targeting igv-like domain immune checkpoint receptors with ... · clinically, the targeted blockade...
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Targeting IgV-like Domain Immune Checkpoint Receptors with Novel Nucleic Acid and Protein-Based Therapeutics
by
Aaron Prodeus
A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy
Department of Medical Biophysics University of Toronto
© Copyright by Aaron Prodeus 2018
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Targeting IgV-like Domain Immune Checkpoint Receptors with
Novel Nucleic Acid and Protein-Based Therapeutics
Aaron Prodeus
Doctor of Philosophy
Department of Medical Biophysics
University of Toronto
2018
Abstract
The immune system is heavily regulated by negative checkpoint pathways; a network of cell
signaling events governed by immune inhibitory ligand-receptor interactions. Physiologically,
these negative checkpoint pathways are crucial for maintenance of self-tolerance in peripheral
tissues. These pathways are often usurped by tumors as a mechanism to dampen anti-tumor
immune responses. Clinically, the targeted blockade of PD-1:PD-L1 and CTLA-4:CD80/86
checkpoint ligand-receptor interactions using monoclonal antibodies has proved to be a viable
means to provoke effective and durable anti-tumor responses. Conversely, agonists that stimulate
these natural immunoinhibitory signaling pathways could potentially serve as immune-
suppressants for treating patients with inflammatory and autoimmune disorders. The central theme
of this thesis was to derive novel biotherapeutics that agonize or antagonize clinically-relevant
checkpoint receptors. Specifically, the thesis focuses largely on targeting checkpoint receptors
with short, single-stranded oligonucleotides, termed DNA aptamers, which bind molecular targets
with affinity and specificity rivalling that of monoclonal antibodies. As a proof-of-principle, I first
describe the derivation of DNA aptamers which bind to murine PD-1 and antagonize the PD-1:PD-
L1 pathway to release anti-tumor immune responses and suppress the growth of a murine colon
carcinoma tumor implant in-vivo. Second, I report the development of aptamers that bind to
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CD200R1 and agonize this inhibitory receptor. Strikingly, these anti-CD200R1 agonists were
found to act as strong immunosuppressant’s in mouse models of transplant rejection and allergy.
Lastly, I described the engineering of a novel multimeric fusion protein, consisting of the
extracellular IgV-like domain of the checkpoint ligand VISTA, fused to a short helical
pentamerization domain derived from the cartilage oligomeric matrix protein. This soluble agonist
readily inhibited T-cell activation in-vitro, and suppressed inflammation in models of transplant
rejection and autoimmune hepatitis. Notably, this construct represents the first agonist targeting
the as-of-yet discovered VISTA-receptor to stimulate immunoinhibitory signaling in-vivo.
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Acknowledgments
I would like to express my immense gratitude and appreciation to everyone who provided support
throughout this journey.
To my supervisor, Dr. Jean Gariepy, I am grateful for your unwavering support and guidance. You
have taught me how to think both big and small, and how to be both diligent and creative in
designing experiments. Your willingness to go above and beyond what is expected for a supervisor
has been critical for me to realize my potential. Thank you for allowing me to attend several
international conferences to provide me an opportunity to present my work, learn, and engage with
the scientific community. Working with you has simply been a wonderful experience, and I look
forward to our continued collaborations.
I am very grateful to my committee members Dr. Reginald Gorczynski and Dr. David Andrews.
Dr. Gorczynski, you have been a critical resource for much of the research presented throughout
this thesis. Not many students are lucky enough to have had the wonderful collaboration and hands
on assistance that you have provided, and I thank you for everything. Dr. Andrews, thank-you for
always keeping me thinking, for asking the important but not-so obvious questions, and
challenging me to do the most effective research possible.
I would also like to extend my gratitude towards a multitude of other collaborators: Dr. Tania
Watts, Dr. Michael Julius, Dr. Ismat Khatri, Dr. Chung-Wai Chow, Dr. Nathalie Vacarresse, Dr.
Alessandra Ferzoco, and Lindsay Woo.
In addition, I would like to extend my appreciation towards both past and present lab members:
Amanda, Ann, Arshiya, Aws, Amirul, Eric, Linda, Marzena, Mays, Nick, and Peter as well as my
summer students Nicholas and Alexandra. A special thank you to Dr. Aws Abdul-Wahid, you were
a critical part of this thesis, thank-you for welcoming to both your field of research and your office.
Your help and advice, from hands-on experimental training, to big-picture talks about the future,
was so important for me. I wish all of my colleagues the best as they continue along their journey
and career paths.
This research would not have been possible without the financial assistance I have received from
the Department of Medical Biophysics in the forms of stipends, awards, and top-ups. In addition,
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I would like to acknowledge the Canadian Breast Cancer Foundation and Ontario Graduate
Scholarship program for providing additional scholarships and financial support over the past five
years.
Lastly, I would like to thank my family and friends. To my Mom and Dad, none of this would have
been possible without your unconditional love and support in everything I do. To my Aunt Joyce,
your loving support is ever present, and I can’t thank you enough for everything you have done.
To my brother Adam, even in different cities you were always a text message away whenever I
needed help. Finally, to my best friend Rachel, without you I am not sure I could have done this.
Thank you for being there, for making me laugh, for keeping me sane, and being there even when
I wasn’t. I am so excited to see what the future has in store for us.
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Table of Contents
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List of Abbreviations
2-F 2-fluoro
32P Phosphorus-32
51Cr Chromium-51
A,T,C,G Adenine, thymine, cytosine and guanine
Allo-MLC allogeneic mixed leukocyte culture
Ang1 Angiopoietin 1
ANOVA analysis of variance
APC Allophycocyanin
APCs Antigen presenting cells
BCA Bicinchoninic acid assay
BSA Bovine serum albumin
cApt control aptamer used in Chapter 3
CD200R1 CD200 receptor 1
CDR Complementarity-determining region
CEA CEACAM5; carcinoembryonic antigen
CEA-N CEA N-domain
CFSE Carboxyfluorescein succinimidyl ester
CIA Collagen induced arthritis
CNS central nervous system
COMP Cartilage oligomeric matrix protein
ConA Concanavalin A
cSeq Control oligonucleotide sequence aptamer used in Chapter 2
CTL cytotoxic T-lymphocyte
CTLA-4 cytotoxic T-lymphocyte-associated protein 4
DCs Dendritic cells
DNA Deoxyribonucleic acid
dT deoxythymidine
EAE experimental autoimmune encephalomyelitis
EAU experimental autoimmune uveitis
ECD extracellular domain
ELISA enzyme-linked immunosorbent assay
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ELISPOT Enzyme-linked immunospot
FACS fluorescence activated cell sorting
FDA U.S. Food and Drug Administration
FITC Fluorescein isothiocyanate
FPLC fast protein liquid chromatography
GE General Electric
GITR Glucocorticoid-induced tumor necrosis factor receptor
GVHD Graft versus host-disease
HABA 4'-hydroxyazobenzene-2-carboxylic acid
HBS HEPES buffered saline
HDM House dust mite
HPLC High performance liquid chromatography
HRP Horseradish peroxidase
I.P. intraperitoneal
I.V. intravenous
ICOS inducible costimulator
IDT Integrated DNA Technology
IFNα Interferon alpha
IFNγ Interferon gamma
Ig immunoglobulin
IgG Immunoglobulin G
IgV Immunoglobulin Variable
IL-10 Interleukin-10
IL-10R Interleukin-10 receptor
IL-12 Interleukin-12
IL-2 Interleukin-2
IL-6 Interleukin-6
IL-6R Interleukin-6 receptor
IMDM Iscove’s Modified Dulbecco Medium
irAEs immune related adverse events
ITIM immunoreceptor tyrosine-based inhibitory motif
ITSM immunoreceptor tyrosine-based switch motif
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KD Dissociation constant
kDa kilodalton
LAG-3 Lymphocyte-activation gene 3
mAb monoclonal antibody
MCh methacholine
MHC Major histocompatibility complex
MWCO molecular weight cut off
NGS Next-generation sequencing
NSCLC Non-small cell lung cancer
nt Nucleotide
ODN oligonucleotide
ORR objective response rate
OVA ovalbumin
PBS Phosphate buffered saline
PCR polymerase chain reaction
PD-1 Programmed cell death protein 1
PD-L1 Programmed death-ligand 1
PD-L2 Programmed death-ligand 2
PE R-phycoerythrin
PEG polyethylene glycol
PGM Personal Genome Machine
PK pharmacokinetic
PKC protein kinase C
PMSA Prostate specific membrane antigen
RIPA Radioimmunoprecipitation assay buffer
RNA ribonucleic acid
Rrs airway respiratory resistance
RT-PCR reverse transcription polymerase chain reaction
SD Standard deviation
SELEX Systematic evolution of ligands by exponential enrichment
SELEX-NGS SELEX coupled to NGS sequencing
SEM Standard error of the mean
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SFU Spot forming unit
SLE Systemic lupus erythematosus
SOMAmers slow-off rate modified aptamers
SPR surface plasmon resonance
SRICR Sunnybrook Research Institute Comparative Research
ssDNA single standed DNA
t1/2 half-life
TAA Tumor-associated antigen
TCR T-cell receptor
TEAA Triethylammonium acetate
TGF-β Transforming growth factor beta 1
Th1 T-helper 1
TILs Tumor infiltrating leukocytes
TLR-9 Toll-like receptor 9
TMB 3,3’,5,5’-Tetramethylbenzidine
TME Tumor microenvironment
VISTA V-domain Ig suppressor of T-cell activation
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List of Tables
Table 1-1. Immune checkpoint molecules currently being targeted in cancer therapy. ................ 9
Table 1-2. Genetic deletion of checkpoint molecules causes an enhancement in the occurrence of
diseases associated with autoimmunity.. ...................................................................................... 10
Table 1-3. Advantages and limitations of aptamers as therapeutic entities compared to
monoclonal antibodies. ................................................................................................................. 31
Table 1-4. Clinical development of aptamers ............................................................................... 32
Table 1-5. Pre-clinical development of functional aptamers targeting immune co-stimulatory or
co-inhibitory receptors. ................................................................................................................. 36
Table 2-1. Variable regions of enriched anti-mPD-1 aptamer families identified after 6 rounds of
SELEX.. ........................................................................................................................................ 51
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List of Figures
Figure 1-1. T-cell responses are regulated by a multitude of co-stimulatory and co-inhibitory
interactions ...................................................................................................................................... 4
Figure 1-2. Structure of PD-1. ...................................................................................................... 13
Figure 1-3. CD200:CD200R1 immunoinhibitory signaling. CD200R1 expressed on the surface
of myeloid and lymphoid cells becomes phosphorylated upon ligation with the widely distributed
CD200 ........................................................................................................................................... 16
Figure 1-4. VISTA structure and function .................................................................................... 19
Figure 1-5. Structure and engineering of modern day therapeutic antibodies. ............................. 21
Figure 1-6. Overlaid crystal structures of an RNA and DNA aptamer bound to thrombin. ......... 25
Figure 1-7. Derivation of Aptamers using SELEX. ...................................................................... 26
Figure 1-8. Tracking the progress of SELEX screens with NGS.. ............................................... 28
Figure 1-9. Chapter Overviews. .................................................................................................... 40
Figure 2-1. Enriched aptamer sequences pull down mPD-1.FcHIS ............................................. 51
Figure 2-2. Secondary structure and binding affinity of highly enriched anti-PD-1 aptamer
sequences MP5 and MP7.. ............................................................................................................ 52
Figure 2-3. Specificity of anti-PD-1 aptamers MP5 and MP7 towards the mPD-1 extracellular
domain........................................................................................................................................... 54
Figure 2-4 Aptamers MP5 and MP7 bind PD-1 expressing cells. ................................................ 55
Figure 2-5. Anti-PD-1 DNA aptamer MP7 antagonizes PD-1/PD-L1 mediated suppression of IL-
2 secretion in-vitro ........................................................................................................................ 56
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Figure 2-6. Anti-PD-1 DNA Aptamers do not stimulate IL-2 secretion in the absence of PD-L1
signalling ....................................................................................................................................... 57
Figure 2-7. MP7 restores PD-L1 suppressed lymphocyte proliferation. CFSE labelled
splenocytes were stimulated by plate bound anti-CD3 antibody in the presence or absence of PD-
L1 and CFSE dilution profiles analysed by flow cytometry......................................................... 59
Figure 2-8. PEGylated MP7 directly blocks PD-1/PD-L1 Binding .............................................. 60
Figure 2-9. PEGylation does not influence the ability of MP7 to block the PD-1:PD-L1
interaction. .................................................................................................................................... 61
Figure 2-10. MC38.CEA cells express low levels of PD-L1 which is upregulated by IFNy
stimulation..................................................................................................................................... 61
Figure 2-11. PEGylated MP7 suppresses the growth of disseminated PD-L1+ colon carcinoma
MC38.CEA cells in-vivo. ............................................................................................................. 62
Figure 2-12. PEGylated MP7 suppresses growth of PD-L1+ MC38.CEA cells in a more
aggressive model. .......................................................................................................................... 63
Figure 2-13. Anti-PD-1 Aptamer PEG-MP7 is not cytotoxic to tumor cells and enhances tumor-
specific T-cell responses in-vivo without induction of a TLR9-mediated innate immune response
....................................................................................................................................................... 64
Figure 2-14. PEGylated MP7 does not induce transcription of genes associated with TLR-9
signaling.. ...................................................................................................................................... 65
Figure 3-1. DNA Aptamers selected to bind to the extracellular domain of murine CD200R1
suppress CTL induction in primary allo-MLC. ............................................................................ 79
Figure 3-2. Truncation of CD200R1 aptamers M49 and M52. .................................................... 80
Figure 3-3. PEGylation does not perturb the ability of M49 and M52 to agonize CD200R1 ...... 81
Figure 3-4. PEG conjugated M49 and M52 induced CD200R1 signaling ................................... 83
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Figure 3-5. PEG-M49 and PEG-M52 function in-vivo ................................................................ 84
Figure 3-6. PEGylated Aptamers do not activate a TLR9 Innate Immune Response. ................. 85
Figure 3-7. PEG-M49 and PEG-M52 prolong the survival of transplanted murine skin grafts ... 86
Figure 3-8. PEGylation of M49 is necessary for prolongation of allograft survival. ................... 87
Figure 3-9. Cross-Species Anti-CD200R1 Aptamers Identified by SELEX. ............................... 88
Figure 3-10. CCS13: A cross species CD200R agonist ................................................................ 90
Figure 3-11. PEGylated CCS13 suppressed allo-immune responses through CD200R1
signalling.. ..................................................................................................................................... 91
Figure 3-12. PEGylated aptamer PEG-CCS13 suppresses house dust mice (HDM)-induced
allergic airway response ................................................................................................................ 92
Figure 4-1. Pentameric VISTA.COMP suppresses T-cell activation and proliferation as a soluble
ligand in-vitro.............................................................................................................................. 106
Figure 4-2. VISTA.COMP suppressed T-cell activation and CTL induction. ........................... 107
Figure 4-3. VISTA.COMP binds to a clonal T-cell line and suppresses its activation .............. 109
Figure 4-4. VISTA.COMP suppresses 2.10 T-cell IL-2 secretion and TCR- phosphorylation
cascades....................................................................................................................................... 110
Figure 4-5. VISTA.COMP suppresses immune responses in-vivo.. ........................................... 112
Figure 4-6. VISTA-Fc does not rescue animals from ConA induced hepatitis .......................... 113
Figure 5-1. VISTA binds to IGSF11. ......................................................................................... 124
Figure 5-2. Differential expression of genes in a T-cell line treated with VISTA.COMP. ........ 125
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Chapter 1: Literature Review
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Literature Review
1.1 Immune Checkpoint Receptors
1.1.1 The Immune System in Human Health and Disease
The immune system plays a critical role in everyday human health by coordinating balanced and
specific responses against foreign pathogens (viruses, bacteria, fungi) and malignant cells while
limiting damage to self. The mammalian immune system involves two fundamental components;
the innate (natural) immune system consisting of cells of the myeloid lineage, which along with
physical barriers provide rapid non-specific responses against foreign invaders; and the adaptive
(acquired) immune system which involves specific antigen-binding mediated responses by
lymphoid cells (Delves & Roitt, 2000a, 2000b). Together, these systems form a highly-regulated
network of organs, tissues, cells, and soluble factors, that maintains a delicate structure of checks
and balances to (i) recognize danger signals, (ii) mount a coordinate response to neutralize threats,
and (iii) downregulate the provoked immune reaction to restore homeostasis. Deregulation of
immune functions is consequential, as uncontrolled or non-specific immune responses can lead to
excessive inflammation, autoimmunity, and/or collateral damage to healthy self (van der Vlist,
Kuball, Radstake, & Meyaard, 2016). Conversely, insufficient immune responses may not protect
against infections or the outgrowth of transformed cells. Consequentially, the ability to
therapeutically modulate immune responses has played a pivotal role in human health and disease.
Indeed, efforts to understand the regulatory mechanisms controlling each arm of the immune
system has led to numerous critical medical advances, including but not limited to, the
development of vaccines, steroidal and non-steroidal immunosuppressant drugs, antihistamines,
adoptive cell therapies, and more recently in the case of cancer immunotherapy, checkpoint
inhibitors.
1.1.2 Immune Co-stimulatory and Co-inhibitory Receptors
T-cells are a central component to the adaptive immune system which through the T-cell receptor
(TCR) specifically recognize foreign peptides in the context of MHC class II (by antigen
presenting cells [APC]) or MHC class I complexes (by other tissues). T-cell mediated immunity
entails the (i) clonal selection of TCR antigen-specific cells, (ii) their activation and expansion in
lymphoid tissues, (iii) transit to sites of inflammation in the periphery to (iv) carry out their
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appropriate effector functions. In the case of CD4+ T-helper cells, these effector functions include
helping local immune cells through cytokine secretion or cell:cell signaling, while the effector
functions of CD8+ cytotoxic T-lymphocytes (CTL) entail their direct killing of infected, damaged
or malignant cells (Janeway, 2005). The amplitude and quality of T-cell responses initiated upon
TCR/MHC engagement is regulated by an intricate balance between additional co-stimulatory and
co-inhibitory signals presented by secondary cell-surface ligand:receptor interactions (Chen &
Flies, 2013). For instance, in the classic two-signal model of T-cell activation, the initial signal
delivered upon ligation of the TCR is complimented by a secondary activation signal upon
engagement of the T-cell co-stimulatory receptor CD28 with CD80 or CD86 expressed on APCs,
leading to the complete activation of naïve T-cells (Greenfield, Nguyen, & Kuchroo, 1998).
Conversely, T-cell responses can be suppressed by co-inhibitory signals transduced by checkpoint
receptors present on the T-cell surface upon binding to ligands on APCs or other cells within the
local microenvironment. For example, the checkpoint receptor CTLA-4 on T-cells can suppress
early stage T-cell activation signals by outcompeting CD28 for binding to CD80 to CD86, and
additionally by transducing immuno-inhibitory signals upon binding to these ligands (Auchincloss
& Turka, 2011). To date, there have been over 75 receptors identified which act as either co-
stimulatory or co-inhibitory molecules to regulate immune function (Figure 1-1). Several of these
receptors, due to their critical role in regulating T-cell development and effector functions,
represent viable targets to clinically modulate a variety of immune-mediated disease processes
(Pardoll, 2012; van der Vlist et al., 2016).
1.1.3 Physiological role of Negative Checkpoint Receptors
The physiological roles of the checkpoint receptors are to protect healthy tissues from damage,
maintain self-tolerance, prevent autoimmunity, and maintain or restore homeostasis after
responding to danger signals. Early evidence of the key role played by immune checkpoint
receptors came from studies on CTLA-4, the first well-characterized and clinically-targeted
checkpoint receptor (Callahan & Wolchok, 2013; Leach, Krummel, & Allison, 1996). As
previously mentioned, CTLA-4 expressed on the surface of T-cells suppresses the activation of
naïve and memory T-cells by competing with CD28 for its binding to the shared ligands CD80
and CD86 resulting in CTLA4:CD80/CD86 interactions that provide inhibitory signals to T-cells
activated through TCR or CD28 signaling cascades (Auchincloss & Turka, 2011). The striking
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Figure 1-1. T-cell responses are regulated by a multitude of co-stimulatory and co-
inhibitory interactions. T-cell activation is initiated upon TCR binding to antigenic peptides
in the context of MHC (signal 1). The amplitude of T-cell responses are further supplemented
by additional co-stimulatory signals (i.e. from CD28, ICOS, and GITR) or conversely
dampened by co-inhibitory receptors (i.e. from CTLA-4, PD-1, and LAG3). These regulatory
interactions can occur in lymph nodes during the initiation of T-cell activation, or in peripheral
tissues to control effector functions. (Figure reproduced from Mahoney, K et al. [2015] Nat.
Rev. Drug. Disc. with permission from Nature Publishing Group)
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physiological role of this interaction in downregulating immune responses is apparent in CTLA-
4-deficient (CTLA-4-/-) mice, which develop severe lymphoproliferative disorders leading to
multi-organ failure and early lethality (Tivol et al., 1995). A similar physiological role for CTLA-
4 in humans is supported by the observation of mild to severe immune related adverse events
(irAEs) in patients treated with ipilimumab (an antagonistic anti-CTLA-4 antibody used in cancer
immunotherapy [see Section 1.1.5]) (Bertrand, Kostine, Barnetche, Truchetet, & Schaeverbeke,
2015; Hodi et al., 2010).
More recently, research on another well-characterized immune checkpoint receptor, PD-1, has
identified a physiological role for this receptor in the maintenance of peripheral self-tolerance. In
contrast to CTLA-4, PD-1 negatively regulates T-cell activity at the later effector stage of the T-
cell immunity cycle (Francisco, Sage, & Sharpe, 2010; Riella, Paterson, Sharpe, & Chandraker,
2012). PD-1 signaling is initiated upon binding to its ligands PD-L1 (B7-H1) or PD-L2 (B7-DC)
on peripheral tissues, functioning to downregulate activated T-cells during an ongoing or resolved
inflammatory response (Freeman et al., 2000; Latchman et al., 2001). PD-1-/- mice were found to
develop spontaneous strain-specific autoimmune disorders, evidence that support its role in
peripheral self-tolerance (Nishimura, Nose, Hiai, Minato, & Honjo, 1999). Importantly, PD-1-/-
mice display a significantly milder phenotype than that of CTLA-4-/- mice. As is the case with
CTLA-4 blockade, antibody-mediated blockade of PD-1 in cancer patients often leads to irAEs,
albeit at a lower extent then that observed by anti-CTLA-4 treatment (16% severe grade 3-4 irAEs
for PD-1 blockade compared to 27% for CTLA-4 blockade) (Hodi et al., 2016). Moreover,
combinatorial blockade of PD-1 and CTLA-4 led to an increased prevalence of severe irAEs
compared to either monotherapy alone (55% grade 3-4 irAEs), supporting a non-redundant role
for these checkpoint receptors. The immunobiology and therapeutic implications of targeting the
PD-1 pathway are further detailed in section 1.2.
1.1.4 Structural Considerations: The IgV-like Domain.
The co-stimulatory and co-inhibitory molecules broadly fall into several structural families: the
TNF superfamily, B7 family, CD28 family, Butyrophilins, Nectin and Nectin-like ligands, and
ILT/CD85 family. The inhibitory checkpoint molecules themselves are highly represented in the
B7 (checkpoint ligands) and CD28 (receptors) structural families. Proteins within these two
families all contain a structurally conserved N-terminal IgV-like domain, that is an Ig-like domain
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which resembles the variable domain found on antibodies (Collins, Ling, & Carreno, 2005) . This
domain is characterized by a sandwich like conformation that is composed of 9 β-strands stabilized
by intramolecular disulphide bonds (Chattopadhyay et al., 2009). Generally, the interactions
between the B7-family ligands and the CD28-like receptors are typically of weak affinity; being
primarily governed by contacts involving residues on the β-sheets or complementarity-determining
region (CDR) loops on the terminal IgV-like domains of each binding partner. For example, PD-
1 interacts specifically with PD-L1 through interactions between the GFCC’ β-sheet interface on
their IgV-like domains (Lin et al., 2008; Lázár-Molnár et al., 2008). The dissociation constant for
this interaction however is modest (KD = 8.2 ± 0.1 µM) (Figure 1-2A) (Cheng et al., 2013). Ligand
binding induces a conformation change in the PD-1 CC’ β-strands to trigger an intracellular
signaling cascade (Zak et al., 2015).
1.1.5 Checkpoint Receptors in Cancer Progression
Cancer immunotherapy is based on the understanding that the immune system can distinguish
malignant from healthy cells by the recognition of tumor-associated antigens (TAA). These TAA
are often a consequence of progressive mutations in protein-coding genes or alterations in protein
post-translational modification profiles within a cancer cell resulting in neoantigens, or can stem
from the differential expression of an open reading frame in a transformed cell relative to its normal
counterpart (Vesely & Schreiber, 2013; Ward, Gubin, & Schreiber, 2016). This immune
surveillance of cancer cells is followed by a phenomenon termed immunoediting, where immune
recognition of TAAs leads to the development of anti-tumor responses capable of eliminating the
malignant cells (Mittal, Gubin, Schreiber, & Smyth, 2014; Schreiber, Old, & Smyth, 2011).
However, cancer cells may resist immune detection and elimination through several evasion
mechanisms leading to tumor progression. One key evasion mechanism, involves the expression
of immune checkpoint ligands either directly on cancer cells, or by accessory cells within the tumor
environment (TME), which function to suppress infiltrating anti-tumor T-cells and create a local
immunosuppressive microenvironment (Drake, Jaffee, & Pardoll, 2006; Pardoll, 2012).
Ultimately, these cancers escape immunoediting and progress unimpeded by the immune system.
The therapeutic blockade of these negative checkpoint receptors has thus become a viable strategy
to reverse immune evasion and restore existing strong TAA-specific anti-tumor T-cell responses
(Callahan & Wolchok, 2013; Mahoney, Rennert, & Freeman, 2015; Pardoll, 2012).
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The first in class checkpoint inhibitor was Ipilimumab (Yervoy), an antagonistic anti-CTLA-4
monoclonal antibody (mAb), which received FDA approval in 2010 after showing an increased
survival benefit for patients with metastatic melanoma. In a three-armed trial, ipilimumab as a
monotherapy or in combination with a gp100 peptide vaccine, led to a 3.5-month progression free
survival benefit compared to a gp100 vaccine alone (Hodi et al., 2010). Importantly, ipilimumab-
treated patients had a 18% long-term survival rate (>2 years) as compared to 5% for the gp100-
vaccine treated group, suggesting that this form of immunotherapy can invoke strong and durable
responses capable of suppressing long-term cancer progression.
The next-generation of immune checkpoint inhibitors to the clinic were antibodies which target
and block the PD-1/PD-L1 pathway. Multiple phase trials with two anti-PD-1 mAbs nivolumab
(Opdivo) and pembrolizumab (Keytruda) have consistently found these antibodies provoke strong
anti-tumor immune responses leading to remarkable objective response rates (ORR) and
significant survival benefits (Hamid et al., 2013; Hodi et al., 2016; Topalian et al., 2012). As a
result, nivolumab and pembrolizumab have several FDA breakthrough designations and approvals
for use in the treatment of metastatic melanoma, non-small-cell lung cancer (NSCLC), Hodgkin’s
lymphoma and bladder cancers (detailed in Section 1.2.2).
The remarkable clinical success and improved survival rates observed in cancer patients treated
with CTLA-4 or PD-1 antagonistic antibodies have spurred dramatic interest into further
development of inhibitors targeting alternate checkpoint pathways. Currently, there are over 100
clinical trials ongoing evaluating PD-1 or CTLA-4 blockade either as a monotherapy, or in
combinations with other therapeutics, in a multitude of cancer types. Moreover, clinical and pre-
clinical studies investigating the potential utility of targeting other checkpoint molecules such as
LAG3, B7-H3, and VISTA, among others (Table 1), are ongoing (Le Mercier, Lines, & Noelle,
2015). In light of the new checkpoints uncovered, it is likely the full potential of checkpoint
blockade in the treatment of cancers has yet to be realized.
1.1.6 Role of Checkpoint Receptors in Autoimmune and Inflammatory Disorders
A large body of work now justifies blocking checkpoint pathways as a strategy to restore anti-
cancer immune responses. However, understanding the physiological roles played by individual
immune checkpoint pairs in the suppression of inflammatory and autoimmune disorders suggests
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an opportunity to stimulate (agonize) these pathways as novel treatment approach for patients
afflicted with either acute and chronic inflammatory conditions (Ceeraz, Nowak, Burns, & Noelle,
2014; Podojil & Miller, 2013; van der Vlist et al., 2016). Support for developing checkpoint
agonists stems from the observations that single checkpoint receptor knockout mice (such as PD-
1-/-, CTLA-4-/-, CD200R1-/-, or VISTA-/- mice) consistently exhibit predisposition to, or
exacerbation of, autoimmune-like disorders. For example, an early study documented that PD-1-/-
mice are predisposed to the developed a lupus-like autoimmune disorder (Nishimura et al., 1999).
A summary of these studies, and their corresponding references, is provided in Table 2.
Protein-based ligands or mAbs serving as checkpoint agonists, have shown efficacy in pre-clinical
rodent models of autoimmunity or inflammation. For instance, treatment of mice with CD200-Fc,
a soluble dimeric form of CD200 the natural ligand for CD200R1, readily activates the CD200R1
inhibitory signaling pathway in myeloid and lymphoid cells, causing a reduction in disease in
models of arthritis (collagen-induced-arthritis [CIA]), multiple sclerosis (experimental
autoimmune encephalitis [EAE]), and transplantation (MHC-mismatched allografts) (Gorczynski
et al., 1999; Gorczynski, Chen, Yu, & Hu, 2001; Liu et al., 2010). CD200:CD200R1 mediated
regulation of immunity is further detailed in Section 1.3.2.
In addition to CD200-Fc, the activity of B7-H4-Fc as a soluble immunosuppressive checkpoint
receptor agonist is under investigation. In-vitro studies have found that B7-H4 suppresses T-cell
activation and pro-inflammatory cytokine production by agonizing an as-of-yet discovered
receptor (Azuma et al., 2009; Sica et al., 2003; Wei, Loke, Zang, & Allison, 2011). In humans
Chen et al, (2009) documented high-levels of soluble B7-H4 in patients with more severe
rheumatoid arthritis, which was proposed to act as a decoy-molecule by inhibiting ongoing B7-H4
mediated immunosuppression. In support, in preclinical arthritic models B7-H4-/- mice develop
accelerated disease, while treatment of mice with a B7-H4-Fc fusion protein attenuates disease
(Azuma et al., 2009). Subsequent, studies have shown that B7-H4-Fc treatment can suppress
immune responses in murine models of MS (EAE), lupus nephritis and type 1 diabetes, leading to
improved outcomes in all cases (Pawar et al., 2015; Podojil et al., 2013; X. Wang et al., 2011). A
B7-H4-Fc fusion developed by Amplimmune (AMP-110) is currently in a Phase I trial for the
treatment of rheumatoid arthritis. Several more agonists targeting checkpoint receptors such as
CD200R, PD-1, VISTA, and VISTA-receptor, are under pre-clinical investigation, and the overall
clinical potential of this approach remains to be discovered.
9
Checkpoint Molecule Therapeutic (type) Stage of clinical
development
CTLA-4 Ipilimumab (mAb)
Tremelimumab (mAb)
FDA approved
Phase III
PD-1 Pembrolizumab (mAb)
Nivolumab (mAb)
PDR001 (mAb)
FDA Approved
FDA Approved
Phase I/II
PD-L1 Atezolizumab (mAb)
Durvalumab (mAb)
Avelumab (mAb)
AMP-224 (PD-L2 Fc fusion)
FDA Approved
FDA Approved
FDA Approved
Phase I
B7-H3 Enoblituzumab (mAb) Phase I
LAG3 REGN3767 (mAb)
BMS-986016 (mAb)
Phase I
Phase I/II
TIM3 TSR-022 (mAb) Phase I
TIGIT OMP-313M32 (mAb)
BMS-986207 (mAb)
MTIG7192A (mAb)
Phase I
Phase I
Preclinical
VISTA JNJ-61610588 Phase I
Preclinical
B7-H4 Preclinical
Table 1-1. Immune checkpoint molecules currently being targeted in cancer therapy.
10
Checkpoint
Knockout
Strain Disease Model
(reference) Result
CTLA-4 -/- C57Bl/6 Naïve mice
(Tivol et al., 1995)
Lymphoproliferative
disorders, multi-organ failure
PD-1 -/- C57Bl/6 Naïve Mice
(Nishimura et al., 1999)
Develop lupus-like
autoimmunity at 10 months
PLP/tg EAE
(Kroner et al., 2009)
Accelerated onset and
exacerbated disease
C57Bl/6 CIA
(Raptopoulou et al., 2010)
Increase incidence and
disease severity
TIGIT -/- 2D2 TCR
transgenic
EAE
(Joller et al., 2011)
Exacerbated disease
B7-H4 -/- DBA/1j CIA
(Azuma et al., 2009)
Accelerated onset and severe
disease
129/SvE EAE
(Wei et al., 2011)
Exacerbated disease
CD200 -/- BALB/c Allograft
(Yu et al, 2013)
Rapid rejection of organ
allografts
C57Bl/6 EAE
(Hoek et al., 2000)
Early onset of disease
C57Bl/6 CIA
(Hoek et al., 2000)
Increased susceptibility to
arthritis
C57Bl/6 EAU
(Broderick et al., 2002)
Accelerated onset of tissue-
specific autoimmunity
VISTA -/- C57Bl/6 Naïve mice
(Wang et al., 2014)
Mild pro-inflammatory
phenotype
C57Bl/6 Naïve mice
(Yoon et al., 2015)
Spontaneous
glomerulonephritis at late age
C57Bl/6 SLE (Sle1,Sle3 mice)
(Ceeraz et al., 2016)
Accelerates development of
fatal lupus nephritis
2D2 TCR
Transgenic
EAE
(Wang et al., 2014)
Enhanced disease incidence
and severity
C57Bl/6 Imiquimod induced
psoriasis
(Li et al., 2017)
Exacerbated psoriasis
inflammation
Table 1-2. Genetic deletion of checkpoint molecules causes an enhancement in the occurrence
of diseases associated with autoimmunity. List of pre-clinical studies highlighting the
consequence of deleting immune checkpoint molecules in terms of predisposition or increased the
severity of autoimmune or inflammatory disorders in rodent models.
11
The context-specific roles of the checkpoint molecules investigated throughout the body of this
thesis (PD-1, CD200R1, and VISTA) are provided in the following sections.
1.2 PD-1:PD-L1-mediated Regulation of Immunity
1.2.1 Structure and Function of PD-1
PD-1 is a type I cell-surface glycoprotein which functions to downregulate T-cell based immune
responses upon binding with its paired ligands PD-L1 or PD-L2. PD-1 is primarily expressed on
T-cells, with increased expression induced throughout T-cell activation (Francisco et al., 2010).
Its expression is also found to be higher on exhausted T-cells in chronic infections such as HIV.
PD-1 is heavily conserved through evolution, with mouse and human PD-1 sharing a 64% amino
acid sequence identity. Structurally, PD-1 is a glycosylated monomeric protein with an apparent
mass of 55kDa, consisting of an N-terminal IgV-like domain, followed by a single pass
transmembrane domain, and a cytoplasmic tail containing two intracellular signaling motifs (ITIM
and ITSM) (Figure 1-2A). The transient interaction between the IgV-like domains of PD-1 and
PD-L1 induces phosphorylation of the ITIM and ISTM motifs on the cytoplasmic tail leading to
the recruitment of the phosphatase SHP2. SHP2 in turn reduces the phosphorylation signals from
the proximal TCR and CD28 signaling pathways leading to a dampening of T-cell activation
signals (Yokosuka et al., 2012). Ultimately, PD-1 ligation by PD-L1 or PD-L2 suppresses T-cell
expansion, proliferation and the secretion of pro-inflammatory cytokine such as IL-2 and IFNγ
(Freeman et al., 2000; Latchman et al., 2001).
The temporal expression patterns of the ligands PD-L1 and PD-L2 provide some insight into the
role of PD-1-mediated immune regulation. For instance, strong inflammatory stimuli can induce
the expression of PD-L1 in most tissues. This expression pattern supports a role for PD-1:PD-L1
signaling as a key mediator in maintaining peripheral tolerance and in controlling damage to self
by regulating the intensity of T-cell effector responses in peripheral organs. In contrast, PD-L2
expression appears to be more restricted to immune cells (Francisco et al., 2010). However, recent
studies have also uncovered its upregulation by non-immune cells depending on the local
microenvironment (Rozali, Hato, Robinson, Lake, & Lesterhuis, 2012).
12
1.2.2 Targeting the PD-1:PD-L1 Pathway in Cancer
Tumor cells often evade elimination by exploiting the PD-1 inhibitory pathway (Drake et al.,
2006). Specifically, PD-L1 expression and signaling is prominent in peripheral tissues where
cancers originate. Within solid tumors, inflammatory conditions can induce PD-L1 expression
either directly on cancer cells or on other accessory cells within the tumor microenvironment
(TME), which binds to and suppresses the anti-tumor activity of PD-1+ tumor infiltrating
leukocytes (TILs) (Pardoll, 2012). Early evidence of the role for PD-1:PD-L1-mediated evasion
came from studies showing that transfecting transplantable tumor cells with PD-L1 led to a
reduction in anti-tumor immune cytotoxicity and an in-vivo growth advantage to these cells
(Hirano et al., 2005). In further pre-clinical studies, mAb-mediated blockade of PD-1:PD-L1
signaling in a multitude of syngeneic mouse models showed a benefit in terms of increasing anti-
tumor T-cell responses and in delaying cancer progression (Hirano et al., 2005; Terawaki et al.,
2011). Multiple blocking anti-PD-1 and anti-PD-L1 mAbs subsequently entered clinical trials
based on the success in pre-clinical tumor models and on the foundation laid by the clinical success
of CTLA-4 blockade.
Early clinical trials with two anti-PD-1 mAbs nivolumab and pembrolizumab demonstrated the
remarkable ability of these biologics to promote strong and durable responses in advanced
melanoma. For instance, a Phase I trial completed in 2012 studying the safety and efficacy of
nivolumab demonstrated a 28% ORR in patients with advanced melanoma, and a 18% ORR rate
in NSCLC, a cancer previously thought to be refractory to immune therapy (Topalian et al., 2012).
In a randomized controlled Phase III trial pembrolizumab treatment led to a 33% ORR in advanced
melanoma patients, compared to 11.9% ORR with ipilimumab (anti-CTLA-4 mAb) (Robert et al.,
2015). As of today, positive results for both anti-PD-1 mAbs have been documented in multiple
Phase trials across several cancer types, leading to FDA breakthrough designations and approvals
in indications including melanoma, NSCLC, Hodgkin’s lymphoma, and bladder cancer. Ongoing
trials are focused on expanding the use of these mAbs across multiple cancer types, and to improve
efficacy using novel combination strategies. Of note, the use of a combination regimen of
nivolumab and ipilimumab treatment led to a 56.6% objective response rate and 11.5-month
progression-free survival in metastatic melanoma patients, compared to 43% and 6.9 months with
nivolumab monotherapy (Larkin et al., 2015). Studies are also underway to identify predictive
biomarkers for this type of therapy. For instance, a recent study demonstrated that PD-1 blockade
13
is especially effective in mismatch repair deficient colorectal cancers (40% ORR) compared to
mismatch repair-proficient colorectal cancers (0% ORR), likely due to the increased neoantigen
load in the former due to diminished DNA repair pathways (Le et al., 2015). Continuing research
to identify novel synergistic combinations, predictive biomarkers, and to delineate the mechanisms
behind PD-1 blockade resistance may further expand the potential of PD-1-based immune-
therapies.
IgV-like domain
Transmembrane
B
Figure 1-2. Structure of PD-1. (A) Ribbon diagrams of the crystallized interaction between the
IgV-like domains of PD-1 (blue ribbon) and PD-L1 (green ribbon), highlighting interaction at
the GFCC’ β-sheet interface. (PDB:4ZQK, Zak et al, 2015) (B) Schematic diagram outlining the
primary structure of PD-1. PD-1 consists of an N-terminal extracellular IgV-like domain,
followed by a transmembrane domain, and cytoplasmic tail containing the ITIM and ITSM
inhibitory motifs responsible for signal transduction.
A
14
1.3 CD200:CD200R1-mediated Regulation of Immunity
1.3.1 Structure and Function of CD200R1
Like PD-1, CD200R1 is a type I glycoprotein expressed on immune cells which functions to
transduce inhibitory signals upon ligation to its widely distributed ligand CD200. In contrast to
PD-1, CD200R1 expression is primarily found on cells of the myeloid lineage and to a lesser extent
on lymphoid cells (Wright et al., 2003; Wright et al., 2000). Structurally, CD200R1 contains two
Ig-like domains (IgV-IgC), a transmembrane region, and a cytoplasmic tail containing a NXPY
motif. CD200, which similarly contains and IgV-IgC extracellular domain, binds relatively weakly
to CD200R1 (KD = 4µM); an interaction that involves their respective N-terminal IgV-like
domains (Hatherley, Cherwinski, Moshref, & Barclay, 2005). The binding of CD200 to CD200R1
results in the rapid phosphorylation of the tyrosine residue within the NPXY motif on the
CD200R1 cytoplasmic tail, leading to the recruitment of the adaptor proteins Dok1 and Dok2
(Figure 1-3). These adapter proteins subsequently recruit SH2 containing inositol phosphatase
(SHIP) and RasGAP, leading to inhibition of Ras/MAPK signaling (Mihrshahi, Barclay, & Brown,
2009; Zhang, Cherwinski, Sedgwick, & Phillips, 2004; Zhang & Phillips, 2006). At the cellular
level, the activation of CD200R1 leads to a polarized cytokine production profile, the inhibition of
cellular effector functions, and the maintenance of tissue homeostasis (Cherwinski et al., 2005;
Hoek et al., 2000).
1.3.2 Role of CD200R1 in Inflammatory Disorders
Studies using CD200R1-/- mice, transgenic mice overexpressing CD200 (CD200Tg), and
recombinant CD200R1 signaling molecules (CD200-Fc) have implicated CD200R1-inhibitory
signaling as a key axis in the regulation of inflammatory disorders such as transplant regulation,
arthritis, autoimmunity, and allergy. CD200-Fc, a potent CD200R1 agonist, significantly prolongs
murine allo- and xenograft survival (Gorczynski et al., 1999). Similarly, allograft survival is
prolonged in CD200Tg mice which overexpress CD200 under control of a doxycycline inducible
promoter. Notably, long-term stimulation of CD200R1-signalling in these mice led to the
induction of full allograft acceptance (Gorczynski et al., 2009). In the rodent collagen-induced
arthritis (CIA) model, treatment of mice with CD200R1 agonists can prevent the induction
(prophylactic regimen) or reduce the severity (treatment regimen) of the disease (Gorczynski,
Chen, Lee, Yu, & Hu, 2002; Gorczynski et al., 2001). CD200R1-signalling has also been
15
implicated in the EAE model of multiple sclerosis, where blockade of CD200:CD200R1
aggravates disease, while CD200-Fc treatment improves outcomes (Hoek et al., 2000).
Furthermore, an agonistic anti-CD200R1 mAb has been shown to suppress the development of
experimental autoimmune uveitis (EAU), a model of tissue-specific neural retina autoimmunity
(Copland et al., 2007). Lastly, therapeutically agonizing CD200R1-signalling has also been
suggested as a potential treatment for allergic disorders. Notably, mast cells express high-levels of
CD200R1 and are functionally suppressed by CD200 (Cherwinski et al., 2005; Zhang et al., 2004).
In an experimental OVA-induced allergic-asthma mouse model, CD200-Fc treatment significantly
decreased airway hyper-responsiveness and the associated immune responses observed upon
allergen challenge (Lauzon-Joset et al., 2015). Together, these pre-clinical studies highlight a
significant therapeutic value in stimulating CD200R1 immunoinhibitory signaling for the
treatment of inflammatory disorders.
16
Figure 1-3. CD200:CD200R1 immunoinhibitory signaling. CD200R1 expressed on the
surface of myeloid and lymphoid cells becomes phosphorylated upon ligation with the widely
distributed CD200. Inhibitory signals are transduced from the phosphoryled NPXY motif on the
CD200R1 tail upon binding to the adapter proteins Dok2 and Dok1. The transduced signals
ultimately downregulated immune functions suppression of cell activation and decreases pro-
inflammatory cytokine secretion.
PDok2
CD200R1
CD200
Inhibitory Signals
(Polarized cytokine production,
suppressed activation,
maintenence of homeostasis)
Endothelial cells Immune Cells
Epithelial cells Cancer Cells
T, B, NK Cells
DC Cells
Macrophages
Mast Cells
17
1.4 VISTA-mediated Regulation of Immunity
1.4.1 Structure and Function of VISTA
VISTA (PD-1H, Dies1, Gi24, DD1α) is a more recently described checkpoint molecule. Originally
identified in 2011 by Wang and colleagues, VISTA was reported to function as a checkpoint ligand
expressed primarily on myeloid cells, functioning to suppress T-cell activation by binding to an
uncharacterized receptor (referred to hereafter as VISTA-receptor) (Wang et al, 2011).
Structurally, VISTA shares significant homology with PD-1 and PD-L1, and as is the case with
these receptors, contains an N-terminal IgV-like domain, followed by a transmembrane domain
and cytoplasmic tail (Figure 1-4A). VISTA-signaling either delivered by an immobilized
recombinant VISTA-Fc fusion, or by its expression on APCs, was found to substantially suppress
the activation, proliferation, and cytokine production in murine T-cells in-vitro (Wang et al, 2011).
Follow-up studies have confirmed that human T-cells are similarly suppressed by VISTA (Lines,
Pantazi, et al., 2014). Interestingly, a more recent study has identified a role for VISTA expressed
on T-cells to act as a checkpoint receptor upon binding to an unknown ligand, suggesting a dual-
functionality of VISTA as both a checkpoint receptor and ligand (Figure 1-4B) (Flies et al., 2014).
Notably, VISTA does contain a cytoplasmic tail with two potential protein kinase C (PKC) binding
sites suggesting that this domain may initiate a signaling cascade following receptor-ligand
engagement. The in-vivo role of VISTA as an immune regulatory receptor was confirmed using
antagonistic anti-VISTA mAbs, as blockade of this pathway led to enhanced T-cell activity and
exacerbated disease in a model of EAE (Wang et al, 2011). Furthermore, anti-VISTA mAbs have
also shown to release potent anti-tumor immune responses both as a monotherapy, and in
combination with anti-PD-1 mAbs, supporting the clinical utility of antagonizing this pathway in
cancer immunotherapy (Le Mercier et al., 2014; Lines, Sempere, Broughton, Wang, & Noelle,
2014). A Phase I clinical trial (NCT02671955) is currently underway to determine the safety and
efficacy of a humanized anti-VISTA mAb in patients with solid tumors.
1.4.2 Role of VISTA Signaling in Autoimmune Diseases
Although less characterized then the aforementioned checkpoint molecules, several studies using
VISTA-/- mice have implicated this pathway as a regulator of disease progression associated with
autoimmune and inflammatory disorders. An initial study reported that VISTA-/- mice bred on a
wild-type C57Bl/6 background displayed a mild inflammatory phenotype, with an increased
18
number of dendritic cells and higher expression of T-cell activation markers relative to wild-type
littermates, but did not develop spontaneous autoimmune or inflammatory disorders (Wang et al.,
2014). In slight contrast, another study reported a more severe phenotype for VISTA-/- C57Bl/6
mice which led to the development of spontaneous glomerulonephritis at ~10 months of age (Yoon
et al., 2015). The discrepancy between these studies is unclear, but could be the result of different
gene targeting approaches, or reflects variations within the animal holding facilities. VISTA-/- mice
bred on lupus prone background (Sle1,Sle3 model) developed accelerated and fatal systemic lupus
erythematosus (SLE) (Ceeraz et al., 2016). Similarly, VISTA-/- 2D2 TCR-transgenic mice develop
exacerbated EAE (Wang et al., 2014). More recently, Wang and colleagues showed that VISTA-
deficient mice were more susceptible to imiquimod-induced psoriasis due to enhanced TLR7
signaling in dendritic cells and increased IL17A secretion by T-cells (Li et al., 2017). While much
of the immunobiology of VISTA remains to be discovered including the identity of its putative
binding partner(s), these studies suggest that agents that promote VISTA:VISTA-receptor
signaling could be clinically useful in treating inflammatory or autoimmune disorders.
19
IgV-like domain
Transmembrane Putative PKC
docking sites
A
B
Figure 1-4. VISTA structure and function. (A) Primary structure of the recently identified
checkpoint molecule VISTA. VISTA contains a IgV-like domain with significant homology to
the PD-1 and PD-L1 and contains a cytoplasmic tail with two putative Protein Kinase C (PKC)
binding sites. (B) VISTA is expressed on both lymphoid and myeloid cells, and has been
identified to act as both as a T-cell immune inhibitory ligand and receptor. The VISTA binding
partners remain to be characterized.
20
1.5 Classes of Therapeutic Agents Targeting Checkpoint Receptors
1.5.1 Humanized Monoclonal Antibodies
Nearly all biotherapeutics targeting the negative checkpoint inhibitors are humanized monoclonal
antibodies. Antibodies are protein-based molecules which bind to their target antigen with high
affinity and specificity. Antibodies can be identified to sterically block receptor: ligand interactions
in the case of antagonists, or conversely can mimic the natural ligand binding thus serving as
agonists to activate cellular signaling pathways. Clinically, FDA-approved anti-PD-1 and anti-
CTLA-4 mAbs checkpoint receptor behave as antagonists by blocking the immune inhibitory
interactions. They have proven to be effective as inhibitors of immunoregulatory signals leading
to enhanced anti-tumor immune responses (Callahan & Wolchok, 2013). Conversely, although
less clinically developed, agonistic checkpoint receptor targeting mAbs hold the potential for
stimulating immune inhibitory signaling pathways to treat inflammatory disorders (Ceeraz et al.,
2014). For example, an agonistic anti-VISTA mAb has been shown to suppress T-cell mediated
immune responses and reduce disease severity in models of acute Concanavalin A (ConA) induced
hepatitis and graft versus host disease (GVHD) (Flies et al., 2014; Flies, Higuchi, & Chen, 2015).
Similarly, an agonistic anti-CD200R1 mAb has been shown to act as an anti-arthritic agent in the
CIA mouse model. Structurally, antibodies are large (~150kDa) multimeric proteins consisting of
two identical light chains linked to two heavy chains by disulphide bonds. Each heavy chain
consists of a variable domain (VH) followed by a constant region (CH1), a hinge region, and the
Fc region containing two constant regions (CH1, CH2). Additionally, the two heavy chains are
linked by disulphide bonds creating a dimeric structure (Figure 1-5). Binding specificity to unique
antigens is conferred by the hypervariable complimentary-determining regions found near the N-
terminal regions on both the light and heavy chains. In terms of the therapeutic use of mAbs, a
major limitation lies in the immunogenicity associated with injecting patients with protein-based
agents harboring non-self determinants (Chames, Van Regenmortel, Weiss, & Baty, 2009; Nelson,
Dhimolea, & Reichert, 2010). Specifically, repeated injections with mAbs can lead to patients
mounting a humoral immune response which can neutralize antibody function and diminish
pharmacokinetic properties. Importantly, advances in the engineering of humanized or fully human
IgGs has led to the next-generation of mAbs which are considerably less immunogenic. However,
their long-term use can still engender immune responses, which becomes problematic for the
21
treatment of lifelong chronic autoimmune or inflammatory disorders (Strand et al., 2017). Another
limitation to antibody therapy is the cost-vs-clinical benefit challenge, as these protein-based
biologics carry inherently high production and commercialization costs, and exhibit poor stability
and shelf-life properties in comparison to most synthetic drugs. Specifically, the high costs are in
part associated with the cultivation and purification of these cell-based products, but more
increasingly due to market pressure (Nelson et al., 2010). One outcome is that their clinical use
may become restricted, despite the fact that synergistic combinations of multiple mAbs may be
identified as more effective treatment options. Finally, another limitation of antibody-based
therapies lies in the observation that some drug targets are not particularly immunogenic, leading
to a minimal repertoire of useful antibodies, which may preclude the discovery of rare functional
mAbs such as those that activate specific receptor signaling pathways (Nelson et al., 2010).
Figure 1-5. Structure and engineering of modern day therapeutic antibodies. mAbs consist
of a dimeric structure, with each monomer containing a light and heavy chain. Advances in
protein engineering techniques have led to the derivation of chimeric (bottom left), humanized
(top right), or fully human mAbs (bottom right) each with increasingly less non-human sequences
(red), and reduced immunogenicity. (Figure reproduced from Carter P., [2001] Nat. Rev. Cancer
with permission from Nature Publishing Group)
22
1.5.2 Fc-based Fusion Proteins
Fc-based fusion proteins are another common class of protein therapeutics that can be engineered
to bind to specific cell-surface receptors. These chimeric proteins contain the immunoglobulin Fc
region linked to any protein-based molecule of interest such as a natural ligand targeting a given
receptor. The fusion of these ligands to the Fc region imparts a number of advantages including
increased solubility and stability, improved pharmacokinetic properties, and ease of purification
(Czajkowsky, Hu, Shao, & Pleass, 2012). An additional advantage is that most Fc-chimeras are
expressed as homodimers, stabilized by intramolecular disulphide bonds from the constant region.
Thus Fc-fusion proteins have increased avidity for their targets relative to a soluble monomeric
ligand. Clinically, Fc-fusions have been developed for both oncology and autoimmune indications.
For instance, Enbrel, a commonly prescribed anti-TNFα therapy for the treatment of rheumatoid
arthritis, consists of the high affinity TNFR2 extracellular domain fused to the Fc region of human
IgG1 (Haraoui & Bykerk, 2007). Limitations for these protein-based therapies parallel those
discussed for mAbs namely high production costs, poor stability and limited shelf-life. Currently,
there are only a few clinical examples of Fc-fusion proteins targeting negative checkpoint
receptors. Notably, a PD-L2 extracellular domain (ECD)-Fc chimera (AMP-224) which functions
as a PD-1 antagonist is in a Phase II trial (NCT02298946). In comparison to mAbs, Fc-fusions
containing checkpoint ligand IgV-like domains tend to more readily act as receptor agonists, likely
due to the presence of the natural ligand binding interface. Indeed, CD200-Fc has been shown to
cause CD200R1-mediation immunosuppression in pre-clinical models of inflammation (Section
1.3). Moreover, VISTA-Fc can suppress T-cell activity in-vitro by agonizing the putative VISTA-
receptor. However, this effect did require immobilization of VISTA-Fc on a solid surface,
suggesting that this chimera may only have limited agonistic activity in-vivo (Wang et al., 2011).
Nonetheless, this example does highlight another unique advantage to the use of Fc-fusion
proteins, being that the receptor for an inhibitory ligand does not need to be characterized to derive
a targeted immune modulator. Fc-fusion proteins may thus prove to be particularly relevant for
targeting novel checkpoint binding agents, as several ligands, such as VISTA, B7-H3 and B7-H4,
have been identified to possess immunosuppressive function but do not have characterized binding
partners. To this end, a B7-H4-Fc fusion which showed promise in preclinical CIA mouse models
is currently in a Phase I trial for rheumatoid arthritis with results pending (NCT02277574) (Azuma
et al., 2009).
23
1.5.3 Small Molecules
Small molecule therapeutics are similarly capable of antagonizing or agonizing cell receptor
signaling pathways. In contrast to biologics, small synthetic compounds have the inherent
advantages of high stability, scalability in terms of chemical synthesis, and lower production costs.
However, limitations come with these advantages in the form of difficulties associated with
discovering a small molecule specific to a given checkpoint target due to the structurally conserved
nature of IgV-folds present across the B7/CD28 family members. The only example to date of a
small molecule checkpoint inhibitor is CA-170, which is reported to bind the IgV-domains of PD-
L1, PD-L2, and VISTA, thereby simultaneously antagonizing both PD-1 and VISTA-mediated
signaling. There is no peer-reviewed publication to date defining the structure of CA-170 or
documenting whether CA-170 binds specifically to these IgV-like domains nor published pre-
clinical evidence confirming its activity. Of note, these receptors share only moderate sequence
identity (34% between PD-L1 vs -L2 and 25% for PD-L1 vs VISTA), and activate distinct cellular
signaling pathways. Despite this, Curis Inc. recently initiated a Phase I study of CA-170 in patients
with advanced tumors and lymphomas (NCT02812875). One motivating factor for exploring the
clinical utility of CA-170, besides its purported blockade of three valid targets, is that it represents
an orally available drug, providing more convenience compared to the injectable administration
required for the anti-PD-1 biologics. If CA-170 does indeed act on several distinct IgV-like
domains and immune signaling pathways, then its safety in terms of potential off-target effects of
CA-170 will have to be carefully evaluated.
1.6 Oligonucleotide Aptamers
Aptamers are short single-stranded oligonucleotides which fold into unique three-dimensional
structures to bind molecular targets of interest with high affinity and specificity (Figure 1-6). Due
to their ability to be developed to target a wide array of molecules (including proteins, peptides,
nucleic acids, and small molecules) aptamers have been used for applications ranging from
diagnostics tools, imaging agents, biosensors, to drug-delivery agents. Importantly, aptamers can
act as agonists and antagonists (Song, Lee, & Ban, 2012). Aptamers specific to a molecular target
are typically identified using an iterative in-vitro selection process called systematic evolution of
ligands by experimental enrichment (SELEX); a strategy first described by two groups in 1990
(Ellington & Szostak, 1990; Tuerk & Gold, 1990). The following sections detail the unique and
24
advantageous properties of oligonucleotide aptamers in the context of their potential as checkpoint
targeting agents.
1.6.1 Derivation of Aptamers Using SELEX
Aptamers (named after the Latin word aptus meaning ‘to fit’) are oligonucleotide motifs selected
using an in-vitro PCR-based process known as SELEX to bind to a molecular target of interest.
The traditional SELEX process is an iterative cyclical panning procedure consisting of 5 major
steps. In the case of DNA aptamers, a starting combinatorial library is first synthesized with an
internal variable region (where any position nucleotide position [N] can be A, T, C, G) flanked by
a 5’ and 3’ constant regions (Ellington & Szostak, 1992). The maximal diversity of any given
library can be described by the theoretical number of different oligonucleotide sequences
composing the library namely 4N elements where N represents the number of random bases
inserted in the variable region of the library. This starting library is incubated with the protein
target of interest immobilized on a solid support such as affinity beads (Figure 1-7, step 1). The
unbound or weakly associated nucleotides are subsequently removed by wash steps (step 2), and
the bound aptamers are subsequently denatured and eluted from the protein target using high
temperature or chemical denaturant (step 3). After recovery, the library now enriched for binding
ligands is amplified by asymmetric PCR to repopulate the pool of single-stranded aptamers (step
4), which serves as the starting library for the next cycle (step 5). This cyclical process is then
repeated for 10-15 cycles. Throughout the process, it is often useful to counter-select the library
against the solid support to prevent expansion of sequences which bind the affinity matrix.
Additionally, throughout the selection cycles, stringency is increased by the addition of extra wash
steps, and decreased library:target incubation times to preferentially select for aptamer sequences
with the highest target efficiency.
25
Figure 1-6. Overlaid crystal structures of an RNA and DNA aptamer bound to
thrombin. Aptamers are short synthetic single stranded oligonucleotides with unique three-
dimensional folds which bind proteins with high affinity and specificity. Depicted is the
overlaid crystal structures of a DNA aptamer (blue, bottom), and an RNA aptamer (purple,
top), bound to the protein thrombin (grey). Figure adapted from Orava, Cicmil and Gariépy
(2010).
26
Figure 1-7. Derivation of Aptamers using SELEX. In the traditional SELEX process, a
combinatorial starting library is chemically synthesized with a random internal region (typically
25-50 nucleotides in length) flanked by two constant primer regions. The initial pool consisting
of at least 1x1015 unique sequences is incubated with the target protein immobilized on a solid
surface using affinity tags (1). Non-bound or weakly associated sequences are removed by
washing (2), and the bound aptamers eluted by denaturation of the tertiary structure with high-
temperature (3). The library now enriched for binding ligands is reamplified by PCR (4), and
used as the starting library for the next selection cycle. Traditionally, this process is carried out
for 10-15 cycles and the enriched aptamers finally identified by DNA sequencing. Figure adapted
from Orava, Cicmil and Gariépy (2010).
27
1.6.2 Next-generation-sequencing analysis of SELEX screens
Traditionally, after 10-15 cycles of SELEX the aptamers would be cloned into a sequencing
plasmid, prepped, and sequenced using the Sanger method to identify the selected aptamers.
However, this method limits researchers to the identification of a finite number of sequences,
which may not be representative of the entire pool. Recent advances in modern high-throughput
sequencing have led to the incorporation of NGS technology to the SELEX process (SELEX-
NGS). Critically, NGS platforms typically yield 106-108 sequence reads of enriched SELEX pools,
allowing for a comprehensive view of the SELEX screens to readily identify enriched sequences
and sequence families (ie. sequences which share a common motif) (Alam, Chang, & Burke,
2015). NGS has led to the identification of high affinity aptamers, in some cases after only 4-5
cycles, drastically reducing the screening time required (Berezhnoy et al., 2012). Furthermore, by
analyzing NGS data from multiple cycles of the same SELEX screen (cycles 3, 7, and 9 for
instance), one can look at the enrichment of certain aptamer sequences over cycle number
reflecting the selection stringency used (Figure 1-8) (Blind & Blank, 2015). Interestingly, it has
been reported that high enrichment of a given sequence (ie. read abundance in cycle [x] /
abundance in cycle [y]) correlates with the selection of higher affinity aptamers. Furthermore, we
and others have recently shown that NGS can be used to identify rare cross-species aptamers by
cross-comparing sequence data from independent SELEX-NGS screens performed against protein
orthologs (Levay et al., 2015).
28
No
rmalized
R
ead
s Increased stringency
Figure 1-8. Tracking the progress of SELEX screens with NGS. Exemplary data of a SELEX
screen towards the beta-2 adrenergic receptor (β2AR) which was analyzed by NGS. Each line
represents the abundance of a unique aptamer sequence across cycles 3, 7 and 9. The black arrow
represents when selection stringency was increased by addition of an extra wash step and
reduction of aptamer pool:target incubation time by half. Note the changes in aptamer
enrichment, with select aptamers continuing to increase in abundance across the screen. Apt1,
Apt2, and Apt3 were confirmed to bind β2AR by pull-down assays.
29
1.6.3 Functional Aptamers as Therapeutics: Advantages
As is the case with antibodies, aptamers derived to target cell-surface receptors can act as
antagonists by sterically blocking receptor:ligand interactions, or conversely behave as agonists to
stimulate receptor signaling pathways. Due to these functional properties, and the fact that the
specificity and affinity of aptamers often rival or supersede that observed with mAbs, these
synthetic ligands have been proposed as useful therapeutic entities (Keefe, Pai, & Ellington, 2010).
In this sense, aptamers carry a number of unique properties that may prove advantageous over
mAbs and other protein-based targeting agents (Table 1-3). One critical advantage lies in that
aptamers are chemically synthesized, in a low cost-chemical process which is readily scalable with
little batch-to-batch variation (Gilboa, McNamara, & Pastor, 2013; Zhou & Rossi, 2017). Another
advantage lies in the relative ease of which aptamers can be post-synthetically conjugated allowing
the tailoring of their pharmacokinetic properties (Haruta et al., 2017; McNamara et al., 2008). For
instance, conjugation of multiple aptamers on a PEG-scaffold can lead to multimeric constructs
with increased avidity and longer circulation half-lives. Further, scaffolds containing both a
targeting and functional aptamer can enhance tissue targeting of a functional ligand. This approach
was demonstrated by Gilboa and his colleagues, who created a dimeric construct of a prostate
specific membrane antigen (PMSA) aptamer linked with an agonistic 4-1BB aptamer (PSMA-4-
1BB), which specifically homed to PSMA-expressing tumors and induced local 4-1BB immune
co-stimulation (Pastor, Kolonias, McNamara Ii, & Gilboa, 2011; Schrand et al., 2015). Another
advantage aptamers carry over mAbs is their smaller size, ranging from 20-80 nucleotides (MW =
8-25 kDa), which allows them to readily penetrate tissues (Healy et al., 2004). Another interesting
and unique feature of aptamer-based therapy is the ability to use the reverse complement of the
aptamer as an in-vivo antidote to rapidly neutralize the activity of bioavailable aptamer, as has
been demonstrated by the reversal of the anticoagulation activity of an anti-factor IXa aptamer
upon injection of its complement antidote both in animal models and humans (Dyke et al., 2006;
Rusconi et al., 2004). Last, but not least, there has been no reports of aptamer-induced
immunogenicity, and it is unlikely that these short oligonucleotides engender neutralizing immune
responses (Keefe et al., 2010).
1.6.4 Therapeutic limitations of Aptamers
Although aptamers possess advantageous properties relative to protein-based biologicals, there are
some critical limitations to their therapeutic use which must be considered (Table 1-3). As is the
30
case with small molecules and peptides, short oligonucleotide aptamers display poor
pharmacokinetic (PK) properties due to (i) rapid removal from circulation due to renal filtration
and (ii) enzymatic cleavage and degradation from plasma-based nucleases. For instance, non-
modified RNA aptamers typically exhibit circulatory half-lives (t1/2) in the range of minutes; a
finding that severely limits their potential for systemic use (Healy et al., 2004). Of note, the only
currently approved aptamer Pegatinib (Macugen), an anti-VEGF aptamer used in the treatment of
age related macular degeneration, is a PEGylated RNA aptamer delivered by local intravitreal
injection (Ng et al., 2006).
Several methods to address the “PK problem” have been proposed, primarily aimed at stabilizing
the nucleotide backbone and increasing the size of these molecules by conjugation to inert high
molecular weight polymers. In the case of RNA aptamers, substitution of ribonucleotides with
nuclease-resistant nucleotide analogues is a common procedure to minimize nuclease-based
degradation (Keefe et al., 2010). Further, the addition of an inverted dT nucleotide to the 3’-end
of DNA or RNA-based aptamers protects oligomers from exonuclease-based cleavage. In addition
to protecting from nuclease degradation, conjugation of aptamers to high MW moieties such as
polyethylene glycol (PEG) significantly extends their circulatory half-life by limiting the rate of
renal filtration. The utility of PEGylating aptamers was demonstrated in a study by Healy, which
showed that conjugating a 40kDa linear PEG chain to a protected RNA-aptamer increased its t1/2
in mice from 4.88 to 11.68 hours and enhanced its tissue distribution (Healy et al., 2004). Further
studies carried out by Haruta and colleagues, focused on optimizing the size and configuration (ie.
branched vs linear) of the PEG group with a view to further improve the PK properties of aptamers.
Notably, a novel conjugation method, termed sBC-PEGylation was used to derivatize an anti-IL-
17 aptamer with a highly branched 80kDa 2x2-arm PEG structure. Remarkably, the sBC-
PEGylated aptamer exhibited a plasma half-life of 102 hours in cynomolgus monkeys, a finding
which may lead to the successful systemic use of this aptamer (Haruta et al., 2017). Currently,
there are several PEGylated-aptamer conjugates in clinical trials for various indications (Table 1-
4). Of note, while PEG is believed to be an inert polymer suitable for human use, there have been
recent reports of adverse PEG-related allergic reactions in trials testing the safety of both
oligonucleotide and protein-based PEG-conjugates (Ganson et al., 2016). Thus, caution with this
approach is necessary and further research into improving the PK properties of aptamers is
warranted.
31
Table 1-3. Advantages and limitations of aptamers as therapeutic entities compared to
monoclonal antibodies.
32
Aptamer
Target Disease Type Modifications Stage
Pegatinib VEGF165 Age related
macular
degeneration
RNA • 2-F, 2-O-methyl
• 3’ inverted dT
• 40kDa PEG
FDA
Approved
ARC1905 C5 Age related
macular
degeneration
RNA • 2-F, 2-O-methyl
• 3’ inverted dT
• 40kDa PEG
Phase II/III
REG-1
anti-
coagulation
system
(aptamer +
antidote)
Factor
IXa
Acute coronary
system
Percutaneous
coronary
intervention
RNA • 2-ribo purine, 2-
F (aptamer)
• 40kDa PEG
(antidote)
Phase III
(halted)
NOX-E36 CCL2 Diabetic
Nephropathy
Oncology
Spiegelmer • L-ribonucleic
acid
• PEGylated
Phase II
NOX A12 CXCL12 Pancreatic and
Colon carcinoma
Glioblastoma
Multiple
Myeloma
Chronic
Lymphoblastic
Leukemia
Spiegelmer • L-ribonucleic
acid
• PEGylated
Phase II
Phase II/III
Phase II/III
Phase II
Table 1-4. Clinical development of aptamers. Shown is a list of aptamers currently in Phase
trials, including the type of aptamer and any post-synthetic modifications.
33
1.6.5 Types of Aptamers: RNA vs DNA
A majority of the clinically and pre-clinically developed aptamers are RNA-based agents. RNA
sequences in nature are well-known to have intrinsic tertiary structure and act as functional ligands
(riboswitches) or catalyze reactions (ribozymes), naturally suggesting a usefulness for the in-vitro
selection of aptamers (Tuerk & Gold, 1990; Wakeman, Winkler, & Dann, 2007). However, the
therapeutic use of RNA oligonucleotides does present practical limitations. First, the chemical
synthesis of RNA oligomers remains relatively challenging relative to the ease of synthesis of
DNA oligonucleotides. Second, and more critically, RNA is inherently unstable, being susceptible
to enzymatic hydrolysis by nucleases. To address this limitation, RNA aptamers derived with
therapeutic intent require post-SELEX substitutions of ribonucleotides on the backbone with
nuclease resistant 2’-amino, 2’-fluoro, or 2’-O-alkyl nucleotides analogues, a process which
requires either a prior knowledge the protein:nucleotide interaction, or substantial additional
exploratory experimentation (Dollins, Nair, & Sullenger, 2008; Zhou & Rossi, 2017).
As is the case with RNA, single-stranded DNA sequences also have the ability to fold into unique
tertiary structures. In nature, this phenomenon is exemplified in the guanine-rich protein-binding
sequences which form stable G-quadruplex structures. In contrast to RNA, the use of DNA as an
aptamer backbone carries a number of advantages. As mentioned above, chemical synthesis of
DNA is an optimized cost-effective process, allowing for inexpensive scalable synthesis of
aptamers. Further, DNA sequences are not as susceptible to nuclease degradation and thus do not
require the use of modified bases (Orava, Cicmil, & Gariépy, 2010). Ultimately, while RNA
aptamers may have a theoretical benefit in terms of higher structural diversity, DNA aptamer have
improved properties for their therapeutic use including increased stability and lower costs.
1.6.6 Types of Aptamers: Novel Nucleotides
Recent advances in aptamer technology have led to the derivation of novel nucleotide-based
aptamers such as Spiegelmers and SOMAmers. In contrast to DNA or RNA aptamers which are
synthesized with the naturally occurring D-stereoisomers, Spiegelmers are built on backbone of
the ‘mirror image’ L-form chiral nucleotides (Eulberg & Klussmann, 2003). This change imparts
substantial resistance from plasma-nucleases as these enzymes are stereospecific. However, as the
enzymes used in the selection process (PCR polymerase and reverse transcriptase) similarly do not
catalyze reactions with these nucleotides, the selection process requires a L-mirror imaged target
34
to select for with D-form nucleotide RNA. Post SELEX, the identified aptamers are synthesized
using L-form nucleotide analogues and assayed for activity on the desired target of normal
chirality. As the generation of proteins with L-configurations is challenging, this approach is
restricted to selecting against small peptides which can be chemically synthesized (Hoffmann,
Hoos, Klussmann, & Vonhoff, 2011; Vater & Klussmann, 2003). Currently, there are two
Spiegelmers in Phase trials, NOX-A12 and NOX-E36, which target the chemokines CXCL12 and
CCL2 respectively (NCT01486797, NCT01547897).
SOMAmers (slow off-rate modified aptamers) are a recently-described aptamer class with exciting
potential. In contrast to DNA or RNA aptamers, SOMAmers incorporate novel synthetic
nucleotides in the SELEX library which contain unique chemical moieties at the 5-carbon position
(ie. benzyl, 2-napthyl, or 3-indoyl-carboxmide) (Rohloff et al., 2014). Critically, these modified
bases do not interfere with enzymes involved in the selection process. The incorporation of these
nucleotides increases the structural diversity of the aptamer library, which when combined with a
modified SELEX method, leads to the derivatization of aptamers with improved affinity.
Moreover, the incorporated nucleotides do impart nuclease resistance and plasma-stability.
SOMAmers are currently commercially developed by SomaLogic, and are available to several
targets as part of proteomic diagnostic platforms (Webber et al., 2014; Wu, Katilius, Olivas,
Dumont Milutinovic, & Walt, 2016). As for their therapeutic utility, SomaLogic has developed an
anti-IL6 SOMAmer (SL1025) which binds to IL-6 with extreme affinity (KD= 0.2nM) and
antagonizes its interactions with the IL6 receptor (Gupta et al., 2014). In a pre-clinical model,
treatment of cynomolgus monkeys with a PEGylated conjugate of SL1025 delayed the onset and
reduced symptoms of CIA (Hirota et al., 2016). Importantly, the administration of this SOMAmer
over the course of 11 days did not engender an immunogenic reaction (ie. absence of anti-SL1025
antibodies). The positive results reported with this unique SOMAmer in a non-human primate
model highlight the therapeutic potential that this aptamer class may possess moving forward.
1.6.7 Immunomodulatory Aptamers
Due to their unique advantages relative to mAbs and other biologicals, functional aptamers
towards the immune co-stimulatory and co-inhibitory receptors have been proposed as novel
therapeutic agents (Dollins, Nair, & Sullenger, 2008; Gilboa et al., 2013). Past work, by our lab
and others, has led to the derivation of functional aptamers which modulate immunity by targeting
35
co-stimulatory receptors or co-inhibitory receptors (Table 1-5). The first checkpoint targeting
aptamer was reported in 2003 by Santulli-Marotto et al., against CTLA-4. This anti-CTLA-4 RNA
aptamer significantly inhibited CTLA-4-mediation immunosuppression of T-cells in-vitro. A
multivalent version of this aptamer was constructed and shown to have improved activity in-vitro,
and enhanced the in-vivo development of anti-tumor immunity in the B16/F.10 melanoma mouse
model (Santulli-Marotto, Nair, Rusconi, Sullenger, & Gilboa, 2003). More recently, aptamers
derived to the checkpoint receptors TIM3 and PD-1 (detailed in Chapter 2) have similarly shown
the ability to provoke anti-tumor immune reactions (Hervas-Stubbs et al., 2016; Prodeus et al.,
2015). Importantly, in the cases of these three aptamers, their potency was comparable to that of
established mAbs, confirming the utility of these aptamers as useful checkpoint antagonists.
In terms of suppressing immune responses using oligonucleotide aptamers, several antagonistic
aptamers towards pro-inflammatory cytokines have shown potential in pre-clinical models,
including the anti-IL-6 SOMAmer described above (Hirota et al., 2016). Similarly, a PEGylated
anti-IL17A antagonistic aptamer inhibited the development of glucose-6-phosphate isomerase-
induced arthritis in mice (Ishiguro, Akiyama, Adachi, Inoue, & Nakamura, 2011). However, there
are no examples of aptamers which agonize checkpoint receptors to suppress immunity, with the
exception of our recent report on the development of CD200R1-specific agonistic aptamers which
can suppress acute inflammatory reactions in mouse models of allergy and transplant rejection
(Prodeus et al., 2014).
36
Target Type Species Function Outcome
Reference
CTLA-4 RNA Murine Antagonist Treatment in melanoma
model
(Santulli-
Marotto et
al., 2003)
TIM3 RNA Murine Antagonist Treatment in colon
carcinoma model in
combination with PD-L1
blockade
(Hervas-
Stubbs et al.,
2016)
PD-1 DNA Murine Antagonist Treatment in colon
carcinoma model
Prodeus et al.
(2015) &
Chapter 2
4-1BB RNA Murine Agonist Treatment in mastocytoma
tumor model
(McNamara
et al., 2008)
CD28 RNA Murine Agonist Vaccine adjuvant in a B-
cell lymphoma model
(Pastor et al.,
2013)
OX-40 RNA Murine Agonist Adjuvant to DC vaccine in
melanoma model
(Dollins,
Nair,
Boczkowski,
et al., 2008)
OX-40 RNA Human Agonist In-vitro proliferation of
human T-cells
(Pratico,
Sullenger, &
Nair, 2013)
CD200R1 DNA Murine Agonist Prolongation of allograft
rejection
Prodeus et al.
(2014) &
Chapter 3
CD200R1 DNA Cross-
species
(human/
mouse)
Agonist Prolongation of allograft
rejection
Suppression of airway
hyperresponsiveness in
acute HDM-allergy model.
Chapter 3
Table 1-5. Pre-clinical development of functional aptamers targeting immune co-stimulatory
or co-inhibitory receptors. Shown are immune receptor targets to which aptamers have been
derived to act as either agonist or antagonists, along with outcome in pre-clinical rodent models.
Acronyms: HDM (House Dust mite), DC (Dendritic Cell)
37
1.7 Thesis Overview
1.7.1 Objectives and Hypotheses
Therapeutically targeting the immune checkpoint receptors with mAbs has shown remarkable
clinical activity, particularly in the ability to antagonize the inhibitory signals which contribute to
immune evasion and provoke clinically effective anti-tumor immune responses. Recent research
has also suggested utility in agonizing these pathways to suppress immune responses in
autoimmune or inflammatory disorders. The overall objective of this thesis was to derive novel
therapeutics, particularly DNA aptamers or novel non-mAb protein-based fusions, which target
the immune checkpoints to either antagonize and agonize immune-inhibitory signaling pathways.
Experimentally, my thesis focused on (i) deriving novel binding ligands targeting negative
checkpoint receptors, (ii) assaying each ligand for agonistic or antagonistic activity using in-vitro
cellular assays, (iii) modifying leads for in-vivo experimentation and (iv) testing each ligand in
relevant tumor or inflammatory mouse models.
The specific conclusions of my thesis are as follows:
1. DNA aptamers can be derived to target and antagonize the PD-1/PD-L1 pathway to
promote anti-tumor immunity.
2. DNA aptamers can be identified which bind CD200R1 and agonize immunoinhibitory
signaling to dampen inflammatory responses in pre-clinical models
3. A soluble agonist targeting the VISTA-receptor can be derived to suppress T-cell mediated
immune responses in-vitro and in-vivo.
38
1.7.2 Specific Aims
To test these hypotheses, the following aims were defined
1. Perform SELEX screens to identify panels of DNA aptamers which bind to the checkpoint
receptors PD-1 and CD200R1.
2. Chemically conjugate linear PEG moieties to the 5’ termini of PD-1 and CD200R1 DNA
aptamers with a view to increase their in-vivo therapeutic activity by extending circulatory half-
life.
3. Identify and characterize from Aims 1 and 2, the anti-PD-1 DNA aptamers in terms of their
ability to block PD-1:PD-L1 interaction and to release in-vivo anti-tumor immune responses in
comparison to a known anti PD-1 antibody.
4. Identify and characterize from Aims 1 and 2, CD200R1-specific aptamers which agonize
immune-inhibitory signaling pathways to reduce inflammatory responses in mouse models of
transplant rejection and allergy.
5. Incorporate NGS technology into SELEX screens to improve aptamer selection to identify
rare cross-species (mouse/human) CD200R1 binding aptamers
6. Design and formulate a novel protein therapeutic based on the VISTA IgV-like domain
which targets the putative VISTA-receptor.
7. Evaluate this VISTA-IgV construct in terms of ability to suppress T-cell activation in-vitro,
and regulate acute inflammatory reactions in-vivo.
39
1.7.3 Chapter Overviews
The studies addressing the aforementioned hypotheses and aims are described in Chapters 2
through 4 and depicted in Figure 1-9.
Chapter 2 describes the selection and identification of a DNA aptamer which binds to PD-1, and
functionally blocks the PD-1:PD-L1 interaction to inhibit PD-L1-mediated suppression of T-cells.
This study also demonstrated that a PEGylated version of this aptamer could inhibit the progressive
growth of PD-L1+ tumors in a MC38 colon carcinoma model with efficacy equivalent to that of a
validated anti-PD-1 mAb.
Chapter 3 details the derivation and characterization of DNA aptamers which bind to CD200R1
and act as agonists in triggering inhibitory signal in immune cells. Murine-specific CD200R1
aptamers induced phosphorylation of the C-terminal tail of CD200R1 to initiate the signaling
cascade, suppressed CTL induction in in-vitro allogeneic mixed leukocyte cultures (allo-MLCs),
and when PEGylated, prolonged the survival of MHC-mismatched skin grafts in a model of
transplant rejection to comparable levels as CD200-Fc. Further, NGS analysis of SELEX
independent screens against human and mouse CD200R1 led to the discovery of a cross-species
CD200R1 aptamer agonist, termed CCS13, which has similar features to the murine specific
aptamers. Importantly, this cross-species aptamer also suppressed CTL induction in allo-MLCs
derived from human PBMCs, and could suppress airway hyper-responsiveness in a mouse HDM-
induced allergy model.
Chapter 4 entails the recent derivation of an engineered pentameric VISTA-IgV construct, termed
VISTA.COMP, which targets and agonizes a T cell-mediated immune inhibitory pathway through
the so far undiscovered VISTA-receptor. Notably, the VISTA-receptor cannot be targeted by
traditional mAbs or aptamers due to its unknown identity. VISTA.COMP readily binds to T-cells
and functionally inhibits their activation in-vitro. Furthermore, VISTA.COMP treatment in mice
suppressed acute inflammatory responses models of transplant rejection and acute hepatitis.
Chapter 5 is a summary of my overall conclusions. The chapter also covers work-in-progress and
a description of what I propose as future aims.
40
Figure 1-9. Chapter Overviews. Visual representation of Chapters 2, 3, and 4. Chapter 2
involves the derivation of antagonistic anti-PD-1 DNA aptamers which provoke anti-tumor
immunity. Chapter 3 details the identification of anti-CD200R1 aptamers which agonize this
immunoinhibitory signaling pathway to dampen immune responses. Lastly Chapter 4 highlights
the engineering of a multimeric VISTA-IgV construct, which binds to T-cells and suppresses
their activation through agonizing the putative VISTA-receptor.
41
42
Chapter 2
Targeting the PD-1/PD-L1 Immune Evasion Axis with DNA aptamers
as a Novel Therapeutic Strategy for the Treatment
of Disseminated Cancers
Reformatted from a similar report published as:
Targeting the PD-1/PD-L1 immune evasion axis with DNA aptamers as a novel therapeutic
strategy for the treatment of disseminated cancers
Aaron Prodeus, Aws Abdul-Wahid, Nicholas W. Fischer, Eric H.-B. Huang,
Marzena Cydzik and Jean Gariépy
Molecular Therapy: Nucleic Acids. 2015; 4e237
Contributions:
AP performed experiments, analyzed data, and wrote the chapter. AP and JG conceptualized the
study. AAW, NWF and EHBH assisted with in-vivo experiments. MC assisted with aptamer
PEGylation. JG supervised the project.
43
Targeting the PD-1/PD-L1 Immune Evasion Axis With DNA Aptamers as a Novel Therapeutic strategy for the Treatment of Disseminated Cancers
2.1 Abstract
Blocking the immunoinhibitory PD-1:PD-L1 pathway using monoclonal antibodies has led to
dramatic clinical responses by reversing tumor immune evasion and provoking robust and durable
anti-tumor responses. As a result, anti-PD-1 antibodies have now been approved for the treatment
of advanced melanoma, non-small cell lung cancer (NSCLC), non-Hodgkin’s lymphoma, and
bladder cancers, and are being clinically tested in a number of other tumor types as both a
monotherapy and as part of combination regimens. Here, we report the development of DNA
aptamers as synthetic, non-immunogenic anti-PD-1 antibody mimics, which bind specifically to
the murine extracellular domain of PD-1 and block the PD-1:PD-L1 interaction. One such aptamer,
MP7, functionally inhibits PD-L1-mediated suppression of IL-2 secretion in primary T-cells. A
PEGylated form of MP7 retains the ability to block the PD-1:PD-L1 interaction, and significantly
suppresses the growth of PD-L1+ colon carcinoma cells in-vivo with effectiveness equivalent to
an antagonistic anti-PD-1 antibody. Importantly, the anti-PD-1 DNA aptamer treatment was not
associated with off-target TLR-9-related immune responses. Due to the inherent advantages of
aptamers including their lack of immunogenicity, low cost, long shelf life, and ease of synthesis,
PD-1 specific antagonistic aptamers may represent an attractive alternative over antibody-based
anti-PD-1 therapeutics.
2.2 Introduction
Cancer cells evade immune surveillance through multiple mechanisms, including increased
secretion of immunosuppressive cytokines (i.e. IL-10 and TGF-β), reduced expression of major
histocompatibility antigens (MHC) on their cell surface, enhanced differentiation of immune
effector cells to a regulatory phenotype, as well as an influx of myeloid-derived suppressor cells
and tumor associated macrophages (Drake et al., 2006). Importantly, tumors also suppress immune
responses by activating immune inhibitory checkpoint pathways. These pathways involve co-
inhibitory ligands (such as PD-L1, B7-H4, VISTA) expressed by cells within the tumor
44
microenviroment (TME) which bind to paired co-inhibitory receptors on infiltrating tumor-
associated antigen (TAA)-specific T-cells to suppress their anti-tumor activities (Ceeraz, Nowak,
& Noelle, 2013; Mahoney et al., 2015; Pardoll, 2012). The combined action of all these
mechanisms can result in the creation of a highly immunosuppressive TME that explains in part
the lack of clinical effectiveness of present-day cancer vaccines aimed at generating TAA-specific
T cell responses (Yaddanapudi, Mitchell, & Eaton, 2013).
One key immune inhibitory pathway involves PD-1, expressed on T-cells, interacting with either
PD-L1 or PD-L2 (Freeman et al., 2000; Latchman et al., 2001). While PD-L2 expression is limited
to cells of the hematopoietic lineage, PD-L1 is inducibly expressed on most tissues acting as a key
player in maintaining peripheral tolerance (Francisco et al., 2010). In solid cancers, PD-L1 is often
adaptively expressed by tumor cells and/or other cells within the TME in response to IFNγ or other
pro-inflammatory cytokines (Juneja et al., 2017; Sanmamed & Chen, 2014). In terms of its
signaling pathway, the engagement of PD-1 on T-cells by its ligands signals for the recruitment of
SHP-2 to the phosphorylated tyrosine of the ITIM motif within the PD-1 cytoplasmic tail
(Yokosuka et al., 2012). The recruited SHP2 functions to dephosphorylate key signals transduced
by the T-cell receptor (TCR) or CD28 pathways, thereby inducing T-cell anergy, exhaustion, or
differentiation to a regulatory phenotype. In-vitro PD-1/PD-L1 signaling suppresses events
associated with T-cell activation including the proliferation and secretion of pro-inflammatory
cytokines (ie. IL-2 and IFNγ) (Francisco et al., 2009; Freeman et al., 2000).
PD-1:PD-L1 blockade by monoclonal antibodies has proven to be an effective modality to
overcome immune evasion and mobilize specific anti-tumor responses in both mouse models and
clinically in patients (Callahan & Wolchok, 2013; Hirano et al., 2005). For instance, recent results
from Phase trial reported a 38% objective response rate in advanced melanoma patients (n=135)
treated with an anti-PD-1 antibody lambrolizumab (Keytruda, MK-3475)(Hamid et al., 2013).
Furthermore, a 15-25% objective response rate has been documented in an anti-PD-1 monotherapy
phase trial in NSCLC patients, a tumor type previously thought to be unresponsive to immune
therapy(Topalian et al., 2012). Currently, anti-PD-1 therapy is approved for the treatment of
several cancers including melanoma, NSCLC, lymphoma and bladder. Looking forward, over 50
clinical trials are currently assessing the potential use of anti-PD-1 antibodies (as a single agent or
in a combination setting) across greater than 25 tumor types.
45
Despite the clear success of these therapeutics, there are a number of disadvantages associated the
long-term use of monoclonal antibodies: such as their immunogenicity (following repeated
administration), and high production costs, which may limit their use and broad availability to
patients (Chames et al., 2009; Harding, Stickler, Razo, & DuBridge, 2010; Nelson et al., 2010). A
potential alternative to antibodies are oligonucleotide aptamers. Aptamers are short single-
stranded synthetic oligonucleotides (DNA or RNA), which bind molecular targets with
comparable affinity and specificity to that of antibodies. Importantly, DNA aptamers have been
shown to lack immunogenicity, and are synthesized using scalable, low-cost solid-phase
chemistry, avoiding the requirement of biological cultivation and purification required for protein-
based therapeutics (Keefe et al., 2010; White, Sullenger, & Rusconi, 2000). Thus, DNA aptamers
which target and antagonize PD-1/PD-L binding, may represent novel therapeutic entities with
improved characteristics relative to anti-PD-1 monoclonal antibodies. In this study, we report as a
proof of principle the development of DNA aptamers which bind specifically to the extracellular
domain of murine PD-1 with nanomolar affinity. We further characterize an anti-PD-1 aptamer,
termed MP7, which functionally blocks PD-1 from interacting with PD-L1 and suppresses the
growth of a PD-L1 positive murine colon carcinoma in-vivo.
2.3 Methods
2.3.1 Mice and Tumor Cell Line
C57BL/6 mice (6–12 weeks old) were kept under pathogen-free conditions at the Sunnybrook
Health Sciences Center Comparative Research Animal facility. All experiments were performed
under the approval of the local animal welfare committee in accordance with the rules and
regulations of the Canadian Council for Animal Care. The murine colon carcinoma cell line MC38
expressing the human carcinoembryonic antigen (MC38.CEA) was a kind gift from Dr. Jeffrey
Schlom (National Cancer Institute, Bethesda, Maryland).
2.3.2 Fusion Proteins and Monoclonal Antibodies
A chimeric protein consisting of the murine PD-1 extracellular domain fused to a human IgG
constant region with a C-terminal histidine tag (mPD-1.FcHIS) was purchased from Sino
Biological Inc (Beijing, China) and used for aptamer selection and in-vitro experiments.
46
Recombinant purified hPD-1.Fc and mPD-L1.Fc were purchased from R&D Systems
(Minneapolis, MN). A blocking anti-mPD-1 antibody (clone RMPI-14) and isotype control 2A3
were purchased from BioXcell (West Lebanon, NH). Flow cytometry was performed with APC
labelled anti-mPD-L1 antibody (clone 10F.9G2, BioLegend, San Diego, CA), FITC labelled anti-
CEACAM5 antibody (clone C365D3 (NCRC23), Cedarlane, Canada), FITC labelled anti-PD-1
antibody (clone 29F.1A12, BioLegend), or FITC-labelled RMPI-14 anti-PD-1 monoclonal
antibody. Histidine-tagged CEA-N domain (CEA-N.HIS) was expressed and purified as
previously described.
2.3.3 DNA Aptamer Selection
Single-stranded DNA aptamers recognizing murine PD-1 were identified using the in-vitro
SELEX method (Ellington & Szostak, 1992; Tuerk & Gold, 1990). A randomized ssDNA library
constructed to have a 25 nucleotide (nt) internal variable sequence flanked by 25nt 5' and 3'
constant primer regions (5’-GACGATAGCGGTGACGGCACAGACGNNNNNNNNNN
NNNNNNNNNNNNNNCGTATGCCGCTTCCGTCCGTCGCTC-3’) was synthesized by
Integrated DNA Technologies. A 4nmol aliquot of this library, representing ~2.5x1015 DNA
sequences, was counter-selected three times against MagneHis Ni-Particles (Promega, Madison,
WI) at 37oC for 1hr in selection buffer (10mM HEPES, 150mM NaCl, 0.05% Tween-20, 1mM
MgCl2, 1mM CaCl2) followed by counter selection to an irrelevant human IgG immobilized onto
Protein A beads (Life Technologies, Burlington, ON). Subsequently, the counter-selected library
was incubated with recombinant mPD-1.FcHIS immobilized on the Ni-Particles for 1hr at 37oC in
binding buffer. After three washes, bound aptamers were eluted by denaturation at 95oC for 5min.
The enriched library was amplified for the next round of SELEX by asymmetric PCR using a 20:1
forward: reverse primer ratio. Five rounds of SELEX were carried forth as described above, each
round consisting of counter selection to both the MagneHIS particles and human IgG before
positive selection to mPD-1.FcHIS. Selection stringency was increased throughout SELEX by
halving the concentration of mPD-1.FcHIS from 10µg/cycle during rounds 1-3 to 5µg/cycle in the
remaining rounds, and by increasing wash time from 10 minutes total to 15 minutes after cycle 2.
47
2.3.4 Next Generation Sequencing and Data Analysis
To identify PD-1 binding aptamers enriched after five rounds of selection, adaptor sequences were
ligated to the selected library using the Ion Plus Fragment Library Kit (Life Technologies) and
next-generation sequencing (NGS) performed using Ion Torrent PGM (SRI Genomics Core
Facility) with the Ion 314 chip (Life Technologies). Data was pre-processed using Galaxy and
analyzed using FASTAaptamer software (Alam et al., 2015).
2.3.5 Nitrocellulose Filter Binding Assay
The binding affinity of selected aptamers towards mPD-1.FcHIS was determined using a dual
membrane filter binding assay. 32P-labelled aptamers were incubated with serial dilutions of mPD-
1.FcHIS in selection buffer supplemented with 0.1% BSA at 37oC for 1 hour. Complexes were
filtered through a double membrane system, containing an upper nitrocellulose layer, and lower
nylon filter. The protein-bound aptamers adsorbed to the nitrocellulose membrane and unbound
aptamers captured on the lower nylon membrane. Membranes were exposed to X-ray film for 16
hours at room temperature, developed, and radiolabel signal quantified using ImageJ software.
2.3.6 Aptamer Flow Cytometry
Fluorescently labelled aptamers MP7, MP5, and cSeq were synthesized with a 3’ terminal FAM
(IDT). 1x106 P815 mastocytoma cells were pre-incubated (blocked) in 100uL PBS supplemented
with 1% FBS, 0.09% NaN3, 1mM MgCl2, 1mM CaCl2, 100µg/mL salmon sperm DNA, 100µg/mL
yeast tRNA, and 1µg Fc blocker (Biolegend) for 15 minutes before addition of 2.5µM aptamer or
FITC-labelled antibody. After a 30-minute incubation at 37oC, cells were washed with PBS and
analyzed using a FACScalibur Cell Analyzer (BD Biosciences).
2.3.7 Surface Plasmon Resonance (SPR)
Aptamer specificity toward the extracellular domain of mPD-1 was evaluated by SPR-based
binding experiments using a Biacore T200 (GE Healthcare). Aptamers were synthesized with a 5’
biotin and immobilized onto separate flow cells of a streptavidin-coated sensor chip at a density
of 50-100RU. Binding analyses were performed by exposing each immobilized aptamer as well as
a blank reference flow cell to injections (120s, 30µL/min) of mPD-1.FcHIS (50nM), hPD-1.Fc
48
(50nM), mPD-L1.Fc (50nM), hIgG (500nM) or CEA-N.HIS (500nM) dissolved in HBS-P (10mM
HEPES pH 7.4, 150mM NaCl, 0.05% v/v Tween 20). Flow cells were regenerated before each
cycle by a 60s pulse with 10mM NaOH/1M NaCl followed by a 5-minute stabilization period.
2.3.8 IL-2 ELISPOT Assay
The antagonistic activity of mPD-1 aptamers was assessed by their ability to restore IL-2 secretion
by splenocytes which were under suppression by PD-L1 signaling. C57BL/6 splenocytes (2x105
cells/well) were cultured in microtiter wells pre-coated with anti-IL2 capture antibody, anti-CD3
antibody (1µg/mL) and either mPD-L1.Fc or an irrelevant hIgG-Fc isotype control (15µg/mL).
Anti-PD-1 aptamers (250nM, 1.15µg, 200µL) or antibody (125nM, 3.75µg, 200µL) were added
to the appropriate wells and the levels of secreted IL-2 was quantified after 48 hours of culture
using a biotinylated anti-IL-2 detection antibody and the ELISPOT Blue Color Module (R&D
Systems, Minneapolis, MN). After color development, spots were enumerated using an automated
Immunospot Analyzer (Cellular Technology Limited). Proliferation experiments were performed
by stimulating CFSE labelled splenocytes in microtiter plates coated as described above.
Splenocytes were labelled with 5µM CFSE as recommended by the manufacturer (Life
Technologies) and dilution profiles analyzed three days after culture using a FACScalibur Cell
Analyzer. When used, aptamers were present in culture at a final concentration of 1.25µM.
2.3.9 Competitive ELISA
The ability of anti-PD-1 aptamers or antibody to block the PD-1/PD-L1 interaction was evaluated
using a competitive ELISA. Recombinant mPD-L1.Fc (3µg/mL) pre-coated onto wells of a 96-
well microtiter plate was incubated with recombinant mPD-1.FcHIS (3µg/mL, 130nM) in
competition with aptamer (2.5µM) or antibody (1µM) for 1-hr. Bound mPD-1.FcHis was detected
using an anti-HIS HRP antibody followed by color development with TMB as the substrate. The
amount of mPD-1.FcHIS bound in each well was quantified using a standard curve generated from
a dilution series of mPD-1.FcHIS and used to determine the percent of PD-1 blocked by each
aptamer or antibody.
49
2.3.10 PEGylation of DNA Aptamers
DNA aptamers with a 5’ hexylamine modifier (IDT) were incubated with a 10-fold excess of a
40kDa mPEG-succinimidyl glutarate ester (NOF America, White Plains, NY) added drop wise
over 3 hours to a 0.5mM solution of aptamer dissolved in 0.1M NaHCO3/CH3CN (1:1, pH 9.0).
PEGylated aptamers were prepared in 0.1M TEAA and purified by reverse phase HPLC using a
C18 OST semi-preparative column (Waters, Milford, MA). The bound PEGylated aptamers were
eluted using a linear gradient going from 5-90% CH3CN over a 60-minute period.
2.3.11 MC38.CEA Tumor Challenge
MC38.CEA colon carcinoma cells (5x105 cells) were implanted intraperitoneally (i.p.) into
C57Bl/6 mice. Mice were subsequently treated i.p. with either PEGylated aptamers (40µg
oligonucleotide weight, 1.3nmol) or antibody (200µg, 1.3nmol) on days 1, 3, 5 and 7 following
tumor implantations. After 21 days, animals were sacrificed for necropsy and their tumor burden
examined by blinded investigators. Longitudinal diameters of tumor masses were measured using
calipers and a modified ellipsoid formula was used to determine tumor volume where
Tumor volume = ½ [(greatest longitudinal diameter) x (smaller longitudinal diameter) 2]
2.3.12 Aptamer Induced TLR9 Immune Responses
PEGylated aptamers (40µg, 1.3nmol) or a TLR9 ligand CpG ODN (ODN 1826, Invivogen, CA)
were administered i.p. into naïve C57BL/6 mice (n=3). Mice were sacrificed three hours later and
serum levels of TNFα and IL-6 quantified using commercial ELISA duosets (R&D Systems). For
in-vitro studies, RAW 264.7 mouse macrophages (ATCC) were treated with 3µM CpG ODN 1585
or PEG-MP7 for three hours. RNA was harvested using an RNeasy Mini Kit (Qiagen) and one-
step RT-PCR performed with optimized primers for IFNα, IL-12, and β-actin (Appendix 1) and
conditions following recommendations from the manufacturer.
2.3.13 Statistics
P-values for ELISPOT and ELISA assays were calculated using Student’s t-test or analysis of
variance (ANOVA) where appropriate. GraphPad PRISM 6 software was used to plot all data,
perform statistical analysis, and fit binding curves using a one site binding model.
50
2.4 Results
2.4.1 Identification of Murine PD-1 Binding DNA Aptamers
In-vitro based SELEX was performed to identify DNA aptamers which specifically recognize the
extracellular domain of murine PD-1 (mPD-1) by screening a random DNA library against a
recombinant chimera consisting of the mPD-1 ECD fused to a histidine-tagged human Fc domain
(hereafter referred to as mPD-1.FcHIS). Five rounds of enrichment were performed consisting of
a counter selection step towards an irrelevant isotype matched IgG, followed by a positive selection
step towards mPD-1.FcHIS. NGS was then performed on the enriched sub-library of DNA
oligonucleotides, revealing a significant selection towards aptamer families harboring sequences
with high sequence identity to a parental highly enriched sequence. Specifically, the two highest
enriched sequence families called MP5 and MP7 (Table 2-1) were observed to be present at 11.7%
and 13% respectively of the total sequences before a substantial decrease in enrichment (<1%) was
observed for the next highest present family member MP23. A pull-down experiment was
performed where biotinylated versions of these aptamers immobilized onto magnetic streptavidin
beads were used to pull-down mPD-1.FcHIS, confirming that these sequences were enriched from
the bulk library during selection due to their ability to bind mPD-1.FcHIS (Figure 2-1). A negative
control sequence (cSeq: CCTGTGTGAGCCTCCTAACCAGAACAGTTAAAGTGTCCCACT
CCCATGCTT ATTCTTGTCTCTC) did not bind to mPD-1.FcHIS.
2.4.2 Anti-PD-1 DNA Aptamers MP5 and MP7 Bind to mPD-1.FcHIS with
low Nanomolar Affinity
The highest enriched parental sequences MP5 and MP7 were chosen for further evaluation.
Structural predictions using Mfold Software (Zuker, 2003)on these aptamer sequences illustrate a
complex hairpin-bulge folding state (Figure 2-2A,C). Neither of these sequences contain a high
prevalence of GG repeats and are not predicted to fold into a G-quadruplex structure. A
nitrocellulose filter binding assay demonstrates that MP5 and MP7 bind to mPD-1.FcHIS in
solution with dissociation constants (KD) of 112 and 167 nmol/L respectively (Figure 2-2 C,D). In
contrast, the reverse complement of these aptamers (Rev-MP5 and Rev-MP7) did not bind mPD-
1.FcHIS.
51
Table 2-1. Variable regions of enriched anti-mPD-1 aptamer families identified after 6
rounds of SELEX. Deep sequencing analysis of round 5 mPD-1 SELEX libraries identified two
major families of aptamers: MP5 and MP7. a Shown sequence is of internal variable 25 nucleotides
excluding 5’ and 3’ constant sequences. b % enrichment calculated by sequence copy number/total
number of sequences x 100. c Dissociation constant (KD) of aptamers to mPD-1.FcHIS determined
using a nitrocellulose filter binding assay (see Figure 2-2).
Family Aptamer N25a Copies (%) b KD (nmol/l) c
MP5 5 GCTACTGTACATCACGCCTCTCCCC 7358 (6.9) 112
5.1 CTACTGTACATCACGCCTCTCCCC 2108 (2.0)
Total ---- 12,415 (11.7)
MP7 7 GTACAGTTCCCGTCCCTGCACTACA 11,858 (11.2) 167
7.1 GTACAGTTCCCGTCCTGCACTACA 434 (0.4)
Total ---- 13,814 (13.1)
Figure 2-1. Enriched aptamer sequences pull down mPD-1.FcHIS. Biotinylated versions of
MP5, MP7, and MP23 when immobilized onto streptavidin magnetic beads pulled-down mPD-
1.FcHIS. Pulled-down mPD-1.FcHIS was identified by western blotting using anti-HIS HRP
antibody. An irrelevant DNA sequence used as a negative control did not pull-down mPD-
1.FcHIS
52
A
C D
B
Figure 2-2. Secondary structure and binding affinity of highly enriched anti-PD-1 aptamer
sequences MP5 and MP7. Mfold secondary structure predictions of the top two enriched
aptamer sequences MP5 (A) and MP7 (C). Concentration-dependent binding of 32P-labeled
aptamers MP5 (B) and MP7 (D) to mPD-1.FcHIS in a nitrocellulose-nylon dual-filter binding
assay. Black circles represent the % DNA bound of the each aptamer at the mPD-1.FcHIS
concentration used. Open squares represent the % DNA Bound of the respective reverse
complement of each aptamer used as a negative binding control.
53
2.4.3 Anti-PD-1 DNA Aptamers Bind Specifically to the Extracellular Region of Murine PD-1
Mouse and human isoforms of PD-1 are highly homologous, sharing 67% amino acid identity
within their extracellular binding domain. Similarly, the IgV-like domains of these molecules share
significant structural homology with each other and other members of the CD28 superfamily
(Figure 2-3A,B) (Lázár-Molnár et al., 2008) . Furthermore, it has been demonstrated that cross-
species binding between mPD-1 and hPD-L1, or hPD-1 and mPD-L1 occurs with comparable
affinity as the same species interaction (Cheng et al., 2013). Yet, SPR-based binding studies
performed with aptamers MP5 and MP7 immobilized onto a solid sensor chip confirmed that both
aptamers bound with specificity towards mPD-1.FcHIS with no detectable binding to hPD-1.Fc
injected at the same concentration (50nmol/L) (Figure 2-3C). Importantly, these aptamers did not
bind to an isotype-matched hIgG (500nmol/L), the mPD-L1.Fc (50nmol/L) nor to the IgV-like N
domain of the carcinoembryonic antigen (CEA-N.HIS) (500nmol/L) confirming that both selected
aptamers bind specifically to the extracellular region of mPD-1 as opposed to binding the histidine
tag, the Fc region, or to structurally-related IgV-like domains. Additionally, fluorescently labelled
versions of MP5 and MP7 as well as two anti-PD-1 monoclonal antibodies, but not cSeq or isotype
control antibodies, readily bound to PD-1 expressed on the surface of P815 mouse mastocytoma
cells (Figure 2-4), confirming the ability of the anti-PD-1 aptamers to bind physiologically
expressed PD-1 on a cell surface.
2.4.4 The Anti-PD-1 Specific Aptamer MP7 blocks PD-1/PD-L1 Mediated Immunosuppression
An IL-2-based ELISPOT assay was performed to determine if aptamers MP5 or MP7 antagonize
PD-1/PD-L1 signaling in primary cells. In this assay, we found that PD-L1.Fc, but not an isotype
matched hIgG Fc control significantly suppresses IL-2 secretion by primary murine T-cells
stimulated with a polyclonal TCR-signal from plate bound anti-CD3 antibody (Figure 2-5A).
Addition of aptamer MP7 (250nmol/L) or a known blocking anti-PD-1 antibody (RMPI-14 mAb),
but not the cSeq nor MP5, significantly restored IL-2 secretion in the presence of PD-L1.Fc (Figure
2-5B). Importantly, the addition of MP7 to splenocytes stimulated with anti-CD3 in the absence
of PD-L1 did not increase the amount of IL-2 secretion above that of anti-CD3 stimulus alone
(Figure 2-6) suggesting that the increase in IL-2 in the presence of PD-L1 is due to the specific
54
blocking of the PD-1:PD-L1 inhibitory signal rather than off-target stimulation of T-cells. In
addition of IL-2 secretion, MP7 restored PD-L1 suppressed proliferation of anti-CD3 stimulated
lymphocytes was confirmed by CFSE dilution assay (Figure 2-7). These data support a hypothesis
that the anti-PD-1 aptamer MP7, but not MP5 acts to block the PD-1/PD-L1 pathway.
A C
B
Mouse PD-1 LEVPNGPWRSLTFYPAWLTVSEGANATFTCSLSNWSEDLMLNWNRLSPSNQTEKQAAFCN
Human PD-1 LDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPE
Consensus *: *: **.. ** ** *.*:** *******:** **.::*** *:******:* *** :
Mouse PD-1 GLSQPVQDARFQIIQLPNRHDFHMNILDTRRNDSGIYLCGAISLHPKAKIEESPGAELVV
Human PD-1 DRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRV
Consensus . *** **.**:: **** :****.:: :****** ******** ***:*:** *** *
Mouse PD-1 TERILETSTRYPS
Human PD-1 TERRAEVPTAHPS
Consensus *** *..* :**
Figure 2-3. Specificity of anti-PD-1 aptamers MP5 and MP7 towards the mPD-1
extracellular domain. (A) Crystal structure overlays highlighting similar Ig folds between the
mouse (blue, PDB ID: 1NPU) and human (grey, PDB ID: 3RRQ) PD-1 extracellular IgV-like
domain. (B) Aligned primary sequences share a 67% amino acid identity within the extracellular
regions of mouse and human PD-1. Grey highlights represent conserved residues. (C) SPR-based
binding responses of mPD-1.FcHIS, hPD-1.Fc, an irrelevant Fc control, histidine-tagged rCEA-
N domain, or mPD-L1.Fc towards immobilized MP5 (filled bars) or MP7 (empty bars).
Histograms represent mean binding response of duplicate injections ± SD (*P<0.001).
*
*
55
2.4.5 PEGylation of DNA Aptamers
Unmodified aptamers exhibit short in-vivo half-lives (<1 hour), primarily due to the rapid renal
filtration of these relatively small molecules (~8-25 kDa). To overcome this limitation, it has been
demonstrated that conjugation of aptamers to high molecular weight PEG groups can limit the rate
of filtration and extend their half-life up to 24-48 hours in mice and 5-7 days in non-human
primates (Haruta et al., 2017). Antagonistic anti-PD-1 DNA aptamers were thus PEGylated prior
to evaluating their in-vivo anti-tumor protective properties. Specifically, an amino group was
synthetically introduced at the 5’ termini of aptamers MP5, MP7 and cSeq (control aptamer) and
subsequently modified with a 40kDa PEG group. The conjugated aptamers were recovered by
reverse-phase HPLC (Figure 2-8A & Appendix 2).
Anti-PD-1 mAb
29F.1A12
Anti-PD-1 mAb
RMPI-14
MP5 MP7
PD-1
Isotype Control
cSeq
Figure 2-4 Aptamers MP5 and MP7 bind PD-1 expressing cells. P815 mouse mastocytoma
cells were stained with FAM-labelled aptamers or FITC-labelled antibodies. Histograms derived
from FACS analyses highlight binding aptamer or antibody tested (blue) relative to unstained
P815 cells (red). Both MP5, MP7, and two known anti-PD-1 antibodies bound P815 cells, while
an irrelevant aptamer and isotype matched antibody showed negligible staining.
56
2.4.6 PEGylated MP7 Directly Blocks PD-1 from Binding to PD-L1
The ability of the PEGylated and non-PEGylated aptamers to directly block the binding of PD-1
with PD-L1 was assessed using a competitive ELISA-based assay where the binding of soluble
mPD-1.FcHIS to immobilized mPD-L1.Fc is inhibited by the addition of aptamer. Consistent with
the IL-2 ELISPOT experiments, both PEG-MP7 and RMPI-14 mAb were able to significantly
block >75% of PD-1/PD-L1 binding in this assay confirming that aptamer MP7 functions as a PD-
1 antagonist (Figure 2-8B). In contrast, neither an isotype matched antibody nor PEG-MP5
inhibited PD-1 binding to PD-L1 while the cSeq weakly blocks ~20% of the interaction, a value
that is not statistically significant in comparison to wells where no aptamer
A B
Figure 2-5. Anti-PD-1 DNA aptamer MP7 antagonizes PD-1/PD-L1 mediated suppression
of IL-2 secretion in-vitro. (A) The effects of PD-L1.Fc or an isotype matched control Fc on IL-
2 secretion by splenocytes upon stimulation with an anti-CD3 antibody were measured using an
IL-2 ELISPOT assay. PD-L1.Fc (15µg/mL) reduced IL-2 SFU by 51% as compared to the Fc
control. Each bar represents the mean IL-2 spot forming units (SFU)/2x105 cells in at least three
replicate wells ± SEM. **P<0.05. (B) PBS, cSeq, anti-PD-1 aptamers (250nM) or an
antagonistic anti-PD-1 antibody (RMPI-14 mAb, 125nM) were added to splenocytes in wells
coated with anti-CD3 + PD-L1.Fc to monitor the blockage of PD-L1 mediated suppression of
IL-2 secretion. Data is represented as % Blockage of PD-L1 suppressed IL-2 Secretion where
IL-2 SFU values in wells without aptamer/antibody are set to 0% and IL-2 SFU counts in wells
supplemented with anti-PD-1 blocking antibody are set to 100% as this anti-PD-1 antibody
restored IL-2 secretion to levels 20% above that of anti-CD3 stimulation alone. Each histogram
bar represents the mean values ± SEM (n=5). NS= not significant.
57
was added. Notably, PEG-MP7 blocked the PD-1/PD-L1 interaction in this assay to a comparable
extent as unmodified MP7 (80% vs. 94%, Figure 2-9) indicating that PEGylation does not overtly
alter the structure or inhibit the antagonistic capabilities of this aptamer in-vitro.
2.4.7 PEGylated anti-PD-1 Aptamer Suppresses the Growth of Disseminated MC38.CEA Colon Carcinoma Cells
Release of PD-1:PD-L1 immunosuppression by anti-PD-1 or PD-L1 antibodies has been shown to
provoke remarkable anti-tumor responses to slow growth of tumors in both animal models and in
human clinical trials. To evaluate the ability of our anti-PD-1 aptamers to similarly promote in-
vivo anti-tumor responses, we used a mouse model where murine colon carcinoma MC38 cells
stably expressing the human CEA (MC38.CEA) as a heterologous antigen were administered
intraperitoneally to wild type C57Bl/6 mice. Consistent with previous studies using MC38 cells,
we found that MC38.CEA cells express low basal levels of PD-L1, which is upregulated 10-fold
upon stimulation with IFNγ (Figure 2-10) (Terawaki et al., 2011). After implantation, the mice
were treated with the PEGylated aptamers MP7 (n=5), cSeq (n=4), RMPI-14 mAb (n=5) or an
isotype matched irrelevant IgG (n=5) (Figure 2-11A). Consistent with our hypothesis, the PD-1
blockade using either the mAb or aptamer MP7 significantly suppressed tumor burden as measured
Figure 2-6. Anti-PD-1 DNA Aptamers do not stimulate IL-2 secretion in the absence of
PD-L1 signalling. Aptamers were added to primary splenocytes which were undergoing
polyclonal stimulation from plate-bound anti-CD3 antibody, and the changes in IL-2 secretion
monitored by ELISPOT. Histograms represent mean SFU ± SD (n=2), with no significant
change present between any anti-CD3 stimulated sample.
58
by the number of peritoneal nodules formed (Figure 2-11B,C) and the total cumulative volume of
all tumors within each animal (Figure 2-11D). Impressively, animals treated with PEG-MP7 (on
average 0.6 nodules/animal with 46mm2 cumulative volume/animal) displayed an equivalent or
higher anti-tumor efficacy as the RMPI-14 mAb (3.2 nodules/animal, 210 mm2 cumulative
volume). As expected, the injection of an irrelevant PEGylated oligonucleotide sequence (PEG-
cSeq) did not alter tumor progression. Notably, we did not observe any overt toxicity upon aptamer
treatment such as splenomegaly or organ hyperplasia in the liver or lymphoid organs. Furthermore,
in a similar but more aggressive tumor model where animals were fed a higher fat diet reaching an
endpoint within 14 days, PEG-MP7 significantly suppressed tumor growth when compared to
animals receiving buffer alone (PBS) or an anti-adhesive PEGylated DNA aptamer specific to
CEA PEG-N54; a DNA aptamer shown to block CEA-mediated, MC38.CEA implantation in the
peritoneal cavity (Figure 2-12A-D) (Orava, Jarvik, Shek, Sidhu, & Gariépy, 2013).
2.4.8 Aptamers Specific to PD-1 are not Cytotoxic and Do Not Trigger TLR9-Innate Immune Signaling
Experiments were carried out to confirm that the reduction in tumor growth in animals treated with
PEG-MP7 is due to its PD-1 antagonistic activity and not as a result of off-target effects. First, we
confirmed that aptamer MP7 was not cytotoxic towards MC38.CEA cells, thereby excluding the
possibility that reduction in tumor growth is a result of off-target aptamer-induced cytotoxicity
towards tumor cells (Figure 2-13A). Secondly, oligodeoxynucleotides (ODN) containing CpG
motifs have been shown to promote Th1 type immune responses by triggering innate immune
signaling through TLR9 expressed on human DC and B cells (Krieg, 2002; Krug et al., 2001). To
test whether MP7, which contains several CG repeats, triggers TLR9 signaling, PEG-MP7 or a
known TLR-9 agonistic CpG were injected in naive mice at a dosage which mirrored the amount
used in our tumor model. Immune response was monitored by quantifying the serum levels of
TNFα and IL-6. As expected, the CpG ODN significantly increased serum cytokine levels, while
PEG-MP7 did not induce cytokine production that could be detected, thus excluding the possibility
of this aptamer as a strong TLR9 agonist (Figure 2-13B). Furthermore, RT-PCR performed on
cultured RAW264.7 mouse macrophages treated with PEG-MP7 or a CpG ODN confirmed that
PEG-MP7 does not induce IFNα1 or IL-12 transcription in-vitro relative to a specific CpG ODN
(Figure 2-13). Taken together, our data suggest that that the anti-PD-1 DNA aptamer PEG-MP7
functions in-vivo as a specific PD-1 antagonist to promote strong anti-tumor immune responses.
59
A B
C D
Figure 2-7. MP7 restores PD-L1 suppressed lymphocyte proliferation. CFSE labelled
splenocytes were stimulated by plate bound anti-CD3 antibody in the presence or absence of PD-
L1 and CFSE dilution profiles analysed by flow cytometry. (A) Comparison of CFSE dilution
profiles from splenocytes cultured in anti-CD3 coated wells (2mg/ml), or wells with anti-CD3
and PD-L1.Fc (15µg/mL). Effect of anti-PD-1 aptamer MP7 (B) or MP5 (C) on PD-L1
suppressed proliferation. MP7, but not MP5, antagonized PD-L-mediated suppression of
lymphocyte cell division (CFSE-low%). (D) Graphical summary of experiment. Each histogram
represents the % of CFSE-low cells in each group of duplicate wells ± SD (*P<0.05 relative to
anti-CD3 + mPD-L1.Fc group).
60
% P
D-1
Blo
ck
ed
No A
ptam
er
Isoty
pe
PEG-M
P5
PEG-c
Seq
PEG
-MP7
RM
PI-1
4 m
Ab
A
B
Figure 2-8. PEGylated MP7 directly blocks PD-1/PD-L1 Binding. (A) Reaction scheme of
site specific aptamer conjugation to a 40kDa linear NHS-PEG at the 5’ terminus. (B) The ability
of PEGylated anti-PD-1 aptamers (PEG-MP5, PEG-MP7), PEG-cSeq, RPMI-14 mAb or an
isotype matched IgG control to inhibit the binding of soluble mPD-1.FcHIS to plate bound mPD-
L1.Fc was determined using a competitive ELISA. PEG-MP7, and the positive control anti-PD-
1 mAb antagonize the PD-1/PD-L1 interaction (P<0.01 relative to controls). Each histogram
represents the mean % of PD-1 blocked from binding PD-L1 ± SD (n=3).
61
Iso
typ
e C
on
tro
l
anti-CEA
Naïve + IFNy
an
ti-P
D-L
1
Figure 2-10. MC38.CEA cells express low levels of PD-L1 which is upregulated by IFNy
stimulation. Flow cytometry analysis MC38.CEA cells co-stained with FITC conjugated anti-
CEA antibody and APC-conjugated anti-PD-L1 antibody, confirming low basal PD-L1
expression which is upregulated by IFNγ stimulation (500units/mL, 24h).
Figure 2-9. PEGylation does not influence the ability of MP7 to block the PD-1:PD-L1
interaction. PEGylated and non-PEGylated MP7 similarly inhibit the binding of soluble mPD-
1.FcHIS from binding to plate-bound mPD-L1.Fc in a competitive ELISA experiment. Each
histogram represents the mean % PD-1 blocked from binding PD-L1 ± SD (n=3, *P<0.001
relative to no aptamer control group).
*
* *
62
Figure 2-11. PEGylated MP7 suppresses the growth of disseminated PD-L1+ colon
carcinoma MC38.CEA cells in-vivo. (A) Experimental outline of in-vivo tumor model.
MC38.CEA cells were injected i.p. into groups of C57Bl/6 mice (n=5; day 0) and animals were
subsequently treated with either RMPI-14 mAb, an isotype matched IgG (Isotype), PEG-MP7 or
PEG-cSeq on days 1,3,5 and 7 (B) Photographs highlighting tumor nodules found in the
intraperitoneal cavity of treated or control mice upon necropsy at day-21 post tumor implantation.
The total number of tumor nodules in each animal (C) and their cumulative tumor volume (D)
was quantified upon necropsy at day 21 to evaluate the anti-tumor effect of each PD-1:PD-L1
blocking agent. The antagonistic anti-PD-1 aptamer (PEG-MP7) and antibody (RMPI-14)
significantly inhibit the progress of MC38.CEA tumors relative to controls.
63
0 1 4 7
20µg Tx (i.p.)
Day
Experimental Groups
1. PBS 2. PEG-N54
3. PEG-MP7 4. RMPI-14 mAb
MC38.CEA
(i.p.)
14
A B
C D
PBS PEG-N54
PEG-MP7 RMPI-14
Figure 2-12. PEGylated MP7 suppresses growth of PD-L1+ MC38.CEA cells in a more
aggressive model. (A) Experimental outline of in-vivo tumor model. MC38.CEA cells were
injected i.p. into groups of C57Bl/6 mice (n=5; day 0) and groups of animals were subsequently
treated with either RMPI-14 mAb, PEG-MP7 or a CEA-specific aptamer PEG-N54 on days 1,
4, and 7. Animals fed a high-fat diet develop increased tumor nodules and growth relative to
standard diet (Figure 2-11), and reach end-point within 14 days. (B) Photographs highlighting
tumor burden in the intraperitoneal cavity of each group after 14 days. The total number of tumor
nodules (C) and cumulative volume of each tumor (D) in the intraperitoneal cavity of each
animal was quantified upon necropsy. Treatment with the anti-PD-1 aptamer MP7 or antibody
(RMPI-14) resulted in significant reductions in tumor burden. *P<0.05, ***P<0.001, NS=not
significant.
64
A
B C
Figure 2-13. Anti-PD-1 Aptamer PEG-MP7 is not cytotoxic to tumor cells and enhances
tumor-specific T-cell responses in-vivo without induction of a TLR9-mediated innate
immune response. (A) MTS cell viability assay was performed after a 24-hour incubation of
MC38.CEA cells with 5µM of PEG-MP7, PEG-cSeq, MP7, or 8M urea. Bars represent the
mean OD490nm ± SD from replicate wells. ***P<0.001. No aptamers exhibited direct cytotoxic
activity. Potential off-target TLR-9 signaling by DNA aptamers was assayed by injecting PEG-
cSeq, PEG-MP7, PBS, or a control CpG ODN into naive C57Bl/6 mice (n=3) followed 3 hours
later by quantifying the serum levels of TNFα (B) and IL-6 (C) by ELISA. Only the injection
of the CpG ODN yielded signal above background levels (dotted line). Each symbol represents
the average cytokine levels from each individual animal measured in duplicate.
65
2.5 Discussion
Immune responses are heavily regulated at the cellular level by a number of ligand:receptor
interactions which transduce either pro-inflammatory, co-stimulatory signals or negative co-
inhibitory signals. These orchestrated signals finely balance the immune system by provoking
appropriate responses to danger signals, while the co-inhibitory ligands, otherwise known as
immunological checkpoints, are key in promoting self-tolerance and preventing autoimmunity
(Francisco et al., 2010; Riella et al., 2012). However, malignant cancer cells often usurp these
checkpoint pathways to limit anti-tumor immune responses and evade immunological deletion
(Pardoll, 2012). Thus, blockade of these checkpoints by monoclonal antibodies has become a
viable therapeutic intervention to prevent evasion and provoke strong and durable anti-tumor
immune responses. The first in-class protein therapeutic approved for used in this manner was an
anti-CTLA-4 monoclonal antibody which functions by antagonizing the T-cell inhibitory signals
delivered through the CTLA-4:B7-1/B7-2 pathway resulting in increased activation of anti-tumor
T-cells (Hodi et al., 2016; Hodi et al., 2010). More recently, the therapeutic potential of blocking
the PD-1:PD-L1 checkpoint pathway has been realized. For instance, antagonistic anti-PD-1
IFNα
IL12
β-actin
CpG ODN
_ PEGMP7
Figure 2-14. PEGylated MP7 does not induce transcription of genes associated with TLR-9
signaling. Agarose gel electrophoresis analysis of IFNα, IL12, and β-actin RT-PCR reactions.
RNA was isolated from a mouse macrophage cell-line (RAW264.7) treated with CpG ODN 1585,
PEG-MP7, or left untreated (-). Only CpG treatment upregulated the levels of these TLR9
associated transcripts.
66
monoclonal antibodies have shown potent anti-tumor effect with objective response rates in 25-
50% of patients tested across various Phase trials (Hamid et al., 2013; Topalian et al., 2012).
Collectively, the preclinical and clinical data validate that blockade of the PD-1 inhibitory signal
delivered by either PD-L1+ tumor cells, APCs, and/or cells within the tumor microenvironment
can mobilize of powerful anti-tumor immune responses capable of eradicating tumor cells.
To date, all biotherapeutics targeting the PD-1:PD-L1 pathway are protein-based monoclonal
antibodies. Currently anti-PD-1 antibodies have been approved for several indications including
melanoma, NSCLC, and are being clinically validated in over 50 trials, across several cancer types,
and both as a monotherapy or in combination with other therapeutic agents or interventions.
However, as these protein-based biologics become widely used, there are key limitations which
may impact their effectiveness and use. First, despite advances in antibody design, the repeated
use of monoclonal antibodies (including humanized and fully human antibodies) can engender a
humoral immune response which may become problematic and limit therapeutic efficacy (Strand
et al., 2017). Another important limitation of these reagents as either a front-line therapy, or in
combination with other protein-based therapies – is the cost associated with manufacturing protein
biologics for human use (Chames et al., 2009; Nelson et al., 2010). Consequently, there is a need
for novel, preferably synthetic agents that can antagonize immune inhibitory pathways while being
cost-effective and non-immunogenic. In the present study, we report for the first time the
development of short synthetic anti-PD-1 DNA aptamers. In contrast to antibodies, aptamers have
been shown to be non-immunogenic, are readily synthesized in a scalable low-cost process with
minimal batch to batch variation, and are stable with a long shelf-life, all properties which
inherently make DNA aptamers attractive alternatives to antibodies.
As a proof-of principle, we have identified DNA aptamers using the in-vitro SELEX technique
that are specific to the murine extracellular domain of PD-1. By coupling our SELEX searches
with NGS, we have identified a number of highly enriched sequence families (Table 2-1), which
led to the identification of high affinity PD-1 binding ligands after 5 rounds of selection. The two
most highly enriched aptamers, termed MP5 and MP7, were synthesized and found to bind to
mPD-1.FcHIS with high affinity, and high specificity, with no detectable binding observed to
highly homologous proteins including human PD-1. In contrast to aptamer MP5 which did not
block PD-L1 induced signal despite its ability to bind PD-1, aptamer MP7 was found to inhibit
PD-L1 mediated immunosuppression (i.e. suppression of IL2 secretion in response to polyclonal
67
anti-CD3 stimulus) on primary T-cells, and was also able to antagonize the binding of PD-1 to PD-
L1 in a competitive ELISA. A major limitation for the use of aptamers as therapeutic entities is
their poor pharmacokinetic profiles, as these short DNA strands are rapidly removed from
circulation due to renal filtration or digested by serum exonucleases. However, the conjugation of
aptamers to high molecular weight polymers such as PEG has been reported to dramatically
increase their half-life by up to 24-48 hours in some cases (Haruta et al., 2017; Healy et al., 2004).
More recently a study demonstrated a significant enhancement of aptamer half-life in non-human
primates of up to 7 days upon aptamer conjugation to novel branched PEG structure (Haruta et al.,
2017). In this study, a 40kDa PEG moiety was attached to the 5’ amino end of MP7, the most
effective PD-1 antagonistic aptamer in our in-vitro studies, before evaluating its in-vivo potential.
In a syngeneic mouse model of disseminated tumors, PEGylated-MP7 was able to restore anti-
tumor immune responses and suppress or completely abrogate the growth of tumors. In this model,
we monitored the growth of PD-L1+ murine MC38 colon carcinoma cells in immunocompetent
mice where tumor growth is susceptible to PD-1 blockade. However, to improve the antigenic
immune response provoked by PD-1 blockade as a monotherapy, we used MC38 cells transfected
to stably express the human CEA (MC38.CEA cells) as a heterologous tumor antigen. Similar to
MC38 cells, the MC38.CEA cells retained low basal levels of PD-L1 expression which is
upregulated by inflammatory stimuli (i.e. IFNγ). Strikingly, we found that treatment of animals
with PEG-MP7 reduced tumor burden to a similar extent as a known antagonistic antibody. Lastly,
aptamer MP7 did not reduce tumor burden due to a direct cytotoxic effect towards tumor cells, nor
through an off-target TLR9 innate immune triggering.
2.6 Conclusions
In summary, we have shown that DNA aptamers directed at PD-1 are capable of antagonizing PD-
L1 signaling, and similar to antibodies, can release host anti-tumor immune responses which are
able to eradicate aggressive disseminated tumors. In addition to PD-1, we and others have
developed functional aptamers (acting as agonists or as antagonists) to other immune regulatory
molecules such as CTLA-4, 4-1BB, OX40, CD28, TNFα, and CD200R1 (McNamara et al., 2008;
Pastor et al., 2013; Prodeus et al., 2014; Santulli-Marotto et al., 2003, Orava et al. 2013). Future
studies are ongoing to identify human-specific anti-PD-1 aptamers and to determine and optimize
the in-vivo biodistribution and pharmacokinetic properties of these reagents.
68
69
Chapter 3
Agonistic CD200R1 DNA Aptamers are Potent Immunosuppressants
that Prolong Allogeneic Skin Graft Survival
Reformatted from similar reports published as:
Agonistic CD200R1 DNA Aptamers are Potent Immunosuppressants that Prolong
Allogeneic Skin Graft Survival
Aaron Prodeus, Marzena Cydzik, Aws Abdul-Wahid, Eric Huang, Ismat Khatri,
Reginald Gorczynski, and Jean Gariépy
Molecular Therapy: Nucleic Acids. 2015; 4e237
&
A synthetic cross-species CD200R1 agonist suppresses inflammatory immune responses
Aaron Prodeus, Amanda Sparkes, Nicholas W. Fischer, Marzena Cydzik, Eric Huang, Ismat
Khatri, Lindsay Woo, Chung Wai Chow, Reginald Gorczynski, and Jean Gariépy
Manuscript in revision for Molecular Therapy: Nucleic Acids
Contributions: AP, RG, JG conceptualized the study. AP performed all experiments unless
otherwise noted. EHBH & NWF performed the SELEX screens. RG performed allo-MLCs and
allograft studies. IK performed phosphorylation experiments. LW performed HDM-allergy
experiments under the supervision of CWC. MC assisted with aptamer PEGylation. AAW
assisted with TLR-9 experiment. AS assisted with editing of the Chapter. Supervision: JG
70
Agonistic CD200R1 DNA Aptamers are Potent Immunosuppressants that Prolong Allogeneic Skin Graft Survival
3.1 Abstract
CD200R1 expressed on the surface of myeloid and lymphoid cells delivers immune inhibitory
signals to modulate inflammation when engaged with its ligand CD200. Signaling through
CD200/CD200R1 has been implicated in a number of immune related diseases including allergy,
infection, cancer and transplantation, as well as several autoimmune disorders including arthritis,
systemic lupus erythematosus and multiple sclerosis. Here we report the development and
characterization of DNA aptamers which bind to murine CD200R1 and act as potent signaling
molecules in the absence of exogenous CD200. These agonistic aptamers suppress cytotoxic T-
lymphocyte induction (CTL) in allogeneic mixed leukocyte cultures (allo-MLCs) and induce rapid
phosphorylation of the CD200R1 cytoplasmic tail to trigger CD200R1-mediated immune-
inhibitory signaling. PEGylated conjugates of these aptamers show significant in-vivo
immunosuppression and enhance survival of allogeneic skin grafts as effectively as soluble
CD200-Fc. Additionally, by using Next-Generation-Sequencing (NGS) to cross-compare
independent SELEX screens against murine and human CD200R1, we identified a number of
putative cross-species anti-CD200R1 aptamers (i.e. aptamers that bind to mouse and human
CD200R1). One such aptamer, termed CCS13, was identified to suppress CTL induction in both
human and mouse allo-MLCs confirming its cross-species CD200R1 agonistic activity. Treatment
with PEGylated-CCS13 led to suppressed immune responses in the context of MHC-mismatch
(murine skin allograft) as well as in a model of acute house-dust-mite (HDM) allergic airway
inflammation. As DNA aptamers exhibit inherent advantages over conventional protein-based
therapeutics including low immunogenicity, ease of synthesis, low cost, and long shelf life, such
CD200R1 agonistic aptamers may emerge as useful and safe non-steroidal anti-inflammatory
therapeutic agents.
3.2 Introduction
Controlled modulation of the immune system using agonistic or antagonistic ligands targeting co-
stimulatory or co-inhibitory cell surface receptors offers great potential for the treatment of
diseases including allergy, infection, autoimmune disorders, transplantation, heart disease, and
71
cancer. One such receptor, CD200R1, a type I glycoprotein expressed on cells of myeloid and
lymphoid lineage, delivers immune inhibitory signals upon ligation to the widely-distributed cell
surface glycoprotein CD200 (Cherwinski et al., 2005; Jenmalm, Cherwinski, Bowman, Phillips,
& Sedgwick, 2006; Wright et al., 2003). Structurally, CD200R contains two Ig-like domains, a
transmembrane region, and a cytoplasmic tail containing a NXPY motif which is phosphorylated
upon CD200 ligation inducing the recruitment of the adaptor protein Dok2 and subsequent signal
transduction events (Mihrshahi et al., 2009; Zhang et al., 2004; Zhang & Phillips, 2006).
The physiological importance of CD200:CD200R1 inhibitory signaling has been established in a
number of diseases including arthritis (Simelyte et al., 2008), transplantation (Gorczynski et al.,
2009; Gorczynski, J. Hu, Z. Chen, Y. Kai, & J. Lei, 2002; Yu, Chen, & Gorczynski, 2013), as well
as central nervous system (CNS) autoimmune diseases such as Parkinson’s disease and multiple
sclerosis (Luo et al., 2010; X. J. Wang, Ye, Zhang, & Chen, 2007; Zhang et al., 2011). A
recombinant CD200-Fc fusion protein has been shown to behave as an in-vivo
immunosuppressant, prolonging allo- and xenograft survival as well as suppressing collagen-
induced arthritis (CIA) in mice(Gorczynski et al., 1999; Gorczynski et al., 2001; Gorczynski, J.
Hu, Z. Chen, Y. Kai, & J. Lei, 2002). Inhibition of CD200:CD200R1 signaling on microglial cells
using a blocking antibody to CD200R1 exacerbated neurodegeneration and disease state in a
murine model of experimental autoimmune encephalomyelitis (EAE) (Wright et al., 2000). These
findings were further supported in a separate EAE study where treatment with CD200-Fc
suppressed microglial accumulation, and decreased the production of pro-inflammatory cytokines
IL-6, TNF-α, and nitric oxide by myeloid cells in the spleen and central nervous system (Liu et al.,
2010). CD200R1 signaling has been implicated in tissue specific autoimmunity as well, as both
systemic and local treatment with an anti-CD200R1 agonistic antibody suppressed experimental
autoimmune uveitis (EAU), a model of CD4+ T-cell organ-specific autoimmunity of the eye
(Copland et al., 2007). Thus, the development of safe and effective immunomodulatory agents
which stimulate CD200R1 signaling are of clinical interest.
Aptamers are short single stranded nucleic acids (RNA or ssDNA) that can be readily developed
to bind a molecular target of interest with affinity and specificity features which compare well with
monoclonal antibodies. As in the case of antibodies, aptamers targeting cell-surface receptors can
either block protein-protein interactions or act as agonists, suggesting the use of such functional
aptamers as immunotherapeutic agents (Sullenger, White, & Rusconi, 2003). In contrast to
72
antibodies and other protein-based agents, aptamers have a number of advantages including a long
shelf life, low immunogenicity, and cost-effective scalable chemical synthesis (Gilboa et al., 2013;
Keefe et al., 2010). However, aptamers as therapeutic entities do display poor pharmacokinetic
profiles as RNA or DNA aptamers are rapidly removed from circulation due to renal filtration and
nuclease degradation. To address this limitation, it has been demonstrated that their
pharmacokinetic properties can be improved upon site-specific conjugation of polyethylene glycol
(PEG) polymers to aptamer termini to reduce renal filtration, as well as the incorporation of
nuclease resistant 2’-F or 2’-Me nucleotides (in the case of RNA aptamers) to impart nuclease
resistance (Healy et al., 2004; Keefe et al., 2010). Functional aptamers which target co-stimulatory
or co-inhibitory receptors represent a new class of targeted immunomodulatory agents with unique
and advantageous properties. Thus far, aptamers with either agonistic or antagonistic function have
been developed which target a number of immune receptors including CTLA-4, 4-1BB, OX-40,
IL-6R, IL-10R, and CD28 with only a few of them being validated for activity in-vivo (Berezhnoy
et al., 2012; Dollins, Nair, Boczkowski, et al., 2008; McNamara et al., 2008; Meyer et al., 2014;
Pastor et al., 2013; Pratico et al., 2013; Santulli-Marotto et al., 2003).
This chapter describes the development and characterization of two DNA aptamers which bind to
and agonize murine CD200R1 to induce immune-inhibitory signaling. These agonistic aptamers,
termed M49 and M52, exhibit in-vitro immunosuppressive properties as measured by their ability
to block CTL induction in allo-MLC. Importantly, PEGylated conjugates of these aptamers retain
their immunosuppressive function in-vivo as treatment with PEG-M49 and PEG-M52 prolongs the
survival of murine skin allografts to a similar extent as CD200-Fc. Additionally, this chapter
includes a summary of unpublished data relating to the discovery and characterization of a cross-
species aptamer, termed CCS13, which agonizes both human and mouse CD200R1 signaling. As
observed with the murine specific anti-CD200R1 aptamers (M49 and M52), PEGylated-CCS13
functions in-vivo to prolong allograft survival. Furthermore, using an acute HDM-induced allergy
model, we show that this therapeutically relevant aptamer has the potential to suppress allergy-
induced inflammation.
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3.3 Methods
3.3.1 Mice
C57BL/6 and BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor ME, USA).
CD200R1-deficient (CD200R1-/-) mice were generated as previously described (Boudakov et al.,
2007) and bred in house. Mice were housed at 5 per cage in an accredited facility at UHN, allowed
food and water ad libitum, and used at 8-12 weeks of age.
3.3.2 Aptamers and Recombinant Proteins
Unless otherwise specified, all aptamers used throughout this study were synthesized and purified
(HPLC-grade) by IDT (Coralville, Iowa). Recombinant full-length human and mouse CD200R1
containing an N-terminal histidine tag were purified from HEK-293 expressing cells using Ni-
NTA affinity resin. A CD200-Fc chimera containing the extracellular domain of CD200
genetically linked to the Fc region of mIgG2a mutated to have reduced complement and FcR
binding (Gorczynski, J. Hu, et al., 2002) was produced using the Expi-293 mammalian cell
expression system (ThermoFisher) according to manufacturer’s directions and purified to
homogeneity using FPLC (Akta) with Protein A HiTrap columns (GE Healthcare) (Appendix 3),
The control aptamer (cApt) used throughout these studies as a negative control shares the 5’ and
3’ region of our aptamers, but contains an irrelevant internal sequence (Listed in Figure 3-1A).
3.3.3 SELEX
Single stranded DNA aptamers (ssDNA) recognizing specifically murine or human CD200R1
were identified using the PCR-based Systematic Evolution of Ligands by Exponential Enrichment
(SELEX) method (Ellington & Szostak, 1992). Briefly, a 25 nucleotide long random synthetic
oligonucleotide library flanked by 25-base long 5' and 3' primer regions (5’-GACGATAGCG
GTGACGGCACAGACGNNNNNNNNNNNNNNNNNNNNNNNNNCGTATGCCGCTTCCG
TCCGTCGCTC-3’) was synthesized (IDT, Coralville, IA USA) along with the corresponding
primer sequences (Forward 5’-GACGATAGCGGTGA CGGCACAGACG-3’ and Reverse 5’
GAGCGACGGACGGAAGCGGCATACG-3’). A 4 nmol aliquot of the library representing 425
or ~ 1.1 x1015 sequences was adsorbed onto MagneHis Ni-Particles (Promega, Fitchburg WI,
USA) at 37oC for 1hr to remove sequences which bound to the solid support. The resulting sub-
library was incubated for 1hr at 37oC with 10µg of a recombinant HIS-tagged murine or human
74
CD200R1 protein immobilized on MagneHis Ni-Particles suspended in 1mL phosphate-buffered
saline (PBS, pH 7.4). Unbound and weakly bound sequences were removed by washing the beads
with PBS for 5 minutes and protein-aptamer complexes were eluted with PBS containing 0.5M
imidazole. Aptamers were recovered (Qiagen Nucleotide Removal kit) and the ssDNA pool
enriched for CD200R1 ligands was amplified for the next round of selection using asymmetric
PCR at a 10:1 forward: reverse primer ratio. Fifteen rounds of selection were performed with the
selection stringency increasing throughout by reducing the concentration of CD200R1 by a factor
of 2 every three rounds while simultaneously increasing the number of wash steps. To identify
murine CD200R1 targeting aptamers, after the 15th cycle, enriched sequences were cloned into
pCR4-TOPO vector (Life Technologies, Burlington, Canada) prior to Sanger sequencing.
3.3.4 Identifying Cross-species anti-CD200R1 Aptamers by Next-Generation-Sequencing (NGS)
DNA oligonucleotide sub-libraries enriched by SELEX for CD200R1 aptamers following 15
rounds of selection towards either mouse or human CD200R1 were sequenced using NGS as
previously described (Chapter 2). Briefly, the enriched sub-libraries recovered after round 15 were
ligated to indexed Ion Torrent adaptor sequences using the Ion Plus Fragment Library Kit
(ThermoFisher) and NGS performed using Ion Torrent PGM (SRI Genomics Core Facility) with
the Ion 314 chip. Data was pre-processed to remove sequences which did not contain the 5’ and
3’ constant regions, and ranked according to % of total read abundance (number of reads for a
unique sequence/total reads sequenced x 100). Sequences which ranked within the top 50 in each
mouse or human selection were manually cross-compared with that of the opposite species to
identify aptamers which shared an identical variable region.
3.3.5 Allogeneic Mouse Mixed Lymphocyte Cultures (allo-MLCs)
Agonistic CD200R1 aptamers were identified and evaluated for their ability to suppress the
induction of CTL in 5-day allo-MLCs. Briefly, 2.5x105 C57BL/6 responder splenocytes were
incubated with an equal number of irradiated BALB/c stimulator cells in the presence of synthetic
aptamers, PEGylated aptamers, or CD200-Fc for 5 days. Levels of CTL induction were assayed
by monitoring the release of 51Cr from loaded P815 mastocytoma target cells over a 5-hour time
period at a 25:1 effector-to-target ratio. Human allo-MLCs were similarly conducted with the
75
exception that human PBMCs isolated from healthy donors were used in place of mouse
splenocytes.
3.3.6 Aptamer:CD200R1 Nitrocellulose Filter Retention Analysis
The binding affinity of M49 and M52 towards CD200R1 was determined using a nitrocellulose
filter binding assay. DNA aptamers were enzymatically labelled at their 5’ end with 32P using T4
Polynucleotide Kinase (Life Technologies, Burlington, Canada). Radiolabeled aptamers were
purified using Micro Bio-Spin 6 desalting columns (Bio-Rad, Mississauga, Canada) and adjusted
to 5000CPM/µL in HBS (20mM HEPES, 150mM NaCl, pH 7.4) supplemented with 1mM MgCl2.
5uL aptamers were incubated with 15uL of increasing concentrations of murine CD200R1 (Sino
Biological Inc.) in HBS containing 1mM MgCl2 and 0.01% BSA at 37oC for 1 hour. After
incubation, complexes were filtered through a dual filter system with an upper 0.4um nitrocellulose
(Bio-Rad, Mississauga, Canada) and lower nylon Hybond N+ membrane (GE Healthcare,
Mississauga, Canada) using a 96 well dot-blot apparatus. Membranes were exposed to film for 16
hours at room temperature, developed, and radiolabel signal quantified using ImageJ software.
3.3.7 PEGylation of DNA Aptamers
The 5’ termini of aptamer M49, M52, and the control aptamer (cApt) (GACGATAGCGGTGAC
GGCACAGACGTCCCGCATCCTCCGCCGTGCCGACCCGTATGCCGCTTCCGTCCGTCG
CTC) were modified with a 20kDa PEG moiety to increase their circulatory half-life. Briefly, a 5’
hexylamino group was incorporated into each DNA aptamer during their synthesis. A 100-molar
excess of a mPEG-succinimidyl glutarate ester (Creative PEGWorks, Winston Salem, NC, USA)
was added stepwise over a period of 10 hours to 25µM solutions of the modified aptamers
dissolved in 100mM NaHC03/CH3CN (1:1 pH 8.5). The PEGylated aptamers were purified by
ultrafiltration using Amicon Ultra Centrifugal Filters with a 30kDa MWCO (Millipore, Billerica,
MA, USA) followed by size exclusion fast protein liquid chromatography (FPLC) using a
Superdex 75 10/300 column (GE Healthcare, Mississauga, Canada) with 100mM NH4CO3 as the
eluent. Purified PEGylated-aptamer conjugates were lyophilized and resuspended in sterile PBS
for subsequent experiments. PEGylated-CCS13 (PEG-CCS13) used in allograft studies was
synthesized as described for M49 and M52. PEG-CCS13 used in the HDM-induced allergy model
contained a 3’ terminal inverted dT to impart increased nuclease resistance[ref]. Large batches of
PEG-CCS13 and the PEGylated control aptamer (PEG-cApt) needed for in-vivo studies were
76
synthesized, PEGylated and purified [>90% purity as determined by RP-HPLC] by BioSpring
GmbH (Frankfurt, Germany).
3.3.8 Detection of CD200R1 Phosphorylation
Intracellular phosphorylation of CD200R1 in response to PEG-M49, PEG-M52, and CCS13 was
detected using a rabbit polyclonal antibody specific to the phosphorylated cytoplasmic tail of
CD200R1 (Khatri et al, 2012). HEK-293 cells stably expressing murine CD200R1 were serum-
starved in OptiMEM (Life Technologies, Burlington, Canada) medium for 3 hours and
subsequently incubated for 30min in OptiMEM medium containing either 2.5µM PEG-M49, PEG-
M52, PEG-cApt, CCS13 or a CD200 positive cell lysate (positive control). Cells were washed
with PBS and lysed in radioimmunoprecipitation (RIPA) buffer (150mM NaCl, 1.0% Igepal, 0.5%
sodium deoxycholate, 0.1% SDS, and 50mM Tris, pH 8.0) containing 50mM NaF, 1mM Na3VO4,
and protease inhibitors cocktail (Sigma). Total CD200R1 was recovered by immunoprecipitation
using an anti-CD200R1 (clone 2A10) monoclonal antibody (overnight 4oC) and Protein G agarose
beads (Pierce). The phosphorylated form of CD200R1 was detected by western blot using the
rabbit polyclonal antibody (1:1000 dilution) and anti-rabbit HRP (1:15,000 dilution).
3.3.9 Activity of PEGylated Aptamers in-vivo
C57BL/6 mice received BALB/c skin allografts followed by tail vein injections at Day 0, 3, 6, 9,
and 12 of the PEG-M49, PEG-M52, or PEG-cApt (650pmol, based on oligonucleotide content) or
CD200-Fc (325pmol) dissolved in 0.2mL PBS, pH 7.4 in combination with low dose (0.5mg/kg)
rapamycin administered intraperitoneally every 48 hours. On Day 14, mice were sacrificed and
their splenocytes used as responder cells in ex-vivo allo-MLCs with or without further aptamer
addition in-vitro.
3.3.10 Analysis of Aptamer-Induced Innate Immune Responses
PBS, PEGylated M49 or M52 (650pmol, based on oligonucleotide content) or a TLR9 ligand CpG
ODN (ODN 1826, Invivogen, CA) was administered by tail vein injection to naïve C57BL/6 mice
(n=3). Mice were sacrificed six hours later and their serum levels of TNFα or IL-6 quantified using
commercial ELISA sets (R&D Systems).
77
3.3.11 Allogeneic Skin Graft Transplantation
PEGylated anti-CD200R1 aptamers were evaluated for their ability to prolong survival of
allogeneic murine skin grafts. C57BL/6 mice (n=6) received BALB/c skin allografts prior to
receiving 6 tail vein injections of the anti-CD200R1 aptamers (650pmol) or CD200-Fc (325nmol)
dissolved in 0.2mL PBS, pH 7.4 once every 3 days over 15 days in combination with low dose
(0.5mg/kg) rapamycin administered intraperitoneally every 48 hours. Graft survival was
monitored daily.
3.3.12 House Dust Mice (HDM)-induced Allergic Airway Response
BALB/c mice were sensitized to HDM (Dermatophagoides pteronyssinus) allergen by repeat daily
intravenous injections on days 1-5 (100µg). Sensitized mice were challenged by HDM injection
(100µg) on day 12 and treated with PEG-CCS13, CD200-Fc, or saline. Airway respiratory system
resistance (Rrs) was measured using the flexiVent system (Scireq Inc. Montreal, Canada) as a
function of increasing doses of methacholine (MCh) as previously described (Salehi, Wang, Juvet,
Scott, & Chow, 2017).
3.3.13 Statistical Analysis
P-values for allograft survival analysis were determined using a Mann-Whitney U test while P-
values for all other experiments were calculated using a Student’s t-test.
3.4 Results
3.4.1 Generation of Murine CD200R1-specific DNA Aptamers Displaying Agonistic Signaling Properties
Over 15 DNA aptamer sequences specifically recognizing murine CD200R1 (mCD200R1) were
identified upon traditional Sanger sequencing of round 15 enriched libraries from a SELEX screen
towards recombinant mCD200R1. These 75-base long sequences along with a scrambled control
aptamer (cApt) (Figure 3-1A) were synthesized and systematically screened for CD200R1
agonistic activity by evaluating their ability to suppress the induction of CTL in a 5-day allo-MLC
assay. Aptamer-induced suppression of CTL induction was monitored using a chromium release
assay of loaded P815 mastocytoma cells serving as target cells for CTL lysis. Four aptamers termed
M21, M48, M49, and M52 (Figure 3-1A) displayed CD200R1 agonistic properties (Figure 3-1B).
78
The negative control aptamer (cApt) consisting of the identical 5’ and 3’ constant regions with an
irrelevant internal sequence did not show activity in this assay. Specifically, aptamers M49 and
M52 suppressed CTL induction at levels comparable to that of a soluble version of the natural
CD200R1 ligand (CD200-Fc) with less than 5% CTL specific lysis of P815 cells occurring at
aptamer concentrations ≥ 325nM. M49 and M52 were chosen for further evaluation. The binding
affinity of each aptamer to the mCD200R1 extracellular domain was derived using a nitrocellulose
filter retention assay. M49 bound to CD200R1 with a dissociation constant (KD) of 390nM ± 86
nM while the KD for M52 interacting with its target was estimated to be > 1µM; a value comparable
with the reported binding affinity of mCD200 to mCD200R1 of 4µM (Hatherley et al., 2005).
The 75-base long M49 and M52 aptamer sequences were truncated based on their predicted
secondary structure derived from mfold software (Figure 3-2A-C) (Zuker, 2003). M49 retained
agonistic activity when truncated to a minimal size of 55 nucleotides while M52 retained activity
down to a length of 44 nucleotides (Figure 3-2D).
3.4.2 PEGylation of M49 and M52
Conjugation of aptamers to high molecular mass polymers such as PEG has been shown to
dramatically improve their circulating half-lives from (Section 1.6.4). Thus, a 20kDa linear PEG
moiety carrying an N-hydroxysuccinimide ester was reacted with a 5’ amine arm introduced during
the synthesis of DNA aptamers M49, M52 and cApt (Figure 3-3A). The resulting PEGylated M49,
M52 and cApt conjugates were recovered by ultrafiltration and further purified by size exclusion
chromatography (Appendix 4).
3.4.3 PEGylated M49 and M52 Retain Agonistic CD200R1 Function
To ensure that the PEGylation of DNA aptamers M49 and M52 did not disrupt their folding and
CD200R1-agonistic activity, these modified aptamers were compared to unconjugated aptamers
for their ability to suppress CTL induction in an allo-MLC assay. Interestingly, both PEG-M49
and PEG-M52 suppressed CTL induction to a greater extent than M49 and M52 (Figure 3-3B)
confirming that PEGylation did not disrupt their immunosuppressive function.
79
A
B
Figure 3-1. DNA Aptamers selected to bind to the extracellular domain of murine CD200R1
suppress CTL induction in primary allo-MLC. (A) Subset of aptamer sequences identified
after 15 iterative SELEX rounds towards recombinant mCD200R1. Full length aptamers consist
of the depicted internal variable region, flanked by two constant regions of 25nt each. A control
aptamer (cApt) composed of a random variable sequence and the same constant regions present
in the library was used throughout this study. (B) To assay for CD200R1 induced
immunosuppression DNA aptamers were added to allo-MLCs consisting of C57BL/6 responder
cells incubated with an equal number of irradiated BALB/c stimulator cells. Induction of cytotoxic
T lymphocytes (CTLs) was monitored by the release of 51Cr from P815 mastocytoma target cells.
Soluble CD200-Fc was used as a positive control. Each bar represents the mean CTL specific
lysis value ± SEM from three replicate wells. Aptamers were added at 975nM (black bars), 325nM
(grey) or 110nM (white). M21, M43, M49 and M52 significantly suppressed CTL induction
(*P<0.01) when compared to cApt or no treatment.
80
M49
M49-T1
A
No A
ptam
er
cApt
M52
M52
-T1
M52
-T2
M49
M49
-T1
M52-T2 M52-T1
M52 C B
D
Figure 3-2. Truncation of CD200R1 aptamers M49 and M52. (A) Table of truncated M49 and
M52 aptamer sequences derived from secondary structure predictions. Shown is aptamer name,
sequence, and length in nucleotides (nt). The constant primer regions on each sequence are
highlighted in red. (B) Secondary structure predictions derived from mfold used to guide
truncation of M49 and M52. (C) Truncated aptamers were added to allo-MLCs and suppression
of CTL induction assayed. Truncated sequences M52-T1 and M49-T1 suppress CTL induction
with comparable efficacy to that of the full-length aptamers. Each bar represents the mean CTL
specific lysis ± SEM from three replicate wells and is representative of two independent
experiments. (*P<0.01, NS=not significant).
81
A
% C
TL
Sp
ecif
ic L
ysis
No A
ptam
er
PEG-c
Apt
PEG
-M49
PEG-M
52M
49M
52
CD20
0Fc
B
Figure 3-3. PEGylation does not perturb the ability of M49 and M52 to agonize CD200R1.
(A) Reaction scheme for PEGylation of anti-CD200R1 aptamers. DNA aptamers were
synthesized with a 5’ hexylamine and reacted with an excess of a monofunctional 20 kDa NHS-
PEG. PEGylated conjugates were recovered by size-exclusion FPLC (Appendix 4) (B)
PEGylated and non-PEGylated aptamers (325nM, based on nucleic acid content) were added to
allo-MLCs and the suppression of CTL induction compared. Similar to M49 and M52, PEG-M49
and PEG-M52 significantly suppress CTL induction. Each bar represents the mean percent CTL
specific lysis ± SEM from three replicate wells. *P<0.01 relative to no treatment, NS = not
significant
82
3.4.4 PEG-M49 and PEG-M52 Induce CD200R1 Phosphorylation
The immediate signaling event following CD200:CD200R1 ligation is the phosphorylation of the
tyrosine residue in the NPXY motif on the C-terminal cytoplasmic tail of CD200R1. The
phosphorylated NPXY motif interacts with adaptor proteins Dok1 and Dok2; binding events that
activate immune inhibitory signal pathways (Mihrshahi et al., 2009). To confirm that the
suppression of CTL induction observed in our allo-MLC assays is indeed a consequence of
aptamer-induced CD200R1 signaling, we asked whether PEG-M49 and PEG-M52 could induce
the phosphorylation of the NPXY motif. HEK-293 cells were stably transfected to express murine
CD200R1 (Figure 3-4A) and treated with aptamers PEG-M49 and PEG-M52. The
phosphorylation of CD200R1 was detected using a phospho mCD200R1-specific antibody. Both
PEG-M49 and PEG-M52 readily induced phosphorylation of the C-terminal tail of CD200R1
(Figure 3-4B), while there was no detectable signal from medium alone or PEG-cApt confirming
that the identified aptamers signal through CD200R1 in a similar manner to CD200.
3.4.5 PEGylated CD200R1 Aptamers Suppress Immune Responses in-vivo
PEG-M49, PEG-M52, PEG-cApt, or CD200-Fc were injected intravenously into C57BL/6 mice
bearing MHC-mismatched BALB/c skin allografts with a view to evaluate the potential in-vivo
therapeutic use of the CD200R1 agonistic DNA aptamers. PEGylated aptamers were administered
at 72hr intervals over 12 days and their immune inhibitory effects evaluated by performing ex-vivo
allo-MLC assays at day 14 (Figure 3-5A). CTL induction was significantly suppressed by treating
animals with PEG-M49, PEG-M52, or CD200-Fc alone but not with PEG-cApt (P<0.01) (Figure
3-5B). Exposure of circulating lymphocytes to PEG-M49 and PEG-M52 both in-vivo and after
their recovery (in-vitro) did not further augment the suppression of CTL induction as compared to
the in-vivo treatment alone (Figure 3-5B).
A common concern regarding the in-vivo use of DNA aptamers is the potential induction of TLR9-
mediated innate immune activation similar to that observed with oligodeoxynucleotides containing
CpG motifs (Krieg, 2002). To address whether PEG-M49 or PEG-M52 induce innate TLR9
responses, naïve C57BL/6 mice were administered PEGylated aptamers or a CpG oligonucleotide
(ODN) and TNFα and IL-6 sera levels quantified after six hours by ELISA. Only the control CpG
ODN exhibited detectable cytokine levels (Figure 3-6) suggesting that these aptamers do not
83
activate a TLR9 mediated innate immune response in mice. Together these findings suggest that
the in-vivo administration of PEGylated CD200R1 agonistic aptamers is sufficient to down
regulate immune responses and that such aptamers may serve as anti-inflammatory agents for
diseases in which CD200:CD200R1 signaling is implicated.
100 101 102 103 104
CD200R1
Co
un
t
0 3.5 - + 0 3.5 µM + 0 2.5 0 5.0 0 5.0
p-CD200R1
A
B
Figure 3-4. PEG conjugated M49 and M52 induced CD200R1 signaling. (A) HEK-293 cells
were transfected to stably express mCD200R1. FACS analysis of parental HEK-293 cells (grey)
or HEK-293 transfectants expressing CD200R1 on the cell surface (white) stained with a FITC
labelled anti-CD200R1 antibody confirms CD200R1 expression. (B) HEK293-CD200R1 cells
were incubated with PEG-M49 or PEG-M52 to monitor aptamer-induced phosphorylation of the
CD200R1 cytoplasmic tail. The phosphorylated form of CD200R1 was detected by western blot
using an antibody specific to phosphorylated-CD200R1 cytoplasmic tail. Supernatant from
CD200-expressing cells was used as a positive control (+). PEG-M49, PEG-M52, and PEG-cApt
were added at the indicated concentrations. (–) represents the medium alone. Both PEG-M49 and
PEG-M52 induce CD200R1 intracellular phosphorylation.
84
3.4.6 PEGylated CD200R1 Aptamers Prolong Allogeneic Skin Graft Survival
The importance of CD200:CD200R1-based immune regulation in transplantation is well
documented. CD200-Fc administration has been shown to significantly prolong the survival of
both allo- and xenografts (Gorczynski et al., 2001). Moreover, transgenic mice overexpressing
CD200 display prolonged allograft survival rates relative to control animals. In contrast, CD200R1
knockout mice reject allografts more vigorously (Gorczynski et al., 2009; Yu et al., 2013). Lastly,
a soluble form of CD200 (sCD200) with immunosuppressive function has been observed in the
Experimental Groups
1. PBS 2. PEG-cApt
3. PEG-M49 4. PEG-M52
5. CD200Fc 0 3 6 9 12
15 µg treatment
Day 14
MLC
No A
ptam
er
PEG-c
Apt
PEG
-M49
PEG
-M52
CD20
0Fc%
C
TL
Sp
ecif
ic L
ysis
A
B
Figure 3-5. PEG-M49 and PEG-M52 function in-vivo. (A) Experimental outline of in-vivo
experiment. BALB/c skin allografts (Day 0) were transplanted onto C57BL/6 mice. These
animals were subsequently treated over a 12-day period with tail vein injections of either 15µg
PEG-M49, PEG-M52, PEG-cApt, or CD200-Fc at 72-hour intervals in combination with a low
dose of rapamycin (0.5mg/kg, 36h, i.p.). (B) After 14 days, mice were sacrificed and their
splenocytes used as responder cells in ex-vivo allo-MLC assays. Cultures were subsequently
treated in-vitro with further addition of aptamers or CD200-Fc in culture (open bars) or left as is
(closed bars). Treatment with anti-CD200R1 aptamers PEG-M49 and PEG-M52 was sufficient
to suppress CTL induction without further addition of aptamer in culture (*P<0.01). Each bar
represents the mean percent of CTL specific lysis ± SEM from three replicate wells and data
shown is representative of two independent experiments.
85
sera of mice after transplantation with serum levels correlating positively with graft survival
(Gorczynski, Chen, Khatri, & Yu, 2013). Therefore, we hypothesized that the administration of
CD200R1 agonistic aptamers could prolong survival of murine skin allografts. To evaluate this
possibility, BALB/c skin grafts were transplanted onto C57BL/6 mice (n=6; Day 0), and the
aptamers PEG-M49, PEG-M52, PEG-cApt (650pmol, based on nucleic acid content) or CD200-
Fc (325pmol) were administered every 3 days over a period of 15 days in combination with low
dose rapamycin (0.5 mg/kg, i.p) given every 48 hours (Figure 3-7A). Rapamycin at this dosage
has been shown to have no effect on graft survival when administered alone (Figure 3-8A).
Strikingly, treatment with PEG-M49 and PEG-M52 protected against allograft rejection when
compared to PBS or PEG-cApt groups (Figure 3-7B) (P<0.05). Importantly, at the time of last
injection (Day 15), all control group animals had rejected their skin grafts while only 16% or 32%
of mice receiving PEG-M49 or PEG-M52 respectively displayed signs of graft rejection. The level
of immunosuppression observed for these aptamers in this transplantation model was comparable
to that of a bivalent form (CD200-Fc) of the natural ligand for CD200R1. Notably, the importance
of conjugation of aptamers to PEG was demonstrated as PEG-M49 prolonged allograft survival to
greater extent than non-PEGylated M49 (Figure 3-8A) and induced increased suppressed of allo-
immune responses in ex-vivo MLCs (Figure 3-8B).
TN
F (
pg
/mL
)
CpG
PEG-M
49
PEG-M
52
PEG-c
Apt
PBS
CpG
PEG
-M49
PEG
-M52
PEG-c
Apt
PBS
A B
Figure 3-6. PEGylated Aptamers do not activate a TLR9 Innate Immune Response. Naïve
C57Bl/6 mice (n=3) received tail vein injections of either PBS, PEGylated aptamer (M49, M52,
or cApt), or CpG ODN and the concentration of serum TNFα (A) or IL-6 (B) six hours later
quantified by ELISA. No aptamer tested engendered detectable levels of cytokines associated
with an innate TLR9 immune response.
86
A
B
0 3 6 9 12 15
15µg treatment
Day
Monitor Graft Survival
Experimental Groups (n=6)
1. PBS 2. PEG-cApt
3. PEG-M49 4. PEG-M52
5. CD200-Fc
Days Post Allograft
% A
llo
gra
ft S
urv
ival
10 12 14 16 18 20 220
20
40
60
80
100
PBS
PEG-cApt
PEG-M52
PEG-M49
CD200Fc
Figure 3-7. PEG-M49 and PEG-M52 prolong the survival of transplanted murine skin
grafts. (A) Experimental outline of allograft model. C57BL/6 mice (n=6) received BALB/c skin
allografts on Day 0 and were treated every 3 days over 15 days with PBS (closed triangles) or
650pmol of either PEG-cApt (open triangles), PEG-M49 (open circles), PEG-M52 (closed
squares) or 325pmol of CD200-Fc (closed circles) in combination with low dose rapamycin
(0.5mg/kg, 36hr, i.p.). (B) Treatment with PEG-M49 and PEG-M52 significantly extended graft
survival (P<0.05, Mann-Whitney U Test) relative to control. Arrow represents time of last
injection. Data shown is representative of two independent experiments.
87
3.4.7 Identification of a Cross-species CD200R1 Agonistic Aptamer
Due to the striking in-vivo immunosuppression observed with murine specific CD200R1
PEGylated aptamers, we investigated whether we could derive cross-species (human/mouse)
aptamers with similar CD200R1 agonistic activity. Such aptamers would be advantageous as it
would enable the characterization of a clinically-relevant therapeutic in accessible pre-clinical
mouse models of inflammatory diseases. To identify putative cross-species anti-CD200R1
aptamers, we performed independent SELEX screens using recombinant human and mouse
CD200R1 as targets and derived DNA oligonucleotide libraries enriched for CD200R1 binders.
NGS was performed on DNA oligonucleotides from the resulting enriched libraries and the highest
enriched sequences within each screen cross-compared with that of the opposite species (Figure
3-9A). This process identified 8 identical aptamer sequences between the two screens which
% A
llo
gra
ft S
urv
ival
% C
TL
Sp
ecif
ic L
ysis
Contr
ol
Rap
amyc
in A
lone
M49
PEG
-M49
CD20
0Fc
A B
Figure 3-8. PEGylation of M49 is necessary for prolongation of allograft survival. (A)
C57BL/6 mice (n=6) received BALB/c skin allografts on Day 0 and were treated every 2 days
over 12 days with PBS (open circles) or 650pmol (by ODN weight) of either PEG-M49 (open
squares), M49 (closed circles) or 325pmol of CD200-Fc (closed triangles) in combination with
low dose rapamycin (0.5mg/kg, 36hr, i.p.). Treatment with PEG-M49 and CD200-Fc
significantly extended graft survival (P<0.05, Mann-Whitney U Test) relative to rapamycin alone
(open circles), PBS or non-PEGylated M49. Arrow represents time of last injection. (B) C57Bl/6
mice bearing BALB/c skin allografts were treated as described in A, before being sacrificed at
day 14. Spleens were harvested and processed for use in allo-MLCs. In-vivo treatment of these
mice with M49, PEG-M49, and CD200-Fc significantly suppressed CTL induction in ex-vivo
MLCs. (*P<0.01).
88
ranked (based on read abundance) within the top 30 overall sequences in each SELEX search
(Figure 3-9B) (Appendix 5). These shared aptamers were synthesized and assayed for CD200R1
agonistic activity in allo-MLCs using either mouse or human leukocytes (Figure 3-10 A,B). While
several aptamers suppressed CTL induction in assays with either human or mouse cultures, only
one aptamer, named CCS13, significantly suppressed CTL induction in both human and mouse
allo-MLCs with activity comparable to CD200-Fc (Figure 3-10C).
3.4.8 PEGylated CCS13 Suppresses Allogeneic Immune Responses Specifically through CD200R1 Signaling
As we previously observed with M49, CCS13 readily induced phosphorylation of the mCD200R1
cytoplasmic tail (Figure 3-10D), and its activity was retained upon PEGylation (Figure 3-11A). To
confirm that CCS13 activity was due to the specific induction of CD200R1 signaling, allo-MLCs
were performed using responder cells derived from CD200R1-deficient mice. Here it was found
that PEG-CCS13 suppressed CTL-induction in responder cells from wild-type, but not
SELEX
mCD200R1
selection
hCD200R1
selection
NGS
Data Analysis
Cross Species
CD200R1 Aptamers
A B
Figure 3-9. Cross-Species Anti-CD200R1 Aptamers Identified by SELEX. (A) At cycle 15,
enriched SELEX pools towards both human and murine CD200R1 were sequenced using
IonTorrent NGS and analysed for common motifs across both species (B) Of the top 50 enriched
sequences (based on fraction of total reads) eight sequences were identified to have the same
variable region in each independent SELEX pool. Shown are the sequence names along with
their enrichment in each SELEX screen
89
CD200R1-/- mice, confirming that this aptamer functions in a CD200R1 specific manner (Figure
3-11B). Critically, this data shows that the immunosuppression observed in allo-MLCs assays is a
specific consequence of aptamer-induced CD200R1 signaling and not related to off-target effects.
3.4.9 PEGylated CD200R1 CCS13 aptamer prolongs allogeneic skin graft survival
To characterize the immunosuppressive effects in-vivo, we tested PEG-CCS13 in a mouse skin-
transplant model, as was previously described for the murine specific anti-CD200R1 aptamers
M49 and M52. Treatment of C57BL/6 mice bearing BALB/c skin grafts with PEG-CCS13 led to
significant prolongation of allograft rejection as compared to PBS or the control aptamer (PEG-
cApt) groups (Figure 3-11C). Notably, the level of immunosuppression elicited by the CCS13
aptamer in this transplantation model was comparable to that observed by CD200-Fc.
3.4.10 PEG-CCS13 Suppresses House Dust Mite (HDM)-induced Allergic Airway Response
In addition to playing a key role in regulating transplant-induced immune responses,
CD200:CD200R1 signaling has also been implicated in the regulation of allergic inflammatory
disorders. For instance, the activation and degranulation of mast cells is inhibited upon CD200
binding of CD200R1. In support of this, the in-vivo treatment of mice with an anti-CD200R1
agonistic antibody inhibited induction of IgE-dependent cutaneous anaphylaxis. Furthermore, in
an experimental model of asthma induced by challenge of sensitized mice with ovalbumin (OVA),
CD200-Fc treatment led to an in inhibition of airway hyper-responsiveness (AHR) and suppression
of the associated inflammatory correlates (Lauzon-Joset et al., 2015). Given the in-vivo success of
CCS13 in prolonging allograft survival, we proceeded to assess whether this aptamer could be
beneficial in an acute model of HDM-induced allergy. In this experiment, HDM-sensitized mice
were treated with PEG-CCS13, and AHR in response to methacholine (MCh) was monitored after
HDM challenge. Airway respiratory resistance (Rrs) increased with escalating doses of MCh given
to HDM-sensitive and challenged mice receiving saline when compared to naïve mice given saline
(P<0.05). Excitingly, PEG-CCS13 treatment led to a significant reduction in Rrs relative to saline
treated controls. This effect was comparable to that observed with CD200-Fc (Figure 3-12A). The
reduction in the MCh-responsiveness was most obvious at the maximal Rrs (Rrs max) following
treatment with CD200-Fc and PEG-CCS13 (Figure 3-12B).
90
No A
ptam
er
CCS1
CCS2
CCS4
CCS5
CCS6
CCS8
CCS10
CCS11
CCS13
0
10
20
30
40
*
**
*
No A
ptam
er
CCS1
CCS2
CCS4
CCS5
CCS6
CCS8
CCS10
CCS11
CCS13
% C
TL
Sp
ecif
ic L
ysis
No A
ptam
er
CCS13
CD20
0Fc
No A
ptam
er
CCS13
CD20
0Fc
0
10
20
30
40
Human allo-MLC
Mouse allo-MLC
**
*
*
p-CD200R1
_
A C
B
Figure 3-10. CCS13: A cross species CD200R agonist (A,B) Each cross-species anti-CD200R1
aptamer was tested for suppression of CTL induction in both mouse (A) and human (B) allo-
MLCs. Aptamer CCS13 showed at least 50% suppression of CTL induction in both human and
mouse allogenic cultures and was chosen for further evaluation. Each bar represents the mean %
CTL specific lysis ± SEM (n=3, *P<0.01 vs no aptamer). (C) Human and mouse allo-MLCs
comparing activity of CCS13 relative to CD200-Fc. (D) To monitor aptamer induced CD200R1
signaling, HEK293 cells stably expressing mCD200R1 were incubated with positive controls
M49 and CD200-Fc, a negative control aptamer cApt (3µM), or the CD200R1 cross species
aptamer CCS13 (all 3µM) and phosphorylation of CD200R1 detected using an antibody specific
to phosphorylated-mCD200R1 cytoplasmic tail. Notably, the cross-species aptamer CCS13,
along with positive controls CD200-Fc and M49, stimulated mCD200R1 phosphorylation.
D
91
A
C
B
Figure 3-11. PEGylated CCS13 suppressed allo-immune responses through CD200R1
signalling. (A) Allo-MLC using both mouse and human leukocytes confirms that upon
PEGylation CCS13 retained the ability to agonize CD200R1 signaling in terms of suppressing
CTL induction in-vitro. Each bar represents the mean % CTL specific lysis ± SEM (n=3, *P<0.05
vs no aptamer). (B) PEG-CCS13 was used in allo-MLCs using responder cells derived from
either wild-type (WT) or CD200R1-deficient mice. The immunosuppressive effects of PEG-
CCS13 is specifically mediated through CD200R1, as exhibited by a lack of suppression of CTL
induction in allo-MLCs derived from CD200R1-null mice (*P<0.05 relative to CD200R1KO).
Each bar represents the mean % CTL specific lysis ± SEM (n=3, *P<0.05 vs no aptamer). (C)
C57BL/6 mice (n=6) received BALB/c skin allografts on day 0 and were treated in 72hr intervals
with PBS (control) or 15µg PEG-cApt, PEG-M49, PEG-M52 or CD200-Fc in combination with
low dose (0.5mg/kg) rapamycin. Treatment with PEG-CCS.13 significantly extended graft
survival (*P<0.05). Arrow represents the time of last aptamer injection (day 15).
*
*
92
Figure 3-12. PEGylated aptamer PEG-CCS13 suppresses house dust mice (HDM)-induced
allergic airway response. (A) Total respiratory system resistance (Rrs) in response to
methacholine (MCh) after challenge of sensitized mice to HDM allergen. Mice were treated with
a single dose of CD200-Fc, PEG-CCS13 or saline after challenge. Rrs increased upon escalating
doses of methacholine (MCh) given to HDM-sensitized mice receiving saline when compared to
naïve mice (*P<0.05, n=6-7). Rrs was decreased significantly in groups treated with either
CD200-Fc and PEG-CCS13 (*P<0.05). (B) Reduction in the MCh-responsiveness was most
obvious at the maximal Rrs (Rrs max) following treatment with CD200-Fc and PEG-CCS13
(n=6-7, *P<0.05 relative to HDM-saline group).
A
B
*
#
93
3.5 Discussion
Ligands which bind to immune co-receptors and promote or impair signaling pathways have
emerged as powerful immunotherapeutics. The best examples to date of such agents are
neutralizing antibodies towards the co-inhibitory molecules CTLA-4 and PD-1 which demonstrate
profound activity in mobilizing immune responses and preventing tumor immune evasion in
humans (Hodi et al., 2010; Topalian et al., 2012). In contrast, agents that impart agonistic activity
to such inhibitory co-receptors may prove useful in clinical situations where an immune response
needs to be repressed such as transplantation, allergy, and autoimmunity. For instance, pre-clinical
studies have shown that soluble forms of CD200 or a monoclonal antibody to CD200R1 can act
as potent agonists to the CD200R1 inhibitory receptor in prolonging the survival of murine
allografts (Gorczynski et al., 1999), reducing the severity of CIA (Simelyte et al., 2008), and
suppressing the progression of EAE (Liu et al., 2010). In the present study, we report the
identification of short synthetic DNA aptamers that can mimic the agonistic properties of the
natural ligand CD200, in binding to CD200R1 and repressing immune cell responses. We first
identified two such aptamers, M49 and M52, which specifically target and agonize murine
CD200R1. These aptamers potently suppress induction of CTLs in allo-MLC assays, and rapidly
induce the phosphorylation of the CD200R1 cytoplasmic tail confirming that the aptamers work
as agonists to induce CD200R1 signaling similar manner to CD200. Strikingly, in vivo
administration of PEGylated M49 and M52 dampened immune activity and prolonged the survival
of murine skin allografts in a transplant model. Of note, the treatment of mice with PEGylated
aptamers or CD200-Fc in transplantation experiments was halted at Day 15 when all mice
receiving either PBS or PEG-cApt had rejected their skin grafts. At that stage, allograft rejection
was observed in only 16% or 32% of mice receiving PEGylated forms of M49 or M52 respectively,
results that were similar to that of the bivalent CD200-Fc (16%). Differences observed in these
groups beyond this point may reflect different pharmacokinetic properties between PEGylated
aptamers and recombinant protein rather than their ability to stimulate CD200R1 signaling.
Previous research has shown that continued CD200R1 stimulation (using a transgenic mouse strain
(CD200Tg) which overexpresses CD200 upon doxycycline administration) can induce full
allograft acceptance, suggesting that sustained treatment with CD200R1 agonists such as these
aptamers may similarly induce long-term allograft acceptance. Furthermore, using the CD200Tg
mice, it was shown that continued transgene expression was not necessary after allograft
94
acceptance suggesting that ongoing or life-long administration of CD200R1 agonists may not be
necessary (Gorczynski et al., 2009).
We have recently shown that functional DNA aptamers targeting TNFα, CEA, and PD-1 do not
trigger cytokine responses linked to innate immunity (Orava, Abdul-Wahid, Huang, Mallick, &
Gariépy, 2013; Orava, Jarvik, et al., 2013; Prodeus et al., 2015). Consistently, the PEGylated
aptamers used in this study did not activate a TLR9 innate immune response. Furthermore,
administration of DNA aptamers have not been associated with the generation of T and B cell
responses (Keefe et al., 2010). These features are especially beneficial for the chronic or long-
term administration of aptamers aimed at treating patients with allergies, arthritis and autoimmune
responses including rejection of tissues following transplantations. In contrast, most protein-based
agents including humanized monoclonal antibodies do engender an immune response when
administered chronically to patients (Strand et al., 2017). Lastly, low production costs and scalable
chemical synthesis make DNA aptamers especially viable agents for therapeutic applications
(Gilboa et al., 2013).
Due to the unique advantages of DNA aptamers and the positive results we observed as a ‘proof-
of-concept’ with the murine CD200R1 specific aptamers M49 and M52, we believed it was
warranted to develop therapeutically relevant anti-human CD200R1 aptamers. However, the
characterization of a human specific aptamer in pre-clinical rodent models could prove
challenging, thus providing rationale to derive a cross-species anti-CD200R1 aptamer. To this end,
by using a systematic SELEX-NGS approach we identified a cross-species mouse/human
CD200R1 agonistic aptamer named CCS13. As seen with M49 and M52, aptamer CCS13 rapidly
induced the phosphorylation of the CD200R1 tail, affirming its agonistic activity. Importantly,
CCS13 does not suppress CTL induction in allo-MLCs which derived responder cells from
CD200R1 knockout mice, indicating that its activity is directly linked to CD200R1 activation.
Importantly, pre-clinical studies in models of MHC-mismatch based skin transplant rejection and
HDM-induced allergy demonstrated that the PEGylated form of aptamer CCS13 displayed potent
anti-inflammatory activity in-vivo.
Currently, there is one FDA approved aptamer, called Pegatinib (Macugen), which is a PEGylated
protected RNA aptamer which binds to VEGF to treat wet macular degeneration (Ng et al., 2006).
There are no immune modulatory aptamers in Phase trials. However, several candidate aptamers
95
have now been reported including aptamers targeting CTLA-4, 4-1BB, OX-40, IL-6R, IL-10R,
CD28 and PD-1 (Berezhnoy et al., 2012; Dollins, Nair, Boczkowski, et al., 2008; McNamara et
al., 2008; Pastor et al., 2013; Prodeus et al., 2015; Santulli-Marotto et al., 2003). Interestingly,
several agonistic aptamers developed to these receptors have required dimerization through
chemical scaffold or using a hybridized DNA technique (Dollins, Nair, Boczkowski, et al., 2008;
McNamara et al., 2008). In fact, the CD200-Fc ligand used in the present study is actually bivalent
in nature harboring two CD200 domains, yet the monovalent PEGylated aptamers used in this
study (CCS13, M49 and M52) have proven to be as effective in suppressing immune responses as
CD200-Fc.
3.6 Conclusions
In summary, we have shown as a proof of concept, that PEGylated aptamers directed at murine
CD200R1 can act in an agonistic function to suppress immune response both in-vitro and in-vivo.
Furthermore, by incorporating NGS into the SELEX process, we can now identify cross-species
reactive aptamers, such as CCS13, which carry therapeutic value and can conveniently be
characterized in our case in validated murine pre-clinical models of inflammatory diseases. CCS13
has thus far been shown to significantly prolong the survival of murine skin-allografts, and
suppress airway hyper-responsiveness in an HDM allergy model, thereby highlighting potential
clinical utility. Further studies are warranted to evaluate the role for CD200R1 agonistic aptamers
in blocking acute and chronic inflammatory responses arising from other clinical situations
including arthritis, trauma and autoimmune conditions. Additionally, pre-clinical studies are
ongoing to further determine the potential in allergic-asthma based disorders and determine
optimal delivery strategies.
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97
Chapter 4
VISTA.COMP: an engineered checkpoint receptor agonist that
suppresses T-cell mediated immune responses.
Reformatted from a similar report published as:
VISTA.COMP: an engineered checkpoint receptor agonist that potently suppresses
T-cell mediated immune responses.
Aaron Prodeus, Aws Abdul-Wahid, Amanda Sparkes, Nicholas W. Fischer, Marzena Cydzik,
Nicholas Chiang, Mays Alwash, Alessandra Ferzoco, Nathalie Vacaresse, Michael Julius,
Reginald M. Gorczysnki, and Jean Gariépy.
Published in JCI Insight, 2 (18). 2017.
Contributions:
AP performed experiments, analyzed data, and wrote the chapter. AP and JG conceptualized the
study. AAW, AS assisted with in-vivo experiments. RMG performed allo-MLCs and allograft
experiments. MC, MA assisted with flow cytometry and protein labelling. NWF and NC assisted
with recombinant protein production. AF, NV, MJ provided critical reagents and expertise.
JG supervised the project.
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VISTA.COMP: an Engineered Checkpoint Receptor Agonist that Suppresses T-cell Mediated Immune Responses.
4.1 Abstract
V-domain Immunoglobulin Suppressor of T-cell Activation (VISTA) is a recently discovered
immune checkpoint ligand that functions to suppress T-cell activity. The therapeutic potential of
activating this immune checkpoint pathway to reduce inflammatory responses remains untapped,
largely due to the inability to derive agonists targeting its unknown receptor. A dimeric construct
of the IgV-like domain of VISTA (VISTA-Fc) was shown to suppress the activation of T-cells in-
vitro. However, this effect required its immobilization on a solid surface, suggesting that VISTA-
Fc may display limited efficacy as a VISTA-receptor agonist in-vivo. Herein we have designed a
stable pentameric VISTA construct (VISTA.COMP) by genetically fusing its IgV-like domain to
the pentamerization domain from the Cartilage Oligomeric Matrix Protein (COMP). In contrast to
VISTA-Fc, VISTA.COMP does not require immobilization to inhibit the proliferation of CD4+
T-cells undergoing polyclonal activation. Furthermore, we show that VISTA.COMP, but not
VISTA-Fc, functions as an immunosuppressive agonist in-vivo, capable of prolonging the survival
of skin allografts in a mouse transplant model, as well as rescuing mice from acute Con-A induced
hepatitis. Collectively, our data demonstrates that VISTA.COMP is a novel checkpoint receptor
agonist and the first agent targeting the putative VISTA-receptor to suppress T-cell mediated
immune response.
4.2 Introduction
T-cell activity is tightly regulated by a spectrum of co-stimulatory and co-inhibitory signals,
allowing for protection against invading pathogens or malignant cells, while maintaining self-
tolerance. To date, several negative checkpoint receptors such as PD-1 and CTLA-4 have been
identified which function to suppress T-cell activity (Freeman et al., 2000; Thompson & Allison,
1997). Antibody-mediated blockade of these pathways has been shown to promote remarkable
anti-tumor immune responses (Hodi et al., 2010; Topalian et al., 2012). Conversely, ligands which
activate these immunoinhibitory pathways regulate the progression of uncontrolled immune
responses in T-cell mediated autoimmune and inflammatory disorders (Francisco et al., 2010;
Raptopoulou et al., 2010; Riella et al., 2012).
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VISTA is a newly characterized checkpoint ligand, expressed primarily on CD11bhigh myeloid
cells, which negatively regulates T-cell responses upon binding to an as-of-yet undiscovered cell
surface receptor (VISTA-receptor) (Lines, Pantazi, et al., 2014; Wang et al., 2011). VISTA is also
expressed on naïve CD4+ and CD8+ T-cells, where it negatively regulates T-cell responses,
suggesting a dual-role of VISTA as both a checkpoint ligand and receptor (Flies et al., 2014).
Structurally, VISTA shares significant homology with PD-1 and PD-L1, consisting of an N-
terminal IgV-like domain followed by a single membrane spanning domain and cytoplasmic tail.
As in the case of the PD-1:PD-L1 pathway, blockade of VISTA using monoclonal antibodies has
been demonstrated to provoke anti-tumor immune responses in several mouse models, suggesting
a role for VISTA:VISTA-receptor signaling in the promotion of tumor immune evasion (Le
Mercier et al., 2014; Lines, Sempere, et al., 2014).
VISTA has also been implicated in regulating the progression of autoimmune diseases. It has been
reported that VISTA-deficient (VISTA-/-) mice bred on a lupus-prone background develop
accelerated and severe systemic lupus erythematosus (SLE) (Ceeraz et al., 2016). Similarly,
VISTA-/- 2D2 T-cell receptor transgenic mice exhibit increased levels of peripheral
encephalitogenic T-cells and develop an exacerbated form of experimental autoimmune
encephalomyelitis (EAE) (Wang et al., 2014). More recently, it was shown that VISTA-deficient
mice exhibited worsened disease in a model of imiquimod induced psoriasis, where enhanced
TLR7 signaling in dendritic cells (DCs) led to augmented IL23 production and increased
expression of IL17A in T-cells (Li et al., 2017). The VISTA-/- mice bred on a wild-type C57Bl/6
background display a mild pro-inflammatory phenotype, exemplified by an increase in DC and a
rise in T-cell activation markers, but did not develop inflammatory disorders (Wang et al., 2014).
In slight contrast, another study described a more severe phenotype, where VISTA-/- C57Bl/6 mice
developed glomerulonephritis at 10 months of age (Yoon et al., 2015). Taken together, these
studies indicate a potential therapeutic advantage in promoting VISTA-mediated
immunosuppression to treat autoimmune or inflammatory diseases.
While agonistic anti-VISTA antibodies have been reported (Flies et al., 2015), the derivation of
novel agonists aimed at the putative VISTA-receptor remains challenging due to its unknown
identity. A dimeric version of VISTA (VISTA-Fc) has been previously reported to suppress both
mouse and human T-cell activation in-vitro (Lines, Pantazi, et al., 2014; L. Wang et al., 2011).
However, this activity requires the immobilization of this ligand on a solid surface. Accordingly,
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no study has reported that a soluble form of the VISTA extracellular domain can act as an effective
agonist to suppress inflammatory responses in-vivo. In this study, we describe the engineering of
a stable pentameric form of the VISTA extracellular domain (VISTA.COMP), which in contrast
to VISTA-Fc effectively inhibits CD4+ T-cell activation as a soluble ligand. Moreover, we present
evidence that this VISTA-receptor agonist suppresses inflammatory responses in-vivo, in the
context of prolonging murine skin allograft survival, and in protecting mice from lethal acute
hepatitis. The results described provide support for continued characterization of VISTA-receptor
agonists (such as VISTA.COMP) as a novel therapeutic strategy to treat autoimmune and
inflammatory disorders.
4.3 Methods
4.3.1 Recombinant Protein Expression and Purification.
VISTA-Fc was produced by cloning a synthetic DNA insert encoding for the extracellular domain
(ECD; residues 16-194) of murine VISTA upstream of the human IgG-1 Fc region (GeneArt;
Thermo Fisher Scientific) into the pcDNA-3.4 expression plasmid (Thermo Fisher Scientific). The
plasmid encoding the murine VISTA.COMP gene was similarly generated by inserting a synthetic
DNA insert coding for the ECD of VISTA, flanked by 5’ and 3’ EcoRI restriction digestion sites,
upstream of the cartilage oligomeric matric protein pentamerization domain (COMP; residues 28-
72) followed by a C-terminal hexahistidine tag. An expression plasmid coding for COMP domain
alone (control) was constructed by excising the VISTA ECD region from the VISTA.COMP
plasmid by EcoRI restriction digestion. All plasmids encoded a 5’ Ig-kappa leader sequence for
high protein secretion in mammalian cells. Recombinant proteins were expressed using the Expi-
293 transient expression system (Thermo Fisher Scientific). Secreted VISTA-Fc was purified from
culture media using HiTrap Protein A HP columns (GE Healthcare), while the histidine tagged
VISTA.COMP and COMP were purified using Ni-NTA resin (Qiagen) and desalted into PBS pH
7.4 using PD-10 columns (GE Healthcare). Proteins were verified for purity using SDS-PAGE,
and protein concentration quantified by BCA assay (Pierce) or A280 measurements. Full
sequences for all constructs used are depicted in Appendix 6.
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4.3.2 Antibodies and ELISA kits
FACS antibodies; PE-anti-hIgG (HP6017), APC-anti-IL2 (JES6-5H4), and PE-streptavidin were
purchased from BioLegend. Anti-CD3e (145-2C11) was purchased from BioXcell. Anti-
phosphotyrosine (4G10) was purchased from EMD Millipore. ELISA duoset antibody pairs for
IL2, IFNγ, IL6 and TNFα were purchased from R&D Systems.
4.3.3 Animals
C57BL/6 mice 8-12 weeks of age (The Jackson Laboratory) used throughout this study were
housed in a pathogen free environment at the Sunnybrook Research Institute Comparative
Research (SRICR) facility. All animal experiments and protocols were approved by the SRICR
animal care committee, accredited by the Canadian Council of Animal Care.
4.3.4 Cell Culture
CD4+ T-cells were isolated from spleens of C57Bl/6 mice using an EasySep Mouse CD4+ T-cell
isolation kit (Stem Cell) and cultured in RPMI-1640 media supplemented with 10% FBS,
penicillin (100U/mL), streptomycin (100 µg/mL) and 0.05mM 2-mercaptoethanol. The murine
2.10 T-cell clone was cultured in complete IMDM supplemented with IL-2 (3.5 ng/mL), lecithin
(20 µg/mL), and BSA (0.5 mg/mL).
4.3.5 2.10 T-cell Clone Activation
96-well microtiter plates were coated with anti-CD3 antibody (3µg/mL in PBS) at 4oC overnight.
To monitor the effects of immobilized checkpoint ligands on a clonal (2.10) T-cell line (Haughn
et al., 1992), the anti-CD3 coated wells were washed and coated with VISTA.COMP or other
recombinant proteins for 1 hour at 37oC in PBS. Wells were then washed with PBS (3x) to remove
residual unbound proteins. 2.10 T-cells grown in culture were recovered, washed in IMDM (x3),
and dispensed into protein-coated wells (1x104 cells/well). To measure proliferation, cells were
pulsed for 6hr with 1µCi of [3H]-Thymidine after 18hr of culture and uptake quantified using a
TopCount NXT scintillation counter (Perkin Elmer). To assay the sensitivity of 2.10 T-cells to
soluble checkpoint ligands, indicated recombinant proteins were diluted in culture media and
added to anti-CD3 antibody-coated wells simultaneously with the 2.10 cells.
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4.3.6 CD4+ T-cell Proliferation and Cytokine Secretion.
Isolated murine CD4+ T-cells were labelled with CFSE following the manufacturers protocol
(Thermo Fisher Scientific) and stimulated in 96-well microtiter plates pre-coated with an anti-CD3
antibody in the presence of either murine VISTA-Fc, VISTA.COMP or COMP alone (coated or
soluble). Cells were harvested 48 or 72 hours later and CFSE-dilution profiles quantified by flow
cytometry (FACScalibur, Becton Dickinson). Culture media was harvested from stimulated CD4+
T-cells at 48 or 72hr and analyzed by ELISA to quantify VISTA.COMP-mediated inhibition of
IL2 and IFNγ secretion.
4.3.7 Flow cytometry
Binding of VISTA.COMP, VISTA-Fc, or control proteins to T-cells was assessed using flow
cytometry. Proteins were first biotinylated using EZ-Link Sulfo-NHS-LC-Biotin reagent (Thermo
Scientific) as directed by the manufacturer. Upon completion of the reaction, the excess biotin
was removed using a PD-10 (GE Healthcare) desalting column. To confirm equivalent levels of
biotinylation of each protein, the quantity of biotin conjugated to each ligand was determined using
HABA/Avidin reagent (Sigma). 2.10 T-cells were incubated with the indicated biotinylated protein
(500nM) or VISTA-Fc for 0.5hr at 4oC in FACS staining buffer (PBS supplemented with 1% FBS
and 0.09% NaN3). After removal of non-bound proteins, cells were incubated with streptavidin-
PE (1:300) or PE-anti-human IgG (1:100) in FACS staining buffer for 15 minutes and the PE-
fluorescence signal analyzed using a FACScalibur cell analyzer. For competition experiments,
VISTA-Fc and unlabelled VISTA.COMP were added to 2.10 cells at equimolar concentrations
(500nM), and bound VISTA-Fc detected using PE-anti-human IgG.
4.3.8 Allogeneic Mouse Mixed Leukocyte Culture Assay (allo-MLC)
VISTA.COMP or CD200-Fc (positive control) were added to allogeneic murine mixed leukocyte
cultures for 5 days and induction of cytotoxic T-lymphocytes (CTLs) assayed as previously
described (Section 3.3.5). Briefly, C57Bl/6 responder splenocytes were incubated with an equal
number of irradiated BALB/c stimulator cells in the presence of each recombinant protein at the
indicated concentration. Induced CTLs were assayed by monitoring the release of 51Cr from
loaded P815 mastocytoma target cells over 5 hours (25:1 effector to target ratio).
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4.3.9 Allogeneic Skin graft Transplant
The immunosuppressive effect of VISTA.COMP was tested in-vivo using a mouse skin allograft
model. BALB/C mice received C57Bl/6 skin grafts (day 0) followed by intravenous treatment with
VISTA.COMP (15 µg) once every 3 days for a total of 5 treatments in combination with low-dose
rapamycin (0.5mg/kg, intraperitoneal injections every 48 hours). A blinded investigator monitored
graft survival daily.
4.3.10 Concanavalin-A Induced Acute Hepatitis
The ability of VISTA.COMP to rescue mice from lethal acute inflammation was evaluated using
the Con-A model of acute hepatitis (Tiegs, Hentschel, & Wendel, 1992). Male C57Bl/6 mice were
treated with intraperitoneal injections of VISTA.COMP (200µg), VISTA-Fc or PBS as indicated,
two hours prior to intravenous injection of a lethal dose (15mg/kg) of Concanavalin A (Con-A,
Sigma-Aldrich). Animals were carefully monitored and considered non-responders and humanely
sacrificed when deemed moribund (displaying excessive lethargy and unresponsiveness) by a
blinded investigator. A subset of animals were sacrificed after 3 hours to quantify serum IL-6 and
TNFα levels by ELISA and the remaining animals were monitored for survival over the course of
24 hours.
4.3.11 Solid Phase Immunoprecipitation Assay.
A solid phase immunoprecipitation assay was performed to assess the inhibitory effects of
VISTA.COMP on TCR phospho-signaling cascades. 2.10 T-cells were exposed to plates coated
with anti-CD3 antibody (with or without VISTA.COMP) for 15 minutes. Residual medium was
removed and cells lysed in situ upon incubation with lysis buffer (50mM Tris pH 7.4, 150mM
NaCl, 1% NP40, 5mM Na4O7P2, 5mM NaF, 2mM Na3VO4, and 1X Sigma Protease Inhibitor
Cocktail) for 30 minutes at 4oC. Wells were vigorously washed 3x with lysis buffer, and adhered
proteins eluted with 3.5% NH4OH. The eluted proteins were lyophilized and resuspended in SDS-
sample buffer, and total phosphorylated proteins visualized by western blot using an anti-
phosphotyrosine antibody.
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4.3.12 Statistics
Statistical analyses were performed using GraphPad Prism software (v6.0.2) using two-tailed
Students T-test unless otherwise stated. A P-value less than 0.05 was considered significant.
Graphs and visuals were created using GraphPad Prism software.
4.4 Results
4.4.1 A Recombinant Pentameric VISTA-IgV Construct Suppresses T-cell Activation as a Soluble Ligand in-vitro.
Consistent with previous reports, we found that dimeric VISTA-Fc constructed by fusing the
VISTA IgV-like domain with the Fc region from IgG1, only suppressed the proliferation of anti-
CD3 stimulated CD4+ T-cells when immobilized on a culture dish (Figure 4-1A) (L. Wang et al.,
2011). This finding suggests that its use in-vivo to suppress T-cell activity may be limited due to
its inability to successfully agonize the putative VISTA-receptor. We hypothesized that the lack
of activity of soluble VISTA-Fc in-vitro may reflect insufficient avidity towards its receptor, or a
lack of ability to cluster the VISTA-receptor on the cell surface. Consequently, we engineered a
higher order VISTA oligomer to address these challenges with a view to generate a soluble agonist
that can effectively suppress T-cell stimulation both in-vitro and in-vivo. Previous studies have
reported on the use of the short pentamerization domain from the Cartilage Oligomeric Matrix
Protein (COMP) to construct stable recombinant pentamers with increased avidity and activity
relative to lesser order multimers. The COMP pentamerization domain is a short 44-amino acid
sequence that spontaneously assembles into a bundle of 5 α-helices arranged in a parallel
orientation and is stabilized by disulphide bridges (Efimov, Lustig, & Engel, 1994). Previously, a
pro-angiogenic factor angiopoietin 1 fused to the COMP domain (COMP-Ang1) showed increased
stability relative to native Ang1 leading to an increased induction of angiogenesis in-vivo (Cho et
al., 2004). Thus, to create a recombinant VISTA pentamer, VISTA.COMP was constructed by
genetically fusing the VISTA IgV-like domain to the COMP pentamerization domain.
Recombinant VISTA.COMP was produced in a mammalian expression system, yielding a
pentameric protein of ~250 kDa (Figure 4-1B).
In contrast to VISTA-Fc, soluble VISTA.COMP substantially suppresses the expansion and
proliferation of isolated anti-CD3 stimulated CD4+ T-cells (Figure 4-1C). The recombinant
COMP domain alone showed negligible effect on T-cell expansion and proliferation suggesting
105
this activity is due to VISTA signaling and not off-target events associated with the COMP
domain. In addition, soluble VISTA.COMP significantly diminished (P<0.01) the secretion of
inflammatory cytokines IL-2 (Figure 4-1D) and IFNγ (Figure 4-1E) by stimulated CD4+ T-cells.
The efficacy of VISTA.COMP suppression was inversely correlated with the strength of TCR
stimulation, as increased anti-CD3 stimulation led to increases in T-cell division in the presence
of VISTA.COMP (Figure 4-2A).
In addition to its ability to suppress T-cell proliferation in response to a polyclonal stimulus,
VISTA.COMP readily suppressed the induction of cytotoxic T-lymphocytes (CTLs), in a dose
dependent manner, in allogenic mixed-leukocyte cultures (Figure 4-2B). These results demonstrate
that this VISTA pentamer represents an effective agonist, capable of activating the VISTA-
receptor on T-cells to regulate their activity without requiring immobilization on a solid surface as
is the case with VISTA-Fc.
106
Figure 4-1. Pentameric VISTA.COMP suppresses T-cell activation and proliferation as a
soluble ligand in-vitro. (A) CFSE-labelled purified murine CD4+ T-cells were activated by
plate-bound anti-CD3 antibody (2.5 µg/mL) in the presence (blue) or absence (red) of
immobilized (left panel) or soluble (right panel) VISTA-Fc (10 µg/mL) for 48 h. VISTA-Fc
suppresses the proliferation of CD4+ T-cells when immobilized, but not when added as a soluble
ligand in the culture media. (B) Recombinant VISTA.COMP purity and pentameric status
confirmed by SDS-PAGE and anti-HIS western blot in the presence or absence of a reducing
agent (DTT). Reduced VISTA.COMP migrates as a single band ~50kDa, while the disulphide-
stabilized pentamer has an apparent mass of 250kDa. (C) Proliferation of CD4+ T-cells
undergoing activation in the presence of coated (9 µg/mL, left panels) or soluble (12 µg/mL,
right panels) VISTA.COMP (blue) or COMP (red). Soluble VISTA.COMP suppresses T-cell
expansion (top, FSC & SSC profiles) and proliferation (bottom, CFSE dilution). Culture
medium was harvested from CD4+ T-cells 48 and 72 hours post anti-CD3 activation in the
presence of COMP or VISTA.COMP and IL-2 (D) and IFNγ (E) secretion quantified by ELISA.
VISTA.COMP was found to significantly suppress IL-2 and IFNγ secretion (Data represents
mean ± SEM, ***P<0.005 relative to COMP control, n=3). All data shown is representative of
at least three independent experiments
DTT + - + -
50 kDa
200 kDa
C
CFSE
anti-CD3 + VISTA.Fc
anti-CD3
Immobilized Soluble
D
E
anti-CD3 + VISTA.COMP
anti-CD3 + COMP
CFSE
Immobilized Soluble
48h
0
100
200
300
400
***
A B
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anti-CD3 mAb
concentration:
anti-CD3 + VISTA.COMP
anti-CD3 + COMP
3 µg/mL 6 µg/mL A
B Wild Type
Concentration ( g/mL)
0.0 2.5 5.0 7.5 10.00
10
20
30
40
**
*
Figure 4-2. VISTA.COMP suppressed T-cell activation and CTL induction. (A) CFSE-
labelled CD4+ T-cells were activated with immobilized anti-CD3 antibody at the indicated
concentration in the presence of COMP (red) or VISTA.COMP (blue). VISTA.COMP
suppression of T-cell proliferation can be overcome by stronger levels of TCR stimulation. (B)
Allogenic MLC assays were performed as described in Supplementary Methods. Addition of
VISTA.COMP significantly suppressed CTL induction in responder cells from wild-type or
CD200R1 -/- mice (Each point represents mean ± SEM with n=3 at a given concentration
(µg/mL), *P<0.05 relative to CD200-Fc in CD200R1-/- mice). CD200-Fc was used as a positive
control allo-MLCs using wild-type responder cells and as a negative control in assays using
CD200R1 -/- responder cells.
108
4.4.2 VISTA.COMP Binds to and Suppresses the Activation of a Clonal T-cell Line.
In addition to primary CD4+ T-cells, we found that a CD4-negative murine IL-2 dependent T-cell
clone (named clone 2.10, (Haughn et al., 1992)) was sensitive to VISTA inhibitory signaling,
providing a controlled system to assay the function of VISTA-receptor agonists. Consistent with
what is observed in primary CD4+ T-cells, VISTA-Fc suppresses anti-CD3 induced proliferation
only when immobilized on a solid surface, while VISTA.COMP completely suppresses
proliferation when both immobilized and soluble in culture medium (P<0.01) (Figure 4-3A).
Titration of soluble VISTA.COMP and VISTA-Fc demonstrates that VISTA.COMP suppresses
anti-CD3 induced 2.10 cell proliferation at concentrations as low as 1 µg/mL (P<0.01) meanwhile
VISTA-Fc has no detectable activity at concentrations as high as 30 µg/mL (Figure 4-3B). In
addition to suppressing proliferation, intracellular flow cytometry shows that soluble
VISTA.COMP, but not VISTA-Fc, suppresses stimulated 2.10 cell IL-2 secretion within 4 hours
of exposure (P<0.05), suggesting an immediate and rapid effect of VISTA.COMP (Figure 4-4A).
As well, VISTA.COMP suppresses the rapid phosphorylation of tyrosine residues within TCR-
complex signaling proteins induced upon anti-CD3 stimulation of the 2.10 cells (Figure 4-4B).
Mechanistically, these results are consistent with the previous finding that exposing naïve CD4+
T-cells to immobilized VISTA-Fc led to long-term suppression of T-cells upon transfer to anti-
CD3 coated wells (in the absence of further VISTA-Fc), which suggests a role for VISTA signaling
as a critical early regulator of T-cell activation. Flow cytometry was then performed on the 2.10
cell line using VISTA-Fc, COMP, or VISTA.COMP, to determine if the inability of soluble
VISTA-Fc to bind to the VISTA-receptor on T-cells contributes to the lack of suppressive activity.
VISTA.COMP and COMP were labelled with an equivalent number of biotins groups, and cell-
bound biotinylated proteins were detected with PE-streptavidin, while bound VISTA-Fc was
detected with PE-anti-IgG. Both VISTA-Fc and VISTA.COMP were found to bind to naïve 2.10
T-cells while the baseline signal observed for COMP confirmed the absence of non-specific
binding arising from the pentamerization domain alone (Figure 4-3C). Supporting a high avidity
interaction of VISTA.COMP with T-cells, a competition assay using VISTA.COMP present at
equimolar concentrations completely inhibited VISTA-Fc binding to 2.10 cells (Figure 4-3D).
Altogether, these findings indicate that a soluble dimeric VISTA construct, while capable of
binding T-cells, does not activate immunoinhibitory signaling through this pathway in-vitro, while
the high-avidity VISTA.COMP is capable of stimulating the VISTA-receptor.
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COMP VISTA.COMP VISTA.Fc
VISTA-receptor
A
C
Immobilized Soluble
3H
-Th
ym
idin
e (
CP
M)
B
*
* *
VISTA.Fc
VISTA.Fc + VISTA.COMP
D
PE-anti-IgG
Figure 4-3. VISTA.COMP binds to a clonal T-cell line and suppresses its activation. (A)
2.10 clonal T-cells were activated in culture with immobilized anti-CD3 antibody (3 µg/mL) in
the presence of immobilized or soluble VISTA-Fc or VISTA.COMP (10 µg/mL) and
proliferation measured by pulsing cells with 3H-Thymidine in the last 6 hours of a 24hr culture.
As observed with primary CD4+ T-cells, pentameric VISTA.COMP suppresses proliferation
both when immobilized or added soluble in culture media, whereas VISTA-Fc only exhibits
suppressive activity when immobilized. (Data represents mean ± SEM, ***P<0.01 relative to
anti-CD3 stimulated control, n=3). Data is representative of at least three independent
experiments. (B) Titration of soluble VISTA.COMP (blue) or VISTA-Fc (red) on 2.10 cells being
activated as described in panel A. Data shows a lack of anti-proliferative effect on cells by soluble
VISTA-Fc at high concentrations (Each point represents mean ± SEM, **P<0.01). (C) FACS
analysis of biotinylated COMP, biotinylated VISTA.COMP, and VISTA-Fc (shaded histogram)
binding to 2.10 clonal T-cells compared to unstained control (empty histograms). Data is
representative of three independent experiments. (D) FACS derived histograms of VISTA-Fc
binding to 2.10 cells (shaded histograms) in the presence or absence of equimolar unlabelled
VISTA.COMP. Clear histograms represent unstained cells. VISTA.COMP readily out-competes
VISTA-Fc binding to these cells.
110
an
ti-I
L2
anti-CD3
anti-CD3 + VISTA.Fc
anti-CD3 + VISTA.COMP
SSC
14.7 13.1 7.32
B
A
SPIP: anti-CD3
IB: anti-pY
anti-CD3: + - +
VISTA.COMP: - - +
11
20 17
25
35
48
63
75 100 135 180 245
Figure 4-4. VISTA.COMP suppresses 2.10 T-cell IL-2 secretion and TCR- phosphorylation
cascades. (A) Anti-CD3 activated 2.10 T-cells were treated with soluble VISTA-Fc or
VISTA.COMP for 4 hours and the production of IL-2 measured by ICFC. Only VISTA.COMP
significantly suppressed the number of IL-2 secreting cells. Data is representative of two
independent experiments. (B) 2.10 clonal T-cells were cultured in a 6-well plate with
immobilized anti-CD3 antibody in the presence or absence of VISTA.COMP for 10 minutes.
Proteins in complex with the T-cell receptor (TCR) were recovered by lysing the cells in each
well and recovering the proteins adhered in each well. Eluted proteins were detected by western
blot using anti-phosphotyrosine antibody (4G10). VISTA.COMP substantially diminished the
phosphorylation of TCR complex proteins induced by TCR signaling.
111
4.4.3 VISTA.COMP Prolongs the Survival of Allografts in a MHC-Mismatched Skin Transplant Model
The ability of VISTA.COMP to suppress T-cell activity in-vitro as a soluble ligand suggests that
it may be a useful agonist to suppress pro-inflammatory responses in-vivo. To this end,
VISTA.COMP was first tested in a murine skin allograft model. Here BALB/C mice received skin
allografts (from C57Bl/6 donors) before receiving treatment with VISTA.COMP, VISTA-Fc, or a
saline control combined with low-dose rapamycin (Figure 4-5A). We have previously
demonstrated that this dose of rapamycin has no effect on graft survival as a monotherapy (see
Chapter 3, Figure 3-8). Strikingly, VISTA.COMP significantly prolonged the survival of skin
allografts with only 1/6 allografts rejected in the VISTA.COMP treatment group at the last day of
treatment (day 15), relative to 6/6 rejected allografts observed in the saline control group (P<0.05,
Mann-Whitney U-test) (Figure 4-5B). Importantly, and in support with our hypothesis, VISTA-Fc
treatment did not prolong allograft survival.
4.4.4 VISTA.COMP Dampens Immune Responses and Reduces Lethality in the ConA-induced Hepatitis Mouse Model
The immunosuppressive effects of VISTA.COMP were also evaluated in an acute inflammatory
hepatic model, namely ConA-induced hepatitis (Tiegs et al., 1992). Here, the administration of
ConA induces acute liver inflammation mediated by a polyclonal activation of CD4+ T and NKT
cells, thereby providing a useful model to assess the suppressive activity of VISTA.COMP on T-
cells in-vivo. Notably, it has been previously shown that agonistic anti-VISTA antibodies directed
towards VISTA on T-cells can rescue mice from lethal ConA-induced hepatic injury. However,
the usefulness of using a VISTA-receptor agonist has not been described. We found that
prophylactic treatment of mice with VISTA.COMP rescued 7 out of 13 (53%) C57Bl/6 mice from
succumbing to a lethal dose of ConA (Figure 4-4C). This treatment correlated with a significant
reduction of TNFα and IL-6 levels in serum 3-hours post ConA injection (P<0.05) (Figure 4-4D).
As seen in the skin allograft model, VISTA-Fc treatment did not have a significant effect on
survival or in reduction of serum TNFα and IL-6 levels (Figure 4-6A,B). The striking results from
these two acute inflammatory disease models suggest that VISTA.COMP may serve as a strong
agonist to suppress inflammatory responses in-vivo.
112
Figure 4-5. VISTA.COMP suppresses immune responses in-vivo. (A) Schematic
representation of the skin allograft rejection model. On day 0, BALB/C animals are engrafted
with skin from C57BL/6 mice and subsequently treated with VISTA.COMP of VISTA-Fc or
PBS over the course of 15 days (arrows). Graft survival was monitored daily by a blinded
investigator. (B) Treatment with VISTA.COMP, but not VISTA-Fc, significantly prolonged
survival of skin allografts. (n=7, *P<0.05 by Mann-Whitney U-Test). VISTA.COMP activity
was confirmed in two independent experiments (pooled n=11, *P<0.01) (C) Treatment with
VISTA.COMP 2 hr prior to Con-A injection rescued C57BL/6 mice from lethal hepatic injury
at 24h (n=13, P<0.05 using Proportion Test). Moribund animals are classified as non-responders
(see Methods). Data is pooled from three independent trials. Treated mice exhibited a significant
decrease in serum (D)TNFα (n=5, *P<0.05) and (E) IL6 (n=5, *P<0.05) 3 hours post Con-A
injection.
113
Pro
po
rtio
n a
t 24h
(%
)
A
TNF
No T
reat
men
t
VIS
TA.C
OM
P
VIS
TA.F
c
0
200
400
600
800
1000
*
IL6
No T
reat
men
t
VISTA
.COM
P
VIS
TA.F
c
0
5000
10000
15000
20000
*
B C
Figure 4-6. VISTA-Fc does not rescue animals from ConA induced hepatitis. (A) Male
C57Bl/6 mice were treated with VISTA.COMP or VISTA-Fc and challenged with a lethal dose
of ConA as described above. Animals were monitored regularly and non-responders were
humanely sacrificed by a blinded investigator when deemed moribund. Treatment with
VISTA.COMP, but not VISTA-Fc, led to an increase in the number of animals surviving
challenge. Additionally, VISTA.COMP treated animals had significantly lower levels of serum
(B) TNFα (C) and IL6 three hours post ConA challenge than the VISTA-Fc or non-treated
controls (*P<0.05, n=4)
114
4.5 Discussion
The activation of immune checkpoint receptors on T-cells is often initiated through the binding of
an IgV-like domain displayed by a protein ligand such as PD-L1 expressed on APCs to a
complementary IgV-like domain on its cognate immune checkpoint receptor PD-1 on T-cells. Past
structural studies have demonstrated that monomeric forms of these IgV-like domains involving
PD1:PD-L1 and CD28:CD80/CD86 interact with each other with modest affinity, reflected by
dissociation constants (KD) typically in the low micromolar (µM) range (Lin et al., 2008; van der
Merwe, Bodian, Daenke, Linsley, & Davis, 1997). To activate checkpoint receptors on T-cells in-
vitro, these immune checkpoint ligands are often expressed as oligomers such as Fc fusion proteins
and are immobilized on a surface; a presentation that mimics avidity events taking place when
such immune checkpoint domains are displayed on the surface of APCs and T-cells. In line with
this avidity requirement, there are currently no soluble agonists directed at VISTA, PD-1 or other
checkpoint receptors in clinical trials.
Here we have shown that the pentamerization of the VISTA IgV-like domain by fusing it to a
short, bundle-forming α-helical peptide derived from the Cartilage Oligomeric Matrix Protein
(COMP) generates a VISTA-IgV scaffold that is sufficient to agonize the putative VISTA-receptor
to suppress T-cells in-vitro, and dampen acute inflammatory reactions in models of MHC-
mismatched skin transplants as well as acute inflammatory hepatic injury. Altogether our data
supports a role for VISTA.COMP as a high-avidity checkpoint receptor agonist. Comparisons
between immobilized and soluble VISTA-Fc and VISTA.COMP show that their ability to act as
VISTA-receptor agonists depends on the level of oligomerization, favoring the higher-avidity
created by immobilization of the VISTA IgV-like domain on a solid surface or by multimerization
using the COMP pentamerization domain. We have found the COMP domain to be a useful
scaffold for expressing stable VISTA pentamers. However, the affinity/avidity and mechanism of
action of VISTA multimers on immune cells is currently difficult to characterize largely due to the
unknown identity of its receptor. Our data, and others, suggest that VISTA functions as an early
regulator of T-cell activation, with VISTA-signaling capable of imprinting long lasting
suppressive effects on T-cells. Indeed, we have shown a dramatic reduction in TCR-triggered
phosphorylation events by VISTA.COMP. Currently, the molecular pathways at play remain to be
elucidated, and may become clearer upon discovery of the putative receptor. Notably, it has been
previously reported that VISTA interacts with itself in a homotypic fashion (Yoon et al., 2015).
115
However, we have not observed a direct homotypic interaction in binding assays using purified
VISTA.COMP and VISTA-Fc (Appendix 7). More importantly, the lack of support for a
physiologically relevant VISTA-homotypic interaction between APC and T-cells comes from a
reported experiment where OVA peptide recognizing T-cells isolated from VISTA-/- mice remain
sensitive to suppression by VISTA expressed on the surface of APCs (Flies et al., 2014). We also
found that the high-avidity VISTA.COMP did not bind to homologous checkpoint receptors such
as PD-1 and PD-L1 (Appendix 7), confirming conclusions reached by others (Flies et al., 2014).
Thus, it seems apparent that VISTA functions as a checkpoint ligand through a putative counter-
receptor expressed on T-cells which has yet to be identified. Research is currently ongoing to
identify the VISTA-receptor and characterize the cell-signaