
both the CD38+ and CD38- fractions, consistent with Taussig et al.(Taussig, Miraki-Moud et al.
2008).
Prior to initiation of our studies, only one group had looked at the in vitro sensitivity of
CD34+CD38- AML cancer stem cells to immune effector cell killing. In that study, lymphokine
activated killer cells and allogeneic lymphocytes exerted a modest cytotoxic effect on AML
cancer stem cells that were intrinsically resistant to the chemotherapeutic agent,
daunorubicin.(Costello, Mallet et al. 2000) Since KG1 had no identifiable stem cell fraction, cell
sorting could not be used, so we utilized functional stem cell readout- the methylcellulose
colony formation assay. This approach is blind to whether a tumour population is driven by a
stem cell hierarchy or a stochastic process, and there is no need to know the molecular features
of a putative stem cell population. To address the question of relative cytotoxicity of immune
effectors to LSCs and bulk leukemia required the development of a properly controlled
methylcellulose assay to facilitate this comparison. We demonstrated that the chromium release
assay significantly underestimated the cytotoxicity of NK-92 against the stem cell fraction in
KG1. At a 10:1 E:T NK-92 had 100% colony inhibition against KG1, but only 78 % lysis by the
chromium release assay. Given that other studies of cancer stem cells show them to be resistant
to chemotherapy(Costello, Mallet et al. 2000), this finding is important in demonstrating that an
immune effector can preferentially kill a leukemic stem cell.
Despite being unable to identify cancer stem cell by immunophenotypic profile within KG1,
using a clonogenic assay we have by-passed this requirement to obtain valuable information on
the cytotoxicity of NK-92 against putative leukemic stem cells within this cell line. This
provides a method for assessment of the impact of any immune effector cell on putative cancer
stem cells of numerous cell lines and primary samples extending beyond leukemia. Further, by
standardizing the methylcellulose cytotoxicity assay as closely as possible with the CRA and by
utilizing the appropriate controls, it is possible to determine if there is differential killing of an
166
effector cell against bulk or leukemic stem cells. This approach is relatively simple, and can be
applied to any tumour that can grow well in semi-solid medium such as methylcellulose.
6.3 Natural cytotoxicity of NK cell lines with and without ADCC
enhancement against leukemic stem cells
While the overall long-term survival for AML is around 40% in adults (Lowenberg, Downing et
al. 1999) and 60% for children, subgroups of AML continue to fare very poorly, including
patients with the recurrent cytogenetic 10 year survival is less than 10% respectively(Grimwade,
Hills et al. 2010). The induction rates for most protocols is 80%, however, the majority still
harbor minimal residual disease, leading to relapse, often with chemotherapy resistant disease.
Therefore, there is a need for novel, non-cross resistant therapies to treat MRD, which contains
LSCs. Immune based therapy, such as NK-92, is an excellent experimental therapeutic option
for AML patients with MRD.
Therefore, we evaluated NK-92 with a focus on using methods that evaluate the impact on
leukemic stem cells and overall survival in an AML xenograft model. We confirmed initial
reports that NK-92 could mediate cytotoxicity in vitro against primary AML(Yan, Steinherz et
al. 1998), and demonstrated that this is done via granule exocytosis. Classically defined sorted
CD34+CD38- LSCs(Lapidot, Sirard et al. 1994) were more sensitive to NK-92 killing than bulk
leukemia at low E:T ratios utilizing a standard chromium release assay. Given the conflicting
reports in the literature of the definitive immunophenotype of the leukemic stem cell in
AML(Lapidot, Sirard et al. 1994; Bonnet and Dick 1997; Taussig, Miraki-Moud et al. 2008;
Goardon, Marchi et al. 2011), we opted to use a clonogenic assay to assess the impact of immune
effectors against leukemic stem cells in a larger set of samples. The comparison of the MCA
with the CRA demonstrated a 2-3 fold higher % colony inhibition than the % lysis, supporting
our initial finding with cell line KG1, that NK-92 can preferentially recognize and kill LSCs over
bulk leukemia. Langencamp et al. demonstrated that single KIR-expressing NK cells,
mismatched for the HLA of primary AML targets, had equivalent killing of LSCs and blasts
using a chromium release assay and a methylcellulose-based cytotoxicity assay,(Langenkamp,
Siegler et al. 2009). By contrast, we demonstrate preferential killing of LSCs by NK-92 relative
to bulk leukemia. This suggests that NK-92 has enhanced ability to kill LSCs relative to LAK
167
cells(Costello, Mallet et al. 2000) or endogenously derived single-KIR expressing NK
cells(Langenkamp, Siegler et al. 2009). This phenomenon is also seen with NK-92 treatment of
multiple myeloma (MM) cell lines which also show preferential killing of clonogenic MM cells
over bulk tumour (Swift, Williams et al. 2012).
Using a secondary transplantation assay, iNK-92 therapy of primary engrafted mice was shown
to impact the LSC fraction in the secondary transplant mice, providing some evidence for in vivo
LSC cytotoxicity using a gold standard assay. AML-xenografted NSG mice were effectively
treated with NK-92 infusions, leading to improvement in survival relative to controls, confirming
previous work (Yan, Steinherz et al. 1998). However, we accomplished this with lower doses of
NK-92 than the original and without the use of IL-2 infusions. Irradiated NK-92 were able to
prolong survival in mice, but were less effective than the non-irradiated NK-92 cells, as expected
due to their inability to in vivo expand. Given the modest impact of iNK-92 on improving
survival in vivo, we attempted to enhance efficacy of this approach using a gene-modified
ADCC-capable NK-92 cell line (Figure 6.1). CD16+NK-92 have been successfully redirected to
kill CD20+ malignant targets using Rituximab, (Binyamin, Alpaugh et al. 2008). CD16+NK-92
cytotoxicity could be enhanced against the CD123+ leukemia line OCI/AML5 using an anti-
CD123 mAb through ADCC.
We then sought to combine iCD16+NK-92 and 7G3 therapy in our AML xenograft model.
Systemic treatment with 7G3 alone had been tested in an AML NOD/SCID xenograft model,
with evidence of impact on primary bone marrow engraftment, but not on survival in
therapeutically relevant animal models(Jin, Lee et al. 2009), indicating a need to optimize this
therapeutic strategy. We were able to increase the efficacy of the iCD16+NK-92 cells and
improve survival by administering 100 µg of 7G3 antibody for five doses relative to use with
isotype control. This demonstrates that the combination of iCD16+NK-92 with 7G3 can
improve survival by ADCC. This is the first demonstration of in vivo efficacy of the CD16+NK-
92 cell line in combination with antibody against any cancer. Recently, CSL362, an Fc-
optimized anti-CD123 humanized monoclonal antibody, was shown to facilitate ADCC from
peripheral blood-derived allogeneic NK cells against primary AML and CD123-expressing cell
line targets.(Busfield, Biondo et al. 2014) Further, CSL362 is in clinical trials as single agent for
relapsed and refractory AML and would make an ideal combination therapy with CD16-NK-92.
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Figure 6.1: Antibody-dependent cell-mediated cytotoxicity (ADCC)
CD16+NK-92 can mediate ADCC via the high affinity CD16A receptor in combination with
anti-CD123 monoclonal antibody (7G3) against CD123 (black antigen) expressing AML cells.
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6.4 Natural cytotoxicity of NK cell lines with and without reverse-
ADCC enhancement against leukemic stem cells
While NK-92 has been tested in phase I trials with minimal toxicities (Arai, Meagher et al. 2008;
Tonn, Schwabe et al. 2013), KHYG-1 has only been assessed against leukemia in vitro,
warranting further study of its potential clinical utility. KHYG-1 cytotoxicity against primary
AML was unreported, and we wished to compare its efficacy to NK-92. We noted that KHYG-1
was less effective than NK-92 at killing both leukemia cell lines and primary AML samples, and
sought to determine the basis for this finding. While we demonstrated that KHYG-1 expressed
more inhibitory KIRs than NK-92, we were unable to implicate them in the role of reduced
relative cytotoxicity.
We pretreated NK-92 and KHYG-1 with antibodies against a panel of activating receptors with
the objective of blocking receptors involved in AML recognition. However, we observed
predominantly stimulation of cytotoxicity from several of these antibodies, with minimal
inhibitory effects. Treatment of NK-92 with antibodies to NKp30 most notably enhanced
cytotoxicity to a modest degree against AML cell lines and to a great degree against primary
AML samples. Flow cytometry demonstrated higher expression of the natural cytotoxicity
receptors (NKp30, NKp44 and NKp46) on KHYG-1 compared with NK-92. Pretreatment of
KHYG-1 with antibodies to all the natural cytotoxicity receptors increased killing of cell line
targets, while anti-NKp30 and anti-NKp44 pretreatment enhanced cytotoxicity against primary
AML. KHYG-1 cytotoxicity against OCI/AML5 was enhanced with effector antibody
pretreatments with an EC50% of 0.01 µg/ml, indicating the potency of this approach.
We postulated that the increased killing was mediated by reverse antibody-dependent cell-
mediated cytotoxicity (R-ADCC), which involves the Fab of an antibody bridging an activating
receptor on the NK cell with an Fcγ receptor on the target (Figure 6.2), leading to NK signal
transduction and degranulation. We did not observe any significant increase in cytotoxicity after
pretreatment of NK-92 or KHYG-1 with anti-NKp30 or anti-NKp44 against Fc gamma negative
esophageal cancer cells. This is consistent with R-ADCC as the mechanism, and not co-
stimulation or co-activation, which would have led to generic enhancement. Regression analysis
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of the delta cytotoxicity with CD32 expression of the cell line targets yielded highly significant
correlations for KHYG-1, further supporting that the enhancement was from R-ADCC.
Figure 6.2: Reverse antibody-dependent cell-mediated cytotoxicity (R-ADCC)
KHYG-1 can mediate reverse ADCC in combination with anti-NKp30 monoclonal antibody
against Fcγ receptor expressing AML cells.
NK cells were first demonstrated to mediate non-ADCC, antibody-induced redirected lysis by
Saxena et al.,(Saxena, Saxena et al. 1982), where the term R-ADCC was first utilized to describe
the phenomenon. R-ADCC has been demonstrated using FcRγ+ P815 murine mastocytoma cell
lines in combination with NK cells pretreated with novel anti-NK cell antibodies to discover the
natural cytotoxicity receptors (NKp30, NKp44 and NKp46). (Vitale, Bottino et al. 1998; Pende,
Parolini et al. 1999) (Pessino, Sivori et al. 1998). Reverse ADCC is a very potent form of
redirected lysis, with an EC50 of ~0.01 µg/ml reported for anti-NKp30 redirected NK cell lysis
against P815 targets(Pende, Parolini et al. 1999), similar to our findings. However, reverse
ADCC has never been demonstrated in vivo.
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A thorough study of the expression of Fcγ receptors on 146 primary AML samples demonstrated
considerable expression of FcγRI (58%), FcγII (67%), and FcγIII (26%). The actual percentage
of samples expressing at least one Fcγ receptor was not presented in this work, but would be
expected to be 90%, assuming independent segregation of receptors, indicating they are relevant
therapeutic targets for AML. One study has demonstrated that lymphokine-activated killer
(LAK) cells could be redirected to kill autogolous AML blasts with anti-CD3 antibodies via
reverse ADDC. Further, this effect was demonstrated to occur through FcγRI (CD64).(Notter,
Ludwig et al. 1993) Precoating LAK cells with anti-CD3 antibodies led to many-fold increases
in cytotoxicity against CD64-expressing targets. Of note, this group also demonstrated that
CD34+ hematopoietic stem cells do not express FcγRs, and are insensitive to anti-CD3
pretreated LAK cells, suggesting that this approach would not be toxic to bone marrow HSCs in
vivo. However, no work was done to assess the impact of R-ADCC on leukemic stem cells. We
show that KHYG-1 was moderately effective at inhibiting OCI/AML5 colony formation, which
could be enhanced three-fold by pretreatment with anti-NKp30 antibody. This provides the first
evidence that R-ADCC can be used to target clonogenic cells in vitro which includes leukemic
stem and progenitor cells. Further, co-incubation of OCI/AML5 with anti-NKp30 pretreated
iKHYG-1 and subsequent infusion in NSG mice improved median survival by 10 days over
control or iKHYG-1 treatment alone, further supporting that reverse ADCC can target leukemic
stem cells within OCI/AML5 cells that have leukemogenic potential. In a therapeutically
relevant, OCI/AML5 NSG xenograft model, we tested the therapeutic efficacy of iKHYG-1 with
or without anti-NKp30 pretreatment. iKHYG-1 was able to improve the median survival by 35
days, which could not be predicted from the results of the chromium release assay which showed
poor cytotoxicity, but was predicted by the clonogenic assay which showed a moderate cytotoxic
effect. This represents the first demonstration of efficacy by KHYG-1 in an in vivo cancer
model, and confirms that the irradiated cells can persist and reduce tumour burden. This builds
on prior work from our group that demonstrated that irradiation has minimal effects on
cytotoxicity in vitro (Suck, Branch et al. 2006). However, while pretreatment of iKHYG-1 with
anti-NKp30 at slight enhanced survival over iKHYG-1-treated OCI/AML5 or primary AML-
xenografted NSG mice, the results were not statistically significant, falling short of a formal
demonstration of in vivo R-ADCC. Given that no antibody was injected directly into these
mice, the antibody likely dissociated in vivo, reducing its potential efficacy.
172
We demonstrate that NK-92 and KHYG-1 have cytotoxicity against a broad range of leukemic
targets that can be enhanced several-fold by pretreatment with anti-NKp30 and anti-NKp44
antibodies. For KHYG-1, reverse ADCC has been formally demonstrated as the mechanism of
enhancement via interaction of antibody coated effectors with FcγRII (CD32) on the target cells.
Furthermore, NKp30 mediated reverse ADCC can enhance cytotoxicity of KHYG-1 against
clonogenic leukemic cells, and affect in vivo proliferation of leukemia. Additional
experimentation is required to fully demonstrate if reverse ADCC can occur in vivo in a
therapeutically relevant model.
6.5 Translational relevance
Adoptive immunotherapy with NK cells represents a novel emerging treatment modality for
patients with AML with several established protocols for allogeneic NK cell therapy for
AML.(Miller, Soignier et al. 2005; Rubnitz, Inaba et al. 2010) Anti-cancer cell-based
immunotherapy with a standardized, highly cytotoxic NK cell lines,(Arai, Meagher et al. 2008;
Tonn, Schwabe et al. 2013) is an attractive alternative to autologous or allogeneic NK cells
which have attendant variability in cytotoxicity and cell manufacturing characteristics. Further,
gene modifications can be done once with cell lines, rather than for each therapeutic attempt with
autologous, patient-derived immune effector cells. As an example, high affinity CD16-
transduced NK-92 cells can be utilized in combination with any humanized monoclonal
antibody, providing a flexible platform for combined cellular and humoral immune therapeutic
approaches. We have demonstrated, proof-in-principle, that this approach can be used to target
LSCs by anti-CD123-facilitated ADCC in vivo and enhance survival in a primary AML
xenograft model. The improvement in survival with this approach was approximately 2 week
extension of median survival. This is consistent with results of CD123 CAR T-cell therapy in a
an AML NSG xenograft model(Mardiros, Dos Santos et al. 2013), however, this model utilized
cell line KG1a, not primary AML cells. Using a different CAR vector, another group tested
CD123 CAR T-cell in a primary AML model with no improvement of median survival, but had
approximately 40% long term survivors at day 100 (~70 days after control median survival),
which was statistically significant.(Gill, Tasian et al. 2014) One other study of CD123 CAR T-
cells demonstrated efficacy against LSCs using primary and secondary engraftment models, but
173
did not study impact on survival(Pizzitola, Anjos-Afonso et al. 2014). Another group utilized
CD123 CAR transduced cytokine induced killer (CIK) cells, and demonstrated in vitro efficacy
against CD123+ cell line and primary AML targets.(Tettamanti, Marin et al. 2013).
We also demonstrate the therapeutic efficacy of KHYG-1 in an AML xenograft model, providing
a rationale to develop this as a novel platform for AML therapy. While KHYG-1 cannot mediate
ADCC because it is FcγRIIIA (CD16) negative, it highly expresses the activating receptors
NKp30 and NKp44, which can facilitate reverse ADCC against Fcγ receptor positive targets.
Given that approximately 90% of primary AML samples express at least one Fcγ receptor, there
is a strong rationale to develop reverse-ADCC targeting of AML as a therapeutic strategy. A
humanized anti-NKp30 antibody is in development, and could be used in combination with
KHYG-1 cell-based therapy for MRD positive AML patients who lack a suitable allogeneic
transplant donor.
174
7 Chapter 7: Conclusions and future directions
Improvements in therapy of AML have been relatively slow, with most new agents being tested
failing at early phase clinical studies. Part of the reason for this failure is that the drug screening
paradigm that has generated most cancer therapeutics has focused on the impact on bulk tumour
cells, rather than cancer stem cells. The most effective therapy for AML remains hematopoietic
stem cell transplantation, the only established cellular immunotherapy, with evidence for a role
in NK cell-mediated benefits in the haplotype transplant setting.
In this thesis, I have focused on the interaction of immune effectors, specifically NK cell lines,
with both bulk leukemia and leukemic stem cells, using established and newly developed
approaches. This has been used in combination with survival analysis in evaluating therapeutic
potential.
In Chapter 3, the classic stem cell markers (CD34+CD38-) established by John Dick’s research
group in 1994, did not hold for the leukemia cell line KG1 or for many other primary AML
samples which have stem cells in the CD34+CD38+ fraction. This indicated the importance of
using a functional readout such as the clonogenic assay to evaluate the impact of immune
effectors on leukemic stem cells. The lower cytotoxic readouts of bulk cytotoxicity assays with
the clonogenic assay suggested that NK-92 was preferentially able to kill leukemic stem cells.
Combining both assays together provided a more complete picture of the cytotoxic impact of
NK-92 against a heterogeneous tumour population.
In Chapter 4, NK-92 was tested against primary AML samples demonstrating moderate
cytotoxicity with the chromium release assay and near complete abolition of colony growth at a
25:1 effector target ratio. Sorted LSCs were also more sensitive to NK-92 by a significant
margin using the chromium release assay. This suggested that the leukemic stem cells were
more sensitive than bulk tumour over a range of samples. We confirmed that non-irradiated NK-
92 can prolong survival in an AML xenograft model, but more importantly demonstrate that the
irradiated cells also have efficacy, though are less effective. Utilization of a high-affinity
CD16+NK-92 cell line, in combination with anti-CD123 mAbs, allowed for ADCC against
175
leukemic stem cells. This approach was validated in an in vivo experiment showing proof-in-
principle that NK cell line-mediated ADCC against leukemic stem cells can enhance survival in
a primary AML xenograft model. To follow-up our findings, we will use a higher dose of 7G3
(300 µg x 5) in combination with CD16+NK-92 in an attempt to improve survival outcomes, and
when available utilize humanized anti-CD123 mAbs in these types of experiments.
In Chapter 5, NK-92 and KHYG-1, another NK cell line was utilized to treat AML cell lines and
primary AML. Both cell lines had no inhibitory regulation by classical KIR ligand interactions
either because of lack of expression of receptors or lack of functionality. However, NK-92
appeared to be more effective overall at killing leukemic cell lines and primary AML than
KHYG-1. Serrendipitously, we discovered that pretreatment of NK-92 and KHYG-1 with
antibodies directed against NK activating receptors led to very large enhancements in
cytotoxicity against leukemic cell lines and primary AML cell lines, but not esophageal lines.
Targets with high FcγRII (CD32) expression were most amenable to this cytotoxic enhancement,
which was highly correlated for KHYG-1 pretreated with either anti-NKp30 or anti-NKp44
antibodies. The mechanism was by reverse ADCC, only described in the context of CD64
positive targets, making the connection with CD32 novel. This enhancement could be mediated
against clonogenic leukemia cells for KHYG-1 only. We also demonstrated that KHYG-1 can
be effective in vivo against OCI/AML5-induced leukemia, which is the first time this cell line
has shown in vivo efficacy against any cancer. While anti-NKp30 pretreatment did not enhance
the efficacy of the iKHYG-1 in an in vivo OCI/AML5 xenograft model, we postulate it was from
dissociation of antibody. We plan to treat an OCI/AML5 xenograft model with both iKHYG-1
and infused anti-NKp30 antibody (50 µg x 6), which will lead to therapeutic serum levels of this
antibody that should facilitate binding to the circulating iKHGY-1.
These studies lay the framework for combination therapies incorporating cell lines and
monoclonal antibody therapies, whereby leukemic stem cells can be targeted via ADCC or
reverse ADCC. Cell line therapy allows for standardization of the dose and schedule, as well as
anticipated efficacy and toxicity. The ongoing development of novel humanized monoclonal
antibodies makes ADCC capable cell lines an ideal adjunct to the more established antibody
therapies.
176
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Appendices
212
Appendix I: NK-92 HTS flow cytometry percent positivity and MFI of antigens
# Antigen % + MFI Antigen name(s)
1 BLTR-1 1.83 133 Leukotriene B4 receptor
2 B2m 99.8 13283 Beta-2 microglobulin
3 CA9 5.39 135 Carbonic anhydrase IX
4 CDH3 3.06 133 Cadherin 3
5 CDH6 5.42 207 Cadherin 6
6 CDH11 1.52 561 Cadherin 11
7 CDw93 4.31 506 C1q-binding protein
8 CDw198 2.02 129 CCR8
9 CDw199 0.0702 188 CCR9
10 CDw210 8.48 172 IL-10 receptor
11 CDw218a 11.9 147 IL-18 receptor
12 CDw329 1.03 524 SIGLEC-9
13 CD1a 0.266 120 T6
14 CD1b 1.19 534 T6
15 CD1c 0.964 123 T6
16 CD1d 0.282 128 CD1d
17 CD2 100 1521 T11, LFA-2, SRBC-R
18 CD3 0.842 133 T3
19 CD3e 0.0639 182 T3
20 CD4 0.345 119 T4
21 CD5 0.324 111 T1, Tp67
22 CD6 56.5 187 T12
23 CD7 99.9 2512 LEU-9
24 CD8 0.198 130 T8, Leu-2, CD8alpha
25 CD8b 1.56 135 CD8beta
26 CD9 0.268 129 p24, MRP-1
27 CD10 0.406 118 CALLA, NEP, gp100
28 CD11a 100 10258 LFA-1, integrin alpha L
29 CD11b 5.67 136 Mac-1, integrin alphaM
30 CD11c 18.2 153 p150, CR4, integrin alphaX
31 CD13 4.09 137 Aminopeptidase N, APN
32 CD14 4.36 134 LPS-R
33 CD15 0.212 128 Lewis-x, Lex
34 CD16 1.78 127 Fcgamma RIIIA
35 CD16b 0.355 122 FcgammaRIIIB
213
36 CD17 3.06 537 Lactosylceramide
37 CD18 100 642 Integrin beta2
38 CD19 0.251 130 B4
39 CD20 0.28 113 B1, Bp35
40 CD21 0.525 133 C3DR, CR2, EBV-R
41 CD22 0.566 131 BL-CAM, Siglec-2
42 CD23 0.345 121 FcepsilonRII
43 CD24 0.617 125 BA-1
44 CD25 8.52 141 IL-2Ralpha, Tac, p55
45 CD26 100 4223 DPP IV
46 CD27 0.401 127 TNFRSF7, T14
47 CD28 41.3 201 Tp44, T44
48 CD29 100 1034 Integrin beta1
49 CD30 88.4 398 Ki-1
50 CD31 0.175 122 PECAM-1
51 CD32 0.759 128 IgSF
52 CD33 1.46 133 p67, Siglec-3
53 CD34 12.1 138 HPCA1
54 CD35 0.0932 139 CR1
55 CD36 5.14 203 GPIV
56 CD37 1.33 533 N/A
57 CD38 99.8 890 T10
58 CD39 1.94 225 ENTPD1
59 CD40 0.522 166 TNFRSF5
60 CD41a 0.225 127 gpIIb
61 CD41b 1.28 536 HPA-3
62 CD42a 0.248 129 GPIX
63 CD42b 0.445 127 GPIba
64 CD43 100 773 Leukosialin, sialophorin
65 CD44 99 1510 Hyalunorate receptor
66 CD45 99.9 5891 LCA
67 CD45RA 89.8 353 LCA
68 CD45RB 99.6 487 LCA, T200, B220
69 CD45RO 100 2149 LCA, UCHL-1
70 CD46 57.8 548 Membrane cofactor protein
71 CD47 98.8 900 IAP
72 CD48 99.6 1195 Blast-1
73 CD49a 97.2 363 VLA-1
74 CD49b 0.614 127 VLA-2
75 CD49c 0.882 126 VLA-3
76 CD49d 100 3227 VLA-4
77 CD49e 2.36 141 VLA-5
214
78 CD49f 100 863 VLA-6
79 CD50 1.11 543 ICAM-3
80 CD51/CD61 2.2 1436 Vitronectin receptor
81 CD52 7.53 137 CAMPATH-1 antigen
82 CD53 92.7 579 MRC OX44
83 CD54 100 4549 ICAM-1
84 CD55 4.2 135 DAF
85 CD56 100 2032 NCAM
86 CD57 1.01 551 HNK-1, Leu-7
87 CD58 99.9 1359 LFA-3
88 CD59 13.7 144 Protectin, MAC- inhibitor
89 CD60b 78.1 2451 9-O-sialyl GD3
90 CD61 6.85 136 GPIIIa
91 CD62E 1.27 130 E-selectin, ELAM-1
92 CD62L 3.29 140 L-selectin, LECAM-1
93 CD62P 3.35 139 P-selectin, PADGEM
94 CD63 99.9 1120 LIMP, LAMP-3
95 CD64 0.827 128 FcgammaRI
96 CD65 1.24 548 VIM2
97 CD65s 1.34 546 VIM2
98 CD66 99.9 998 BGP-1, NCA-160
99 CD66b 1.11 572 CD67, CGM6
100 CD66c 9.48 141 NCA
101 CD66d 98.7 427 CGM1; CEACAM-3
102 CD66e 72.6 249 CEACAM-5
103 CD69 0.946 132 AIM
104 CD70 0.467 118 Ki-24
105 CD71 99.6 1372 T9
106 CD72 1.39 552 Lyb-2
107 CD73 1.48 128 L-VAP-2
108 CD74 1.5 543 Invariant chain
109 CD75 1.36 518 LN-1
110 CD77 1.86 530 Gb3, Pk blood group
111 CD79a 4.46 133 Iga
112 CD79b 2.45 140 Igb
113 CD80 16.9 172 B7, B7-1, BB1
114 CD81 100 2072 TAPA-1
115 CD82 42 193 R2
116 CD83 0.881 130 HB15
117 CD84 2.58 136 SLAMF5
118 CD85a 1.94 127 LILRB3
119 CD85d 12.2 141 LIRB2, ILT-4, LIR-2
215
120 CD85g 27.5 156 ILT-7
121 CD85h 4.29 133 LILRA-2, ILT-1, LIR-7
122 CD85j 95.7 370 LIRRB-1, ILT-2
123 CD86 100 1359 B70, B7-2
124 CD87 0.334 124 UPA-R
125 CD88 1.12 135 C5aR
126 CD89 1.37 129 FcalphaR
127 CD90 28.3 190 Thy-1
128 CD91 4.86 135 TGFBR5
129 CD92 22.9 151 CTL1B
130 CD94 100 1876 NKG2C, KP43
131 CD95 99 410 Apo-1, Fas
132 CD96 99.5 650 TACTILE
133 CD97 65.9 172 AURA51
134 CD98 100 4383 4F2
135 CD99 35.1 150 MIC2, E2
136 CD100 89.4 865 SEMA4D
137 CD101 0.478 129 V7, p126
138 CD102 29 161 ICAM-2
139 CD103 0.21 109 HML-1, alpha6, integrin alphaE
140 CD104 0.462 131 Beta4 integrin
141 CD105 2.94 135 Endoglin
142 CD106 0.608 129 VCAM-1
143 CD107a 34 149 LAMP-1
144 CD107b 1.98 575 LAMP-2
145 CD108 95.4 468 SEMA7A
146 CD109 0.812 132 7D1, 8A3
147 CD110 46.1 199 MPL, TPO-R
148 CD111 37.6 168 PRR1, Nectin-1
149 CD112 21.8 178 PRR2, Nectin-2
150 CD114 6.19 216 G-CSFR
151 CD115 1.61 141 M-CSFR, c-fms
152 CD116 18.8 217 GM-CSFR alpha
153 CD117 8.48 250 c-kit, SCFR
154 CD118 2.55 131 LIFR, gp190
155 CD119 42.3 185 IFNgammaR
156 CD120a 1.99 131 TNFR-I
157 CD120b 98.7 773 TNFR-II
158 CD121b 20 147 IL-1R, type II
159 CD122 96 328 IL-2Rbeta
160 CD123 10.5 145 IL-3Ralpha
161 CD124 1.72 129 IL-4Ralpha
216
162 CD125 1.8 141 IL-5Ralpha
163 CD126 1.6 131 IL-6Ralpha
164 CD127 27.9 1015 IL-7Ralpha
165 CD129 0.33 112 IL-9R
166 CD130 6.51 133 IL-6Rbeta, gp130
167 CD131 0.821 132 IL-3Rbeta
168 CD132 85.7 280 Common gamma
169 CD133 0.189 184 AC133, prominin-like 1
170 CD134 3.56 134 OX-40
171 CD135 1.2 130 Flt3/Flk2
172 CD136 0.377 124 MSP-R, RON
173 CD137 0.471 119 4-1BB, TNRFSF9
174 CD137L 29.5 159 4-1BB L
175 CD138 0.434 131 Syndecan-1
176 CD140a 0.22 113 PDGFRalpha
177 CD140b 82.1 215 PDGFRbeta
178 CD141 0.265 119 Thrombomodulin
179 CD142 0.852 129 Tissue Factor
180 CD143 2.18 249 ACE
181 CD144 0.306 127 VE-Cadherin, Cadherin-5
182 CD146 0.307 126 MUC18, S-endo
183 CD147 100 3806 Neurothelin, basoglin
184 CD148 99.9 630 HPTP-eta
185 CD150 0.992 130 SLAM
186 CD151 92 381 PETA-3
187 CD152 17.3 141 CTLA-4
188 CD153 4.88 158 CD30L
189 CD154 0.428 125 CD40L, gp39, TRAP
190 CD155 33.9 160 PVR
191 CD156b 1.48 131 ADAM8
192 CD157 0.365 130 BST-1
193 CD158a 0.243 112 KIR2DL1, p58.1
194 CD158b 0.263 130 KIR2DL2, p58.2
195 CD158b2 0.395 130 KIR2DL3
196 CD158d 37.3 156 KIR2DL4
197 CD158e2 0.225 131 KIR3DL1
198 CD158f 6.73 138 KIR2DL5
199 CD158i 6.01 135 KIR2DS4
200 CD159a 1.82 131 NKG2A
201 CD159c 0.971 128 NKG2C
202 CD160 73.2 427 BY55
203 CD161 8.76 144 NKR-P1
217
204 CD162 99.9 2457 PSGL-1
205 CD163 0.453 106 Ber-MAC3, M130
206 CD164 4.53 138 MGC-24
207 CD165 0.203 128 AD2, gp37
208 CD166 13.6 142 ALCAM
209 CD167 12.1 138 DDR1
210 CD169 1.74 131 Sialoadhesin, Siglec-1
211 CD170 2.18 127 Siglec-5, CD33-like2
212 CD171 0.905 129 L1
213 CD172a 2.55 134 SIRPgamma
214 CD172b 0.208 124 SIRPbeta, SIRB1
215 CD172g 84.6 259 SIRPgamma, SIRPB2
216 CD175s 87.5 807 Sialyl-Tn
217 CD177 1.08 544 NB1
218 CD178 30.4 152 FasL, CD95L
219 CD179a 25 146 VpreB
220 CD180 1.18 131 RP-105
221 CD181 11.5 146 CXCR1, IL-8RA
222 CD182 5.38 32217 CXCR2, IL-8RB
223 CD183 36.5 156 CXCR3
224 CD184 12.4 144 CXCR4, fusin
225 CD185 2.32 473 CXCR5, BLR1
226 CD186 0.97 130 CXCR6, BONZO
227 CD191 6.22 142 CCR1, MIP-1alphaR, RANTES-R
228 CD192 0.789 192 CCR2, MCP-1-R
229 CD193 30.4 164 CCR3, CKR3
230 CD194 5.34 209 CCR4
231 CD195 0.969 133 CCR5
232 CD196 20.1 159 CCR6, LARC receptor, DRY6
233 CD197 0.658 128 CCR7
234 CD200 0.68 127 OX-2
235 CD201 0.341 129 EPC-R
236 CD202b 17.3 148 Tie2, Tek
237 CD203c 9.65 141 NPP3 / PDNP3, ENpp1, PD-1b
238 CD204 6.17 311 Macrophage scavenger-R
239 CD205 93.2 238 DEC-205
240 CD206 0.3 108 Macrophage mannose-R
241 CD207 58.9 301 Langerin
242 CD208 1.02 128 DC-LAMP, LAMP-3
243 CD209 0.0858 137 DC-SIGN
244 CD212 24 146 IL-12-R beta1
245 CD213a2 0.189 110 IL-13-R alpha2
218
246 CD215 6.78 141 IL-15R alpha
247 CD217 16.9 208 IL-17-R
248 CD218b 3.31 140 IL-18Rbeta, IL18RAP
249 CD220 11.9 215 Insulin-R
250 CD221 0.544 128 IGF-1 R
251 CD222 2.99 525 IGF-II R
252 CD223 10.4 140 Lag3
253 CD226 75.1 207 DNAM-1, PTA-1, TLiSA1
254 CD227 2.08 542 MUC1, EMA
255 CD229 4.48 139 Ly-9
256 CD230 98.3 517 PRNP
257 CD231 30.8 161 TALLA-1, A15
258 CD234 6.3 144 Duffy, DARC
259 CD235a 24.7 154 Glycophorin A
260 CD243 (BC) 24.8 147 MDR-1, p170, P-gp
261 CD243 (BD) 0.313 136 MDR-1, p170, P-gp
262 CD244 6.79 134 2B4
263 CD245 99.1 756 p220/240
264 CD249 40.6 156 Aminopeptidase A
265 CD252 15.8 1046 OX-40Ligand, gp34
266 CD253 36.3 182 TRAIL, TNFSF10
267 CD254 2.59 130 TRANCE, RANKL, OPGL
268 CD255 8.64 139 TWEAK
269 CD256 25.3 187 APRIL, TALL-2
270 CD257 49.5 198 BLyS, BAFF, TALL-1
271 CD258 4.51 137 LIGHT, HVEM-L
272 CD261 7.58 137 TRAIL-R1, DR4
273 CD262 99.3 386 TRAIL-R2, DR5
274 CD263 0.79 127 TRAIL-R3, DcR1, LIT
275 CD264 1.84 130 TNFRSF10D, TRAILR4
276 CD267 70.6 323 TACI, TNFR SF13B
277 CD268 26.2 362 BAFFR, TR13C
278 CD269 1.55 136 BCMA, TNFRSF13B
279 CD270 30.4 152 TNFSF14
280 CD271 0.746 133 NGFR, p75 (NTR)
281 CD272 4 7770 BTLA
282 CD273 0.301 113 B7DC, PD-L2, PDCD1L2
283 CD274 0.988 131 B7-H1, PD-L1
284 CD275 10.4 140 B7-H2, ICOSL, B7-RP1
285 CD276 22 149 B7-H3
286 CD277 0.832 129 BT3.1, butyrophilin SF3 A1
287 CD278 1.41 127 ICOS, AILIM
219
288 CD279 2.25 133 PD1, SLEB2
289 CD281 37 166 TLR1
290 CD282 1.04 557 TLR2
291 CD283 37.9 170 TLR3
292 CD284 3.91 135 TLR4
293 CD286 32.7 164 TLR6
294 CD288 34.4 166 TLR8
295 CD289 24.6 151 TLR9
296 CD290 49.5 187 TLR-10
297 CD292 1.56 562 BMPR1A, ALK3
298 CD294 10.3 207 CRTH2. GPR44
299 CD295 21.7 147 LeptinR, LEPR
300 CD298 100 1588 Na/K ATPase beta3 subunit
301 CD299 28.4 164 DC-SIGN-related, LSIGN, DC-SIGN2
302 CD300a 25.3 174 CMRF35H, IRC1, IRp60
303 CD300c 42.9 174 CMRF35A, LIR
304 CD300e 33.3 160 CMRF35L
305 CD301 0.186 141 MGL, HML
306 CD303 51.6 184 BDCA2, HECL
307 CD304 8.14 137 BDCA4, neuropilin 1
308 CD305 32 177 LAIR1
309 CD307 7.07 141 IRTA2
310 CD309 34.2 167 VEGFR2, KDR
311 CD312 30.7 181 EMR2
312 CD314 78.9 298 NKG2D, KLR
313 CD317 99.9 2764 BST2, HM1.24
314 CD318 1.86 131 CDCP1, SIMA135
315 CD319 92.8 316 CRACC, SLAMF7
316 CD321 94.1 346 JAM1, F11 receptor
317 CD322 2.14 557 JAM2, VE-JAM
318 CD324 7.62 140 E-Cadherin, Uvomorulin
319 CD325 2.22 130 N-Cadherin, NCAD
320 CD326 0.25 129 Ep-CAM, Ly74
321 CD328 3.46 133 SIGLEC7, AIRM-1
322 CD332 13 204 FGFR2, BEK, KGFR
323 CD333 2.18 138 FGFR3, ACH, CEK2
324 CD334 0.65 123 FGFR4, JTK2, TKF
325 CD335 36.9 155 NKp46, Ly-94 homolog
326 CD336 4.55 145 NKp44, Ly-95 homolog
327 CD337 41.6 556 NKp30, Ly117
328 CD338 25.5 156 ABCG2, BCRP, Bcrp1, MXR
220
329 CD339 1.61 518 Jagged-1, JAG1, JAGL1, hJ1
330 CD340 0.366 192 erbB2, HER-2, EGFR-2
331 CD344 51.8 199 EVR1
332 CD351 73.2 493 FCAMR
333 CD352 100 1094 SLAMF6, NTB-A
334 CD354 19.5 150 TREM-1
335 CD355 10.1 139 CRTAM
336 CD357 16.1 219 TNFSF18, GITR
337 CD358 11.9 145 TNFSF21, DR6
338 CD360 (BD) 21.5 152 IL-21RA
339 CD360 (BL) 18.9 147 IL-21RA
340 CD362 1.15 233 SDC2, HSPG-1
341 CD363 6.95 139 S1PR1
342 CLA 22.6 502 CLA
343 CLIP 1.33 534 CLIP
344 DCIR 2.02 192 DCIR
345 EGF-R 0.533 123 EGF-R
346 FMC7 1.16 552 FMC7
347 HLA-ABC 100 4234 HLA-ABC
348 HLA-A2 0.947 129 HLA-A2
349 HLA-DM 0.337 124 HLA-DM
350 HLA-DR 0.304 128 HLA-DR
351 HPC 3.54 132 HPC
352 ITGB7 99.9 1153 ITGB7
353 LTBR 20.3 168 LTBR, TNFRSF3
354 Lgr-5 1.54 133 Lgr-5
355 MIC A/B 0.506 143 MIC A/B
356 Notch1 81.5 334 Notch1
357 Notch2 91.3 489 Notch2
358 Notch3 15.1 215 Notch3
359 Notch4 1.09 129 Notch4
360 PAC-1 1.12 549 PAC-1
361 Podoplanin 6.11 136 PDPN
362 SSEA-3 1.01 133 SSEA-3
363 SSEA-4 0.232 110 SSEA-4
364 Stro-1 34 257 Stro-1
365 TCR alpha beta 0.279 127 TCR alpha beta
366 TCR gamma delta 18.1 158 TCR gamma delta
367 TPBG 0.378 214 TPBG
368 VB8 TCR 3.93 319 VB8 TCR
369 VD2 TCR 14.4 163 VD2 TCR
370 fMLP-R 10.8 142 fMLP-R
221
Appendix II: KHYG-1 HTS flow cytometry percent positivity and MFI of antigens
# Antigen % + MFI Antigen name(s)
1 BLTR-1 39.8 320 Leukotriene B4 receptor
2 B2m 100 14037 Beta-2 microglobulin
3 CA9 28.3 355 Carbonic anhydrase IX
4 CDH3 6.38 261 Cadherin 3
5 CDH6 0.0527 252 Cadherin 6
6 CDH11 0.1 367 Cadherin 11
7 CDw93 55.6 425 C1q-binding protein
8 CDw198 62.4 453 CCR8
9 CDw199 0.508 394 CCR9
10 CDw210 34 322 IL-10 receptor
11 CDw218a 94.4 468 IL-18 receptor
12 CDw329 0.445 374 SIGLEC-9
13 CD1a 0.0827 263 T6
14 CD1b 1.41 371 T6
15 CD1c 0.959 243 T6
16 CD1d 0.29 233 CD1d
17 CD2 100 6723 T11, LFA-2, SRBC-R
18 CD3 0.265 289 T3
19 CD3e 0.033 452 T3
20 CD4 0.224 237 T4
21 CD5 0.126 239 T1, Tp67
22 CD6 26.2 469 T12
23 CD7 100 8271 LEU-9
24 CD8 99.9 3641 T8, Leu-2, CD8alpha
25 CD8b 14.4 278 CD8beta
26 CD9 6.38 280 p24, MRP-1
27 CD10 11.8 274 CALLA, NEP, gp100
28 CD11a 100 52431 LFA-1, integrin alpha L
29 CD11b 52.7 346 Mac-1, integrin alphaM
30 CD11c 100 2357 p150, CR4, integrin alphaX
31 CD13 38.7 312 Aminopeptidase N, APN
32 CD14 67.7 353 LPS-R
33 CD15 0.0246 303 Lewis-x, Lex
34 CD16 16.5 279 Fcgamma RIIIA
35 CD16b 0.23 274 FcgammaRIIIB
222
36 CD17 12 380 Lactosylceramide
37 CD18 100 2422 Integrin beta2
38 CD19 0.0926 223 B4
39 CD20 0.851 250 B1, Bp35
40 CD21 16.6 274 C3DR, CR2, EBV-R
41 CD22 1.27 251 BL-CAM, Siglec-2
42 CD23 0.183 258 FcepsilonRII
43 CD24 7.28 274 BA-1
44 CD25 35.4 307 IL-2Ralpha, Tac, p55
45 CD26 100 1974 DPP IV
46 CD27 1.65 257 TNFRSF7, T14
47 CD28 84.7 2340 Tp44, T44
48 CD29 99.9 1903 Integrin beta1
49 CD30 86.2 551 Ki-1
50 CD31 0.0854 253 PECAM-1
51 CD32 24.8 289 IgSF
52 CD33 61.2 759 p67, Siglec-3
53 CD34 0.0692 241 HPCA1
54 CD35 0.0919 228 CR1
55 CD36 0.268 235 GPIV
56 CD37 0.22 345 N/A
57 CD38 22.8 375 T10
58 CD39 0.0554 281 ENTPD1
59 CD40 0.0725 208 TNFRSF5
60 CD41a 0.012 352 gpIIb
61 CD41b 0.23 350 HPA-3
62 CD42a 0.524 258 GPIX
63 CD42b 1.21 257 GPIba
64 CD43 100 3931 Leukosialin, sialophorin
65 CD44 100 6773 Hyalunorate receptor
66 CD45 100 2087 LCA
67 CD45RA 13.6 290 LCA
68 CD45RB 99.9 5508 LCA, T200, B220
69 CD45RO 99.9 917 LCA, UCHL-1
70 CD46 99.9 979 Membrane cofactor protein
71 CD47 99.7 779 IAP
72 CD48 100 4416 Blast-1
73 CD49a 97.5 1757 VLA-1
74 CD49b 65.9 406 VLA-2
75 CD49c 67.4 417 VLA-3
76 CD49d 100 6316 VLA-4
77 CD49e 1.88 240 VLA-5
223
78 CD49f 97.8 1488 VLA-6
79 CD50 100 2690 ICAM-3
80 CD51/CD61 2.15 244 Vitronectin receptor
81 CD52 99.8 966 CAMPATH-1 antigen
82 CD53 97.3 707 MRC OX44
83 CD54 100 10759 ICAM-1
84 CD55 100 6623 DAF
85 CD56 88.7 7373 NCAM
86 CD57 0.128 390 HNK-1, Leu-7
87 CD58 100 2710 LFA-3
88 CD59 85.2 393 Protectin, MAC- inhibitor
89 CD60b 17.2 386 9-O-sialyl GD3
90 CD61 1.23 243 GPIIIa
91 CD62E 72.7 515 E-selectin, ELAM-1
92 CD62L 3.53 284 L-selectin, LECAM-1
93 CD62P 8.12 274 P-selectin, PADGEM
94 CD63 98.7 849 LIMP, LAMP-3
95 CD64 0.583 243 FcgammaRI
96 CD65 1.41 396 VIM2
97 CD65s 8.88 378 VIM2
98 CD66 80.7 1314 BGP-1, NCA-160
99 CD66b 0.128 366 CD67, CGM6
100 CD66c 49.3 337 NCA
101 CD66d 61.8 384 CGM1; CEACAM-3
102 CD66e 91 839 CEACAM-5
103 CD68 6.46 259
104 CD69 93.7 498 AIM
105 CD70 100 1241 Ki-24
106 CD71 99.5 1757 T9
107 CD72 0.278 356 Lyb-2
108 CD73 1.89 258 L-VAP-2
109 CD74 10.1 434 Invariant chain
110 CD75 0.183 324 LN-1
111 CD77 11.6 375 Gb3, Pk blood group
112 CD79a 0.254 224 Iga
113 CD79b 59.6 402 Igb
114 CD80 31.2 321 B7, B7-1, BB1
115 CD81 100 2518 TAPA-1
116 CD82 99.8 3500 R2
117 CD83 50.9 324 HB15
118 CD84 29.2 317 SLAMF5
119 CD85a 0.894 259 LILRB3
224
120 CD85d 38.3 323 LIRB2, ILT-4, LIR-2
121 CD85g 26.1 295 ILT-7
122 CD85h 14.6 279 LILRA-2, ILT-1, LIR-7
123 CD85j 81.8 519 LIRRB-1, ILT-2
124 CD86 56.4 345 B70, B7-2
125 CD87 1.23 251 UPA-R
126 CD88 2.79 256 C5aR
127 CD89 11.1 271 FcalphaR
128 CD90 71.3 740 Thy-1
129 CD91 3.25 257 TGFBR5
130 CD92 41.4 321 CTL1B
131 CD94 98.8 1212 NKG2C, KP43
132 CD95 98 541 Apo-1, Fas
133 CD96 98.6 1069 TACTILE
134 CD97 94.8 533 AURA51
135 CD98 100 8916 4F2
136 CD99 23.3 279 MIC2, E2
137 CD100 99.8 1017 SEMA4D
138 CD101 3.27 272 V7, p126
139 CD102 100 2213 ICAM-2
140 CD103 0.0205 279 HML-1, alpha6, integrin alphaE
141 CD104 9.83 274 Beta4 integrin
142 CD105 22.1 296 Endoglin
143 CD106 16.3 272 VCAM-1
144 CD107a 19 299 LAMP-1
145 CD107b 2.09 375 LAMP-2
146 CD108 65.1 376 SEMA7A
147 CD109 0.276 271 7D1, 8A3
148 CD110 73.1 589 MPL, TPO-R
149 CD111 11.6 281 PRR1, Nectin-1
150 CD112 37.1 325 PRR2, Nectin-2
151 CD114 71.5 723 G-CSFR
152 CD115 9.08 272 M-CSFR, c-fms
153 CD116 74.1 1049 GM-CSFR alpha
154 CD117 8.04 25938 c-kit, SCFR
155 CD118 36.9 327 LIFR, gp190
156 CD119 13.5 71350 IFNgammaR
157 CD120a 19.2 279 TNFR-I
158 CD120b 99.4 2052 TNFR-II
159 CD121b 39.2 329 IL-1R, type II
160 CD122 95.8 794 IL-2Rbeta
161 CD123 58.5 430 IL-3Ralpha
225
162 CD124 12.8 282 IL-4Ralpha
163 CD125 25.4 311 IL-5Ralpha
164 CD126 34 319 IL-6Ralpha
165 CD127 53.6 543 IL-7Ralpha
166 CD129 0.0833 217 IL-9R
167 CD130 6.12 263 IL-6Rbeta, gp130
168 CD131 1.68 268 IL-3Rbeta
169 CD132 92.6 1049 Common gamma
170 CD133 0.0177 326 AC133, prominin-like 1
171 CD134 45.5 340 OX-40
172 CD135 24.7 302 Flt3/Flk2
173 CD136 20 296 MSP-R, RON
174 CD137 13.5 285 4-1BB, TNRFSF9
175 CD137L 76.6 596 4-1BB L
176 CD138 0.192 284 Syndecan-1
177 CD140a 4.29 263 PDGFRalpha
178 CD140b 14.4 280 PDGFRbeta
179 CD141 1.97 245 Thrombomodulin
180 CD142 0.68 270 Tissue Factor
181 CD143 15.8 289 ACE
182 CD144 0.558 277 VE-Cadherin, Cadherin-5
183 CD146 0.0478 305 MUC18, S-endo
184 CD147 100 9563 Neurothelin, basoglin
185 CD148 99.8 1719 HPTP-eta
186 CD150 0.635 260 SLAM
187 CD151 100 6606 PETA-3
188 CD152 50.9 364 CTLA-4
189 CD153 29.3 305 CD30L
190 CD154 4.4 276 CD40L, gp39, TRAP
191 CD155 83.9 873 PVR
192 CD156b 56.5 346 ADAM8
193 CD157 0.232 231 BST-1
194 CD158a 0.175 280 KIR2DL1, p58.1
195 CD158b 0.193 285 KIR2DL2, p58.2
196 CD158b2 14.5 277 KIR2DL3
197 CD158d 73.5 381 KIR2DL4
198 CD158e2 0.162 275 KIR3DL1
199 CD158f 13.5 275 KIR2DL5
200 CD158i 18.8 298 KIR2DS4
201 CD159a 17 290 NKG2A
202 CD159c 47.9 359 NKG2C
203 CD160 28.6 462 BY55
226
204 CD161 30.5 305 NKR-P1
205 CD162 100 7888 PSGL-1
206 CD163 12.7 284 Ber-MAC3, M130
207 CD164 16.4 315 MGC-24
208 CD165 0.0872 238 AD2, gp37
209 CD166 3.48 261 ALCAM
210 CD167 19 286 DDR1
211 CD169 26.3 289 Sialoadhesin, Siglec-1
212 CD170 34 327 Siglec-5, CD33-like2
213 CD171 1.4 245 L1
214 CD172a 17.3 289 SIRPgamma
215 CD172b 0.706 269 SIRPbeta, SIRB1
216 CD172g 39.2 332 SIRPgamma, SIRPB2
217 CD175s 53 412 Sialyl-Tn
218 CD177 1.47 263 NB1
219 CD178 31.9 311 FasL, CD95L
220 CD179a 57.2 382 VpreB
221 CD180 1.32 266 RP-105
222 CD181 64.8 471 CXCR1, IL-8RA
223 CD182 50.8 409 CXCR2, IL-8RB
224 CD183 94.2 507 CXCR3
225 CD184 17.8 309 CXCR4, fusin
226 CD185 1.8 370 CXCR5, BLR1
227 CD186 5.9 401 CXCR6, BONZO
228 CD191 11.8 281 CCR1, MIP-1alphaR, RANTES-R
229 CD192 0.172 304 CCR2, MCP-1-R
230 CD193 80.8 1139 CCR3, CKR3
231 CD194 3.49 421 CCR4
232 CD195 11.5 276 CCR5
233 CD196 92.1 911 CCR6, LARC receptor, DRY6
234 CD197 0.0369 269 CCR7
235 CD200 0.359 252 OX-2
236 CD201 0.0709 292 EPC-R
237 CD202b 73 595 Tie2, Tek
238 CD203c 6.82 266 NPP3 / PDNP3, ENpp1, PD-1b
239 CD204 22.5 286 Macrophage scavenger-R
240 CD205 80.6 493 DEC-205
241 CD206 0.837 257 Macrophage mannose-R
242 CD207 3.12 398 Langerin
243 CD208 23.9 295 DC-LAMP, LAMP-3
244 CD209 0.0316 238 DC-SIGN
245 CD212 40.1 313 IL-12-R beta1
227
246 CD213a2 0.314 303 IL-13-R alpha2
247 CD215 50.8 359 IL-15R alpha
248 CD217 29.9 434 IL-17-R
249 CD218b 59 417 IL-18Rbeta, IL18RAP
250 CD220 5.88 401 Insulin-R
251 CD221 3.16 277 IGF-1 R
252 CD222 4.87 369 IGF-II R
253 CD223 99 6858 Lag3
254 CD226 82.6 398 DNAM-1, PTA-1, TLiSA1
255 CD227 95.7 1046 MUC1, EMA
256 CD229 99.3 758 Ly-9
257 CD230 99.9 1172 PRNP
258 CD231 73.8 602 TALLA-1, A15
259 CD234 16.9 279 Duffy, DARC
260 CD235a 56.3 411 Glycophorin A
261 CD243 (BC) 19.7 282 MDR-1, p170, P-gp
262 CD243 (BD) 0.094 268 MDR-1, p170, P-gp
263 CD244 3.64 261 2B4
264 CD245 99.2 1105 p220/240
265 CD247 19.4 291
266 CD249 18.3 320 Aminopeptidase A
267 CD252 17.2 496 OX-40Ligand, gp34
268 CD253 59.9 514 TRAIL, TNFSF10
269 CD254 51.4 370 TRANCE, RANKL, OPGL
270 CD255 30.6 325 TWEAK
271 CD256 18 287 APRIL, TALL-2
272 CD257 73.4 476 BLyS, BAFF, TALL-1
273 CD258 36 302 LIGHT, HVEM-L
274 CD261 46.4 313 TRAIL-R1, DR4
275 CD262 86.7 460 TRAIL-R2, DR5
276 CD263 9.81 268 TRAIL-R3, DcR1, LIT
277 CD264 10.5 269 TNFRSF10D, TRAILR4
278 CD267 17.6 479 TACI, TNFR SF13B
279 CD268 10.9 11864 BAFFR, TR13C
280 CD269 31.1 316 BCMA, TNFRSF13B
281 CD270 72.3 486 TNFSF14
282 CD271 1.08 279 NGFR, p75 (NTR)
283 CD272 4.53 68697 BTLA
284 CD273 0.316 231 B7DC, PD-L2, PDCD1L2
285 CD274 2.03 277 B7-H1, PD-L1
286 CD275 38.4 337 B7-H2, ICOSL, B7-RP1
287 CD276 76.3 863 B7-H3
228
288 CD277 11.8 277 BT3.1, butyrophilin SF3 A1
289 CD278 15.3 289 ICOS, AILIM
290 CD279 27.2 303 PD1, SLEB2
291 CD281 47 350 TLR1
292 CD282 0.231 373 TLR2
293 CD283 76.5 1102 TLR3
294 CD284 42.4 334 TLR4
295 CD286 63.4 419 TLR6
296 CD288 72.9 623 TLR8
297 CD289 35.9 323 TLR9
298 CD290 70.1 549 TLR-10
299 CD292 1.46 357 BMPR1A, ALK3
300 CD294 11 412 CRTH2. GPR44
301 CD295 40.8 340 LeptinR, LEPR
302 CD298 100 7447 Na/K ATPase beta3 subunit
303 CD299 57.2 405 DC-SIGN-related, LSIGN, DC-SIGN2
304 CD300a 100 8067 CMRF35H, IRC1, IRp60
305 CD300c 72.3 481 CMRF35A, LIR
306 CD300e 12.4 274 CMRF35L
307 CD301 0.812 397 MGL, HML
308 CD303 2.99 4223 BDCA2, HECL
309 CD304 21.2 289 BDCA4, neuropilin 1
310 CD305 92.1 634 LAIR1
311 CD307 28.5 308 IRTA2
312 CD309 42.7 339 VEGFR2, KDR
313 CD312 48 400 EMR2
314 CD314 94.7 2723 NKG2D, KLR
315 CD317 100 2482 BST2, HM1.24
316 CD318 75 355 CDCP1, SIMA135
317 CD319 82.2 469 CRACC, SLAMF7
318 CD321 90.7 1311 JAM1, F11 receptor
319 CD322 0.535 367 JAM2, VE-JAM
320 CD324 1.1 370 E-Cadherin, Uvomorulin
321 CD325 38.5 321 N-Cadherin, NCAD
322 CD326 1.05 404 Ep-CAM, Ly74
323 CD328 61.8 436 SIGLEC7, AIRM-1
324 CD332 0.416 354 FGFR2, BEK, KGFR
325 CD333 36.3 307 FGFR3, ACH, CEK2
326 CD334 6.89 266 FGFR4, JTK2, TKF
327 CD335 50.8 327 NKp46, Ly-94 homolog
328 CD336 99.5 1039 NKp44, Ly-95 homolog
229
329 CD337 99.3 4130 NKp30, Ly117
330 CD338 80.5 756 ABCG2, BCRP, Bcrp1, MXR
331 CD339 0.539 362 Jagged-1, JAG1, JAGL1, hJ1
332 CD340 0.38 403 erbB2, HER-2, EGFR-2
333 CD344 69.1 546 EVR1
334 CD349 7.02 409
335 CD351 81 2159 FCAMR
336 CD352 100 3164 SLAMF6, NTB-A
337 CD354 66.3 481 TREM-1
338 CD355 47.3 337 CRTAM
339 CD357 4.37 399 TNFSF18, GITR
340 CD358/DR6 69.9 589 TNFSF21, DR6
341 CD360 (BD) 74.4 461 IL-21RA
342 CD360 (BL) 54.7 367 IL-21RA
343 CD362 0.281 485 SDC2, HSPG-1
344 CD363 58.9 389 S1PR1
345 CLA 81.2 871 CLA
346 CLIP 0.601 368 CLIP
347 DCIR 1.24 394 DCIR
348 EGF-R 1.39 264 EGF-R
349 FMC7 0.119 374 FMC7
350 FOXP3 76.8 446
351 Galectin-3 0.549 248 352 HLA-ABC 100 3164 HLA-ABC
353 HLA-A2 1.01 248 HLA-A2
354 HLA-DM 1.16 272 HLA-DM
355 HLA-DR 0.383 260 HLA-DR
356 HPC 0.288 229 HPC
357 ITGB7 98.2 1395 ITGB7
358 LTBR 57 655 LTBR, TNFRSF3
359 MIC A/B 4.6 266 MIC A/B
360 NPM-ALK 0.143 272 361 Notch1 0.0578 401 Notch1
362 Notch2 21 283 Notch2
363 Notch3 37.5 464 Notch3
364 PAC-1 0.169 387 PAC-1
365 Podoplanin 7.72 262 PDPN
366 SSEA-3 18 279 SSEA-3
367 SSEA-4 0.368 230 SSEA-4
368 Stro-1 4.45 402 Stro-1
369 TCR alpha beta 0.26 242 TCR alpha beta
370 TCR gamma delta 9.29 17690 TCR gamma delta
230
371 TPBG 0.0174 265 TPBG
372 VB8 TCR 41.4 362 VB8 TCR
373 VD2 TCR 66.7 552 VD2 TCR
374 fMLP-R 29 302 fMLP-R
231
Appendix III: OCI/AML3 HTS flow cytometry percent positivity and MFI of antigens
# Antigen % + MFI Antigen name(s)
1 BLTR-1 1.65 1286 Leukotriene B4 receptor
2 B2m 98.9 3133 Beta-2 microglobulin
3 CA9 0.89 604 Carbonic anhydrase IX
4 CDH3 0.198 490 Cadherin 3
5 CDH6 1.57 427 Cadherin 6
6 CDH11 8.12 707 Cadherin 11
7 CDw93 15 816 C1q-binding protein
8 CDw198 74.4 5413 CCR8
9 CDw199 56.2 438 CCR9
10 CDw210 0.186 547 IL-10 receptor
11 CDw218a 0.128 1277 IL-18 receptor
12 CDw329 0.28 847 SIGLEC-9
13 CD1a 0.0733 1206 T6
14 CD1b 0.212 945 T6
15 CD1c 0.695 620 T6
16 CD1d 0.152 376 CD1d
17 CD2 0.132 1429 T11, LFA-2, SRBC-R
18 CD3 0.0687 1147 T3
19 CD3e 0.0163 839 T3
20 CD4 69.3 1346 T4
21 CD5 0.0462 1280 T1, Tp67
22 CD6 1.29 1180 T12
23 CD7 0.0707 898 LEU-9
24 CD8 0.0649 849 T8, Leu-2, CD8alpha
25 CD8b 0.174 1024 CD8beta
26 CD9 0.186 596 p24, MRP-1
27 CD10 0.0898 1336 CALLA, NEP, gp100
28 CD11a 98.4 8983 LFA-1, integrin alpha L
29 CD11b 0.613 602 Mac-1, integrin alphaM
30 CD11c 0.208 730 p150, CR4, integrin alphaX
31 CD13 68.6 2149 Aminopeptidase N, APN
32 CD14 0.164 1150 LPS-R
33 CD15 0.27 408 Lewis-x, Lex
34 CD16 0.214 598 Fcgamma RIIIA
35 CD16b 0.128 1126 FcgammaRIIIB
232
36 CD17 77.1 12014 Lactosylceramide
37 CD18 12.9 738 Integrin beta2
38 CD19 0.0783 1250 B4
39 CD20 0.0769 998 B1, Bp35
40 CD21 0.205 898 C3DR, CR2, EBV-R
41 CD22 0.211 1596 BL-CAM, Siglec-2
42 CD23 0.147 839 FcepsilonRII
43 CD24 0.176 1262 BA-1
44 CD25 0.125 588 IL-2Ralpha, Tac, p55
45 CD26 0.202 768 DPP IV
46 CD27 0.0803 1339 TNFRSF7, T14
47 CD28 0.219 839 Tp44, T44
48 CD29 78.3 2476 Integrin beta1
49 CD30 0.0464 1342 Ki-1
50 CD31 31.5 1840 PECAM-1
51 CD32 64.5 1183 IgSF
52 CD33 19.7 827 p67, Siglec-3
53 CD34 0.0794 998 HPCA1
54 CD35 2.63 1800 CR1
55 CD36 42.2 1554 GPIV
56 CD37 0.0675 1005 N/A
57 CD38 29.5 913 T10
58 CD39 0.284 291 ENTPD1
59 CD40 0.0697 348 TNFRSF5
60 CD41a 0.119 596 gpIIb
61 CD41b 0.0506 621 HPA-3
62 CD42a 0.131 833 GPIX
63 CD42b 0.13 1197 GPIba
64 CD43 99.9 3605 Leukosialin, sialophorin
65 CD44 99.8 6039 Hyalunorate receptor
66 CD45 99.8 4828 LCA
67 CD45RA 82.2 2003 LCA
68 CD45RB 37.3 885 LCA, T200, B220
69 CD45RO 1.71 948 LCA, UCHL-1
70 CD46 98.7 1238 Membrane cofactor protein
71 CD47 15.4 752 IAP
72 CD48 0.0854 991 Blast-1
73 CD49a 0.213 656 VLA-1
74 CD49b 4.01 959 VLA-2
75 CD49c 0.133 1463 VLA-3
76 CD49d 99.7 6428 VLA-4
77 CD49e 98.3 24850 VLA-5
233
78 CD49f 8.34 1029 VLA-6
79 CD50 0.127 766 ICAM-3
80 CD51/CD61 0 ¥ Vitronectin receptor
81 CD52 0.0884 808 CAMPATH-1 antigen
82 CD53 1.74 610 MRC OX44
83 CD54 42.9 1699 ICAM-1
84 CD55 99.9 4757 DAF
85 CD56 0.944 575 NCAM
86 CD57 0.0926 673 HNK-1, Leu-7
87 CD58 91.7 2218 LFA-3
88 CD59 0.909 1950 Protectin, MAC- inhibitor
89 CD60b 0.472 728 9-O-sialyl GD3
90 CD61 0 ¥ GPIIIa
91 CD62E 0 ¥ E-selectin, ELAM-1
92 CD62L 0.129 1241 L-selectin, LECAM-1
93 CD62P 0.0783 705 P-selectin, PADGEM
94 CD63 0.808 471 LIMP, LAMP-3
95 CD64 0.757 522 FcgammaRI
96 CD65 99.7 9119 VIM2
97 CD65s 90 2295 VIM2
98 CD66 0.155 805 BGP-1, NCA-160
99 CD66b 0.0543 928 CD67, CGM6
100 CD66c 4.42 1081 NCA
101 CD66d 0.725 728 CGM1; CEACAM-3
102 CD66e 69.6 1876 CEACAM-5
103 CD69 0.134 447 AIM
104 CD70 30.2 1034 Ki-24
105 CD71 70.9 1298 T9
106 CD72 0.0881 970 Lyb-2
107 CD73 58 1715 L-VAP-2
108 CD74 0.784 592 Invariant chain
109 CD75 0.0637 959 LN-1
110 CD77 0.125 943 Gb3, Pk blood group
111 CD79a 0.349 473 Iga
112 CD79b 0.46 968 Igb
113 CD80 0.174 1139 B7, B7-1, BB1
114 CD81 79.9 5074 TAPA-1
115 CD82 87.3 5399 R2
116 CD83 0.121 1031 HB15
117 CD84 5.47 699 SLAMF5
118 CD85a 0.267 954 LILRB3
119 CD85d 0.951 493 LIRB2, ILT-4, LIR-2
234
120 CD85g 1.83 579 ILT-7
121 CD85h 2.19 937 LILRA-2, ILT-1, LIR-7
122 CD85j 5.14 1139 LIRRB-1, ILT-2
123 CD86 7.4 1311 B70, B7-2
124 CD87 16.3 945 UPA-R
125 CD88 0.28 1271 C5aR
126 CD89 0.644 1474 FcalphaR
127 CD90 12.3 966 Thy-1
128 CD91 1.44 939 TGFBR5
129 CD92 2.4 745 CTL1B
130 CD94 0.0415 1203 NKG2C, KP43
131 CD95 5.79 829 Apo-1, Fas
132 CD96 0.186 675 TACTILE
133 CD97 39.2 902 AURA51
134 CD98 99.9 8271 4F2
135 CD99 1.6 472 MIC2, E2
136 CD100 0.879 609 SEMA4D
137 CD101 28.5 1569 V7, p126
138 CD102 9.74 697 ICAM-2
139 CD103 0.0848 1015 HML-1, alpha6, integrin alphaE
140 CD104 0.154 937 Beta4 integrin
141 CD105 31.7 900 Endoglin
142 CD106 0.0799 952 VCAM-1
143 CD107a 0.184 837 LAMP-1
144 CD107b 0.111 952 LAMP-2
145 CD108 1.46 558 SEMA7A
146 CD109 4.28 807 7D1, 8A3
147 CD110 2.19 568 MPL, TPO-R
148 CD111 70.8 1333 PRR1, Nectin-1
149 CD112 0.241 522 PRR2, Nectin-2
150 CD114 2.56 155000 G-CSFR
151 CD115 0.728 663 M-CSFR, c-fms
152 CD116 16.3 1022 GM-CSFR alpha
153 CD117 3.27 2229 c-kit, SCFR
154 CD118 0.321 708 LIFR, gp190
155 CD119 27.4 934 IFNgammaR
156 CD120a 9.38 732 TNFR-I
157 CD120b 8.15 792 TNFR-II
158 CD121b 1.69 584 IL-1R, type II
159 CD122 1.45 532 IL-2Rbeta
160 CD123 16.1 754 IL-3Ralpha
161 CD124 0.165 812 IL-4Ralpha
235
162 CD125 0.0869 1691 IL-5Ralpha
163 CD126 22.1 790 IL-6Ralpha
164 CD127 8.74 1180 IL-7Ralpha
165 CD129 0.0382 532 IL-9R
166 CD130 0.116 458 IL-6Rbeta, gp130
167 CD131 0.112 510 IL-3Rbeta
168 CD132 2.87 1292 Common gamma
169 CD133 0.0558 436 AC133, prominin-like 1
170 CD134 3.62 600 OX-40
171 CD135 11.9 847 Flt3/Flk2
172 CD136 0.129 661 MSP-R, RON
173 CD137 0.102 961 4-1BB, TNRFSF9
174 CD137L 3.85 661 4-1BB L
175 CD138 0.0771 1311 Syndecan-1
176 CD140a 0.103 1346 PDGFRalpha
177 CD140b 0.167 742 PDGFRbeta
178 CD141 81.6 7263 Thrombomodulin
179 CD142 0.131 1034 Tissue Factor
180 CD143 0.906 503 ACE
181 CD144 0.0641 672 VE-Cadherin, Cadherin-5
182 CD146 0.163 799 MUC18, S-endo
183 CD147 100 9563 Neurothelin, basoglin
184 CD148 91.4 1619 HPTP-eta
185 CD150 0.16 1120 SLAM
186 CD151 82.4 6069 PETA-3
187 CD152 0.145 765 CTLA-4
188 CD153 0.523 1107 CD30L
189 CD154 0.0938 1336 CD40L, gp39, TRAP
190 CD155 93.7 3457 PVR
191 CD156b 32 975 ADAM8
192 CD157 0.222 1158 BST-1
193 CD158a 0.0807 1178 KIR2DL1, p58.1
194 CD158b 0.0645 623 KIR2DL2, p58.2
195 CD158b2 0.189 562 KIR2DL3
196 CD158d 4.8 754 KIR2DL4
197 CD158e2 0.165 937 KIR3DL1
198 CD158f 0.492 558 KIR2DL5
199 CD158i 0.436 705 KIR2DS4
200 CD159a 0.291 687 NKG2A
201 CD159c 0.299 522 NKG2C
202 CD160 1.68 515 BY55
203 CD161 0.174 930 NKR-P1
236
204 CD162 95.8 2318 PSGL-1
205 CD163 0.0548 1241 Ber-MAC3, M130
206 CD164 0.244 667 MGC-24
207 CD165 0.121 777 AD2, gp37
208 CD166 2.67 623 ALCAM
209 CD167 0.355 1346 DDR1
210 CD169 1.59 1235 Sialoadhesin, Siglec-1
211 CD170 0.0975 1092 Siglec-5, CD33-like2
212 CD171 0.186 737 L1
213 CD172a 98.3 4029 SIRPgamma
214 CD172b 0.0731 564 SIRPbeta, SIRB1
215 CD172g 0.349 524 SIRPgamma, SIRPB2
216 CD175s 65 934 Sialyl-Tn
217 CD177 0.0385 957 NB1
218 CD178 3.22 572 FasL, CD95L
219 CD179a 0.219 575 VpreB
220 CD180 0.213 648 RP-105
221 CD181 0.923 683 CXCR1, IL-8RA
222 CD182 2.96 184000 CXCR2, IL-8RB
223 CD183 0.0995 667 CXCR3
224 CD184 2.05 697 CXCR4, fusin
225 CD185 26.2 810 CXCR5, BLR1
226 CD186 0.243 618 CXCR6, BONZO
227 CD191 9.37 725 CCR1, MIP-1alphaR, RANTES-R
228 CD192 0.205 301 CCR2, MCP-1-R
229 CD193 1.81 610 CCR3, CKR3
230 CD194 34.2 389 CCR4
231 CD195 0.114 1195 CCR5
232 CD196 2.17 552 CCR6, LARC receptor, DRY6
233 CD197 0.089 335 CCR7
234 CD200 0.114 984 OX-2
235 CD201 0.0791 1164 EPC-R
236 CD202b 1.76 596 Tie2, Tek
237 CD203c 4.61 1359 NPP3 / PDNP3, ENpp1, PD-1b
238 CD204 0.293 1139 Macrophage scavenger-R
239 CD205 64.1 1175 DEC-205
240 CD206 0.074 678 Macrophage mannose-R
241 CD207 0.149 610 Langerin
242 CD208 0.135 1372 DC-LAMP, LAMP-3
243 CD209 0.275 1153 DC-SIGN
244 CD212 0.0685 369 IL-12-R beta1
245 CD213a2 0.132 689 IL-13-R alpha2
237
246 CD215 1.2 634 IL-15R alpha
247 CD217 98.5 1102 IL-17-R
248 CD218b 0.61 826 IL-18Rbeta, IL18RAP
249 CD220 30.2 361 Insulin-R
250 CD221 9.18 599 IGF-1 R
251 CD222 1.49 556 IGF-II R
252 CD223 4.84 1153 Lag3
253 CD226 0.12 474 DNAM-1, PTA-1, TLiSA1
254 CD227 20 740 MUC1, EMA
255 CD229 3.02 592 Ly-9
256 CD230 77.4 3614 PRNP
257 CD231 7.34 744 TALLA-1, A15
258 CD234 4.57 885 Duffy, DARC
259 CD235a 11 934 Glycophorin A
260 CD243 (BC) 0.116 1280 MDR-1, p170, P-gp
261 CD243 (BD) 0.123 2482 MDR-1, p170, P-gp
262 CD244 0.407 427 2B4
263 CD245 68.9 1456 p220/240
264 CD249 0.309 613 Aminopeptidase A
265 CD252 18.8 1305 OX-40Ligand, gp34
266 CD253 12.8 801 TRAIL, TNFSF10
267 CD254 0.32 541 TRANCE, RANKL, OPGL
268 CD255 1.1 497 TWEAK
269 CD256 14.8 1298 APRIL, TALL-2
270 CD257 16.1 941 BLyS, BAFF, TALL-1
271 CD258 1.11 571 LIGHT, HVEM-L
272 CD261 0.61 478 TRAIL-R1, DR4
273 CD262 12.5 989 TRAIL-R2, DR5
274 CD263 1.63 681 TRAIL-R3, DcR1, LIT
275 CD264 1.77 636 TNFRSF10D, TRAILR4
276 CD267 45.4 1142 TACI, TNFR SF13B
277 CD268 25.9 1546 BAFFR, TR13C
278 CD269 0.167 855 BCMA, TNFRSF13B
279 CD270 12.8 1217 TNFSF14
280 CD271 0.419 636 NGFR, p75 (NTR)
281 CD272 20.4 1120 BTLA
282 CD273 0.269 661 B7DC, PD-L2, PDCD1L2
283 CD274 0.102 1150 B7-H1, PD-L1
284 CD275 2.21 564 B7-H2, ICOSL, B7-RP1
285 CD276 81.5 4223 B7-H3
286 CD277 0.514 539 BT3.1, butyrophilin SF3 A1
287 CD278 0.13 892 ICOS, AILIM
238
288 CD279 0.459 761 PD1, SLEB2
289 CD281 4.33 607 TLR1
290 CD282 21 699 TLR2
291 CD283 5.05 717 TLR3
292 CD284 27.5 1320 TLR4
293 CD286 4.04 712 TLR6
294 CD288 7.11 859 TLR8
295 CD289 0.475 514 TLR9
296 CD290 1.9 533 TLR-10
297 CD292 0.334 1017 BMPR1A, ALK3
298 CD294 1.24 310 CRTH2. GPR44
299 CD295 1.59 512 LeptinR, LEPR
300 CD298 99.9 6207 Na/K ATPase beta3 subunit
301 CD299 2.86 810 DC-SIGN-related, LSIGN, DC-SIGN2
302 CD300a 1.04 1049 CMRF35H, IRC1, IRp60
303 CD300c 1.47 599 CMRF35A, LIR
304 CD300e 3.58 643 CMRF35L
305 CD301 0.158 1453 MGL, HML
306 CD303 7.64 807 BDCA2, HECL
307 CD304 1.3 1333 BDCA4, neuropilin 1
308 CD305 99.6 9684 LAIR1
309 CD307 4.12 1051 IRTA2
310 CD309 10.6 717 VEGFR2, KDR
311 CD312 62.3 1382 EMR2
312 CD314 9.95 808 NKG2D, KLR
313 CD317 83.8 5346 BST2, HM1.24
314 CD318 0.224 831 CDCP1, SIMA135
315 CD319 5.13 689 CRACC, SLAMF7
316 CD321 29 1059 JAM1, F11 receptor
317 CD322 2.9 33040 JAM2, VE-JAM
318 CD324 2.64 869 E-Cadherin, Uvomorulin
319 CD325 0.225 623 N-Cadherin, NCAD
320 CD326 0.148 865 Ep-CAM, Ly74
321 CD328 0.0697 1346 SIGLEC7, AIRM-1
322 CD332 0.372 277 FGFR2, BEK, KGFR
323 CD333 9.75 890 FGFR3, ACH, CEK2
324 CD334 0.151 488 FGFR4, JTK2, TKF
325 CD335 0.15 968 NKp46, Ly-94 homolog
326 CD336 0.14 1097 NKp44, Ly-95 homolog
327 CD337 8.92 869 NKp30, Ly117
328 CD338 1.32 1051 ABCG2, BCRP, Bcrp1, MXR
239
329 CD339 0.0956 839 Jagged-1, JAG1, JAGL1, hJ1
330 CD340 0.468 1481 erbB2, HER-2, EGFR-2
331 CD344 12.3 779 EVR1
332 CD351 45.9 1283 FCAMR
333 CD352 0.168 1134 SLAMF6, NTB-A
334 CD354 12 984 TREM-1
335 CD355 0.771 520 CRTAM
336 CD357 43 415 TNFSF18, GITR
337 CD358 5.71 673 TNFSF21, DR6
338 CD360 (BD) 1.49 521 IL-21RA
339 CD360 (BL) 1.88 607 IL-21RA
340 CD362 93.8 2352 SDC2, HSPG-1
341 CD363 3.51 1172 S1PR1
342 CLA 1.26 735 CLA
343 CLIP 0.0982 518 CLIP
344 DCIR 0.176 344 DCIR
345 EGF-R 0.0654 950 EGF-R
346 FMC7 0.115 979 FMC7
347 HLA-ABC 79.4 1372 HLA-ABC
348 HLA-A2 94.2 2154 HLA-A2
349 HLA-DM 0.0945 539 HLA-DM
350 HLA-DR 0.128 717 HLA-DR
351 HPC 0.132 1268 HPC
352 ITGB7 10.5 1118 ITGB7
353 LTBR 86.7 1978 LTBR, TNFRSF3
354 Lgr-5 0.643 740 Lgr-5
355 MIC A/B 0.201 737 MIC A/B
356 Notch1 86.4 694 Notch1
357 Notch2 36.4 1097 Notch2
358 Notch3 7.33 320 Notch3
359 Notch4 1.04 1333 Notch4
360 PAC-1 0.034 1094 PAC-1
361 Podoplanin 0.105 759 PDPN
362 SSEA-3 0.164 730 SSEA-3
363 SSEA-4 0.0964 1183 SSEA-4
364 Stro-1 13.6 342 Stro-1
365 TCR alpha beta 0.0803 1268 TCR alpha beta
366 TCR gamma delta 6.17 833 TCR gamma delta
367 TPBG 0.0689 346 TPBG
368 VB8 TCR 4.75 119000 VB8 TCR
369 VD2 TCR 8.77 1425 VD2 TCR
370 fMLP-R 0.105 712 fMLP-R
240
Appendix IV: OCI/AML5 HTS flow cytometry percent positivity and MFI of antigens
# Antigen % + MFI Antigen name(s)
1 BLTR-1 18.6 368 Leukotriene B4 receptor
2 B2m 99.8 14251 Beta-2 microglobulin
3 CA9 16.4 362 Carbonic anhydrase IX
4 CDH3 20.7 376 Cadherin 3
5 CDH6 0.311 250 Cadherin 6
6 CDH11 6.36 422 Cadherin 11
7 CDw93 46 456 C1q-binding protein
8 CDw198 66.2 533 CCR8
9 CDw199 0.0214 280 CCR9
10 CDw210 19.2 377 IL-10 receptor
11 CDw218a 24.9 388 IL-18 receptor
12 CDw329 2.89 467 SIGLEC-9
13 CD1a 1.34 552 T6
14 CD1b 2.54 411 T6
15 CD1c 16 497 T6
16 CD1d 26.7 410 CD1d
17 CD2 0.237 318 T11, LFA-2, SRBC-R
18 CD3 0.13 323 T3
19 CD3e 0.0249 264 T3
20 CD4 0.243 326 T4
21 CD5 0.13 314 T1, Tp67
22 CD6 0.815 298 T12
23 CD7 0.846 326 LEU-9
24 CD8 0.695 312 T8, Leu-2, CD8alpha
25 CD8b 0.541 325 CD8beta
26 CD9 0.476 318 p24, MRP-1
27 CD10 0.288 326 CALLA, NEP, gp100
28 CD11a 100 41148 LFA-1, integrin alpha L
29 CD11b 52.5 620 Mac-1, integrin alphaM
30 CD11c 25.4 440 p150, CR4, integrin alphaX
31 CD13 15.9 391 Aminopeptidase N, APN
32 CD14 0.376 297 LPS-R
33 CD15 6.47 512 Lewis-x, Lex
34 CD16 15.3 448 Fcgamma RIIIA
35 CD16b 0.232 325 FcgammaRIIIB
241
36 CD17 52.8 717 Lactosylceramide
37 CD18 99.9 1654 Integrin beta2
38 CD19 0.136 323 B4
39 CD20 0.157 334 B1, Bp35
40 CD21 3.76 340 C3DR, CR2, EBV-R
41 CD22 1.14 343 BL-CAM, Siglec-2
42 CD23 2.88 346 FcepsilonRII
43 CD24 1.19 305 BA-1
44 CD25 17.3 453 IL-2Ralpha, Tac, p55
45 CD26 0.834 315 DPP IV
46 CD27 0.618 323 TNFRSF7, T14
47 CD28 2.98 328 Tp44, T44
48 CD29 100 4151 Integrin beta1
49 CD30 82 827 Ki-1
50 CD31 58.2 712 PECAM-1
51 CD32 98.9 4029 IgSF
52 CD33 100 3483 p67, Siglec-3
53 CD34 0.273 316 HPCA1
54 CD35 3.39 438 CR1
55 CD36 4.91 643 GPIV
56 CD37 0.354 412 N/A
57 CD38 99.8 1889 T10
58 CD39 3.36 442 ENTPD1
59 CD40 0.138 309 TNFRSF5
60 CD41a 0.177 313 gpIIb
61 CD41b 0.289 426 HPA-3
62 CD42a 4.05 328 GPIX
63 CD42b 6.1 334 GPIba
64 CD43 99.8 2658 Leukosialin, sialophorin
65 CD44 100 12728 Hyalunorate receptor
66 CD45 99.9 5413 LCA
67 CD45RA 100 27005 LCA
68 CD45RB 98.6 797 LCA, T200, B220
69 CD45RO 1.5 318 LCA, UCHL-1
70 CD46 98.1 835 Membrane cofactor protein
71 CD47 99.8 1912 IAP
72 CD48 91.4 952 Blast-1
73 CD49a 1.58 365 VLA-1
74 CD49b 67.5 461 VLA-2
75 CD49c 0.527 322 VLA-3
76 CD49d 100 16612 VLA-4
77 CD49e 100 7084 VLA-5
242
78 CD49f 0.819 325 VLA-6
79 CD50 0.518 416 ICAM-3
80 CD51/CD61 1.56 303 Vitronectin receptor
81 CD52 3.09 356 CAMPATH-1 antigen
82 CD53 85 623 MRC OX44
83 CD54 100 9468 ICAM-1
84 CD55 99.8 1849 DAF
85 CD56 72.8 742 NCAM
86 CD57 0.384 410 HNK-1, Leu-7
87 CD58 100 4351 LFA-3
88 CD59 99.4 890 Protectin, MAC- inhibitor
89 CD60b 9.2 422 9-O-sialyl GD3
90 CD61 0.359 320 GPIIIa
91 CD62E 1.78 317 E-selectin, ELAM-1
92 CD62L 2.64 329 L-selectin, LECAM-1
93 CD62P 1.33 332 P-selectin, PADGEM
94 CD63 62.4 421 LIMP, LAMP-3
95 CD64 0.415 309 FcgammaRI
96 CD65 99.7 5891 VIM2
97 CD65s 95.3 1215 VIM2
98 CD66 4.57 323 BGP-1, NCA-160
99 CD66b 0.36 424 CD67, CGM6
100 CD66c 27.7 371 NCA
101 CD66d 3.89 338 CGM1; CEACAM-3
102 CD66e 98.4 2128 CEACAM-5
103 CD69 3.46 335 AIM
104 CD70 99.3 1912 Ki-24
105 CD71 95.1 1178 T9
106 CD72 0.369 400 Lyb-2
107 CD73 0.35 317 L-VAP-2
108 CD74 15.3 431 Invariant chain
109 CD75 0.225 393 LN-1
110 CD77 55.3 452 Gb3, Pk blood group
111 CD79a 72.2 607 Iga
112 CD79b 5.2 339 Igb
113 CD80 8.84 461 B7, B7-1, BB1
114 CD81 99.9 6192 TAPA-1
115 CD82 37.4 468 R2
116 CD83 1.23 331 HB15
117 CD84 87.5 1686 SLAMF5
118 CD85a 4.82 318 LILRB3
119 CD85d 34.8 420 LIRB2, ILT-4, LIR-2
243
120 CD85g 45.1 441 ILT-7
121 CD85h 14.2 357 LILRA-2, ILT-1, LIR-7
122 CD85j 43.7 432 LIRRB-1, ILT-2
123 CD86 39.4 424 B70, B7-2
124 CD87 0.559 334 UPA-R
125 CD88 2.34 334 C5aR
126 CD89 4.8 328 FcalphaR
127 CD90 36.4 561 Thy-1
128 CD91 14.8 338 TGFBR5
129 CD92 92 1049 CTL1B
130 CD94 0.124 323 NKG2C, KP43
131 CD95 93.2 742 Apo-1, Fas
132 CD96 30 372 TACTILE
133 CD97 90.1 547 AURA51
134 CD98 100 37957 4F2
135 CD99 78.1 475 MIC2, E2
136 CD100 59.1 511 SEMA4D
137 CD101 40.4 559 V7, p126
138 CD102 96.3 1180 ICAM-2
139 CD103 0.156 279 HML-1, alpha6, integrin alphaE
140 CD104 3.07 325 Beta4 integrin
141 CD105 9.3 336 Endoglin
142 CD106 2.12 316 VCAM-1
143 CD107a 1.76 346 LAMP-1
144 CD107b 0.765 409 LAMP-2
145 CD108 4.91 374 SEMA7A
146 CD109 14.7 399 7D1, 8A3
147 CD110 50.7 495 MPL, TPO-R
148 CD111 97.3 3605 PRR1, Nectin-1
149 CD112 16.8 357 PRR2, Nectin-2
150 CD114 5.78 426 G-CSFR
151 CD115 55.6 574 M-CSFR, c-fms
152 CD116 51.3 566 GM-CSFR alpha
153 CD117 15.3 624 c-kit, SCFR
154 CD118 12.7 367 LIFR, gp190
155 CD119 94.2 950 IFNgammaR
156 CD120a 86.4 869 TNFR-I
157 CD120b 25 372 TNFR-II
158 CD121b 70.4 604 IL-1R, type II
159 CD122 38.1 449 IL-2Rbeta
160 CD123 96.4 1099 IL-3Ralpha
161 CD124 3.69 367 IL-4Ralpha
244
162 CD125 36.9 451 IL-5Ralpha
163 CD126 86.4 723 IL-6Ralpha
164 CD127 23.3 3989 IL-7Ralpha
165 CD129 0.277 318 IL-9R
166 CD130 3.43 316 IL-6Rbeta, gp130
167 CD131 1.9 315 IL-3Rbeta
168 CD132 44.8 518 Common gamma
169 CD133 0.0134 206 AC133, prominin-like 1
170 CD134 20.8 350 OX-40
171 CD135 84.1 824 Flt3/Flk2
172 CD136 2.4 333 MSP-R, RON
173 CD137 1.5 340 4-1BB, TNRFSF9
174 CD137L 70.9 609 4-1BB L
175 CD138 0.359 337 Syndecan-1
176 CD140a 0.376 322 PDGFRalpha
177 CD140b 9.12 348 PDGFRbeta
178 CD141 59.5 466 Thrombomodulin
179 CD142 0.987 315 Tissue Factor
180 CD143 45.5 496 ACE
181 CD144 0.444 311 VE-Cadherin, Cadherin-5
182 CD146 0.182 328 MUC18, S-endo
183 CD147 100 18791 Neurothelin, basoglin
184 CD148 98.9 1686 HPTP-eta
185 CD150 14.3 376 SLAM
186 CD151 99.8 1964 PETA-3
187 CD152 10.1 366 CTLA-4
188 CD153 8.31 341 CD30L
189 CD154 0.965 332 CD40L, gp39, TRAP
190 CD155 98.6 1740 PVR
191 CD156b 92 1110 ADAM8
192 CD157 7.88 340 BST-1
193 CD158a 0.186 333 KIR2DL1, p58.1
194 CD158b 0.182 302 KIR2DL2, p58.2
195 CD158b2 4.68 337 KIR2DL3
196 CD158d 22.1 380 KIR2DL4
197 CD158e2 0.193 322 KIR3DL1
198 CD158f 14.9 352 KIR2DL5
199 CD158i 24 380 KIR2DS4
200 CD159a 4.09 316 NKG2A
201 CD159c 33.4 403 NKG2C
202 CD160 2.66 282 BY55
203 CD161 4.96 328 NKR-P1
245
204 CD162 99.8 8460 PSGL-1
205 CD163 3.2 310 Ber-MAC3, M130
206 CD164 21.7 370 MGC-24
207 CD165 0.612 307 AD2, gp37
208 CD166 99.7 1561 ALCAM
209 CD167 20.3 353 DDR1
210 CD169 4.8 328 Sialoadhesin, Siglec-1
211 CD170 93 831 Siglec-5, CD33-like2
212 CD171 2.24 331 L1
213 CD172a 99.8 9468 SIRPgamma
214 CD172b 0.458 340 SIRPbeta, SIRB1
215 CD172g 11.6 344 SIRPgamma, SIRPB2
216 CD175s 96.8 1089 Sialyl-Tn
217 CD177 0.154 406 NB1
218 CD178 42.6 465 FasL, CD95L
219 CD179a 24.8 361 VpreB
220 CD180 39.6 393 RP-105
221 CD181 26.4 402 CXCR1, IL-8RA
222 CD182 13.7 130000 CXCR2, IL-8RB
223 CD183 13.1 350 CXCR3
224 CD184 1.66 337 CXCR4, fusin
225 CD185 8.59 445 CXCR5, BLR1
226 CD186 3.17 320 CXCR6, BONZO
227 CD191 37.8 438 CCR1, MIP-1alphaR, RANTES-R
228 CD192 0.406 284 CCR2, MCP-1-R
229 CD193 41.8 455 CCR3, CKR3
230 CD194 10.6 334 CCR4
231 CD195 0.812 331 CCR5
232 CD196 38.7 489 CCR6, LARC receptor, DRY6
233 CD197 0.231 320 CCR7
234 CD200 0.738 298 OX-2
235 CD201 0.437 361 EPC-R
236 CD202b 27.5 411 Tie2, Tek
237 CD203c 23.2 375 NPP3 / PDNP3, ENpp1, PD-1b
238 CD204 6.05 455 Macrophage scavenger-R
239 CD205 99.9 3440 DEC-205
240 CD206 0.631 398 Macrophage mannose-R
241 CD207 68.3 413 Langerin
242 CD208 3.65 328 DC-LAMP, LAMP-3
243 CD209 0.194 329 DC-SIGN
244 CD212 14.3 331 IL-12-R beta1
245 CD213a2 85 466 IL-13-R alpha2
246
246 CD215 29.2 402 IL-15R alpha
247 CD217 87.7 616 IL-17-R
248 CD218b 14.1 375 IL-18Rbeta, IL18RAP
249 CD220 59.2 378 Insulin-R
250 CD221 97.5 799 IGF-1 R
251 CD222 10.8 408 IGF-II R
252 CD223 44.9 424 Lag3
253 CD226 2.01 393 DNAM-1, PTA-1, TLiSA1
254 CD227 48.4 522 MUC1, EMA
255 CD229 50 427 Ly-9
256 CD230 98.6 1940 PRNP
257 CD231 41.2 473 TALLA-1, A15
258 CD234 18.5 373 Duffy, DARC
259 CD235a 52.5 471 Glycophorin A
260 CD243 (BC) 34.8 423 MDR-1, p170, P-gp
261 CD243 (BD) 0.251 342 MDR-1, p170, P-gp
262 CD244 22.3 345 2B4
263 CD245 96 1192 p220/240
264 CD249 34 371 Aminopeptidase A
265 CD252 9.23 5522 OX-40Ligand, gp34
266 CD253 36.9 456 TRAIL, TNFSF10
267 CD254 7.84 353 TRANCE, RANKL, OPGL
268 CD255 17 353 TWEAK
269 CD256 43.8 497 APRIL, TALL-2
270 CD257 55.4 581 BLyS, BAFF, TALL-1
271 CD258 72.8 712 LIGHT, HVEM-L
272 CD261 35 372 TRAIL-R1, DR4
273 CD262 98.7 1398 TRAIL-R2, DR5
274 CD263 31.7 403 TRAIL-R3, DcR1, LIT
275 CD264 48.1 536 TNFRSF10D, TRAILR4
276 CD267 65.3 896 TACI, TNFR SF13B
277 CD268 50.7 720 BAFFR, TR13C
278 CD269 10.8 394 BCMA, TNFRSF13B
279 CD270 45.3 448 TNFSF14
280 CD271 4.98 425 NGFR, p75 (NTR)
281 CD272 2.4 25873 BTLA
282 CD273 2.02 327 B7DC, PD-L2, PDCD1L2
283 CD274 0.65 326 B7-H1, PD-L1
284 CD275 20.9 369 B7-H2, ICOSL, B7-RP1
285 CD276 99.6 3027 B7-H3
286 CD277 4.99 356 BT3.1, butyrophilin SF3 A1
287 CD278 0.604 320 ICOS, AILIM
247
288 CD279 6.26 346 PD1, SLEB2
289 CD281 79.4 758 TLR1
290 CD282 0.368 402 TLR2
291 CD283 57.7 524 TLR3
292 CD284 22.1 353 TLR4
293 CD286 80.5 666 TLR6
294 CD288 58.4 596 TLR8
295 CD289 15.9 356 TLR9
296 CD290 30.5 397 TLR-10
297 CD292 2.38 405 BMPR1A, ALK3
298 CD294 2.52 284 CRTH2. GPR44
299 CD295 43.2 389 LeptinR, LEPR
300 CD298 100 8169 Na/K ATPase beta3 subunit
301 CD299 29.5 393 DC-SIGN-related, LSIGN, DC-SIGN2
302 CD300a 99.7 3911 CMRF35H, IRC1, IRp60
303 CD300c 42.2 433 CMRF35A, LIR
304 CD300e 48.6 467 CMRF35L
305 CD301 0.111 355 MGL, HML
306 CD303 54 519 BDCA2, HECL
307 CD304 91.1 725 BDCA4, neuropilin 1
308 CD305 99.9 5228 LAIR1
309 CD307 5.91 329 IRTA2
310 CD309 70.4 607 VEGFR2, KDR
311 CD312 92 1449 EMR2
312 CD314 33.6 442 NKG2D, KLR
313 CD317 99.1 1770 BST2, HM1.24
314 CD318 7.57 330 CDCP1, SIMA135
315 CD319 14.9 356 CRACC, SLAMF7
316 CD321 100 5563 JAM1, F11 receptor
317 CD322 5.83 403 JAM2, VE-JAM
318 CD324 19.7 373 E-Cadherin, Uvomorulin
319 CD325 5.15 325 N-Cadherin, NCAD
320 CD326 0.585 330 Ep-CAM, Ly74
321 CD328 11.2 340 SIGLEC7, AIRM-1
322 CD332 2.16 285 FGFR2, BEK, KGFR
323 CD333 76.5 740 FGFR3, ACH, CEK2
324 CD334 2 320 FGFR4, JTK2, TKF
325 CD335 0.759 300 NKp46, Ly-94 homolog
326 CD336 0.261 316 NKp44, Ly-95 homolog
327 CD337 4.88 19861 NKp30, Ly117
328 CD338 46.2 448 ABCG2, BCRP, Bcrp1, MXR
248
329 CD339 2.47 397 Jagged-1, JAG1, JAGL1, hJ1
330 CD340 0.655 444 erbB2, HER-2, EGFR-2
331 CD344 35.3 411 EVR1
332 CD351 59.2 633 FCAMR
333 CD352 0.838 318 SLAMF6, NTB-A
334 CD354 25.2 398 TREM-1
335 CD355 54.3 1283 CRTAM
336 CD357 77.1 566 TNFSF18, GITR
337 CD358 49.6 512 TNFSF21, DR6
338 CD360 (BD) 42.2 414 IL-21RA
339 CD360 (BL) 23.9 384 IL-21RA
340 CD362 0.345 359 SDC2, HSPG-1
341 CD363 22.6 380 S1PR1
342 CLA 99.2 1056 CLA
343 CLIP 79 669 CLIP
344 DCIR 0.638 290 DCIR
345 EGF-R 0.324 285 EGF-R
346 FMC7 0.251 425 FMC7
347 HLA-ABC 99.8 4471 HLA-ABC
348 HLA-A2 0.415 321 HLA-A2
349 HLA-DM 0.628 318 HLA-DM
350 HLA-DR 68 477 HLA-DR
351 HPC 0.748 316 HPC
352 ITGB7 93.9 1298 ITGB7
353 LTBR 23.8 420 LTBR, TNFRSF3
354 Lgr-5 4.56 331 Lgr-5
355 MIC A/B 1.65 313 MIC A/B
356 Notch1 84 526 Notch1
357 Notch2 73.3 875 Notch2
358 Notch3 11.2 298 Notch3
359 Notch4 21.5 368 Notch4
360 PAC-1 0.916 470 PAC-1
361 Podoplanin 8.51 342 PDPN
362 SSEA-3 26.3 349 SSEA-3
363 SSEA-4 0.507 350 SSEA-4
364 Stro-1 12.3 305 Stro-1
365 TCR alpha beta 0.521 326 TCR alpha beta
366 TCR gamma delta 52.1 686 TCR gamma delta
367 TPBG 0.141 251 TPBG
368 VB8 TCR 7.86 343 VB8 TCR
369 VD2 TCR 12.5 401 VD2 TCR
370 fMLP-R 14.8 370 fMLP-R
249
Copyright Acknowledgements
250
Chapter 3: Reprinted with minor modifications from: B.A. Williams, X.-H. Wang and A.
Keating. Clonogenic assays measure leukemia stem cell killing not detectable by chromium
release and flow cytometric cytotoxicity assays. Cytotherapy 2010: 12(7);951-60.
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Section 1.3.3: Reprinted with minor modifications from: B. A. Williams, B. E. Swift, R. Cheng
and A. Keating. 2013. NK-92 cytotoxicity against cancer stem cells in hematologic
malignancies. (Springer) Stem Cells and Cancer Stem Cells: Therapeutic Applications in Disease
and Injury, Ed. M.A. Hayat, 9 (24); 249-257.
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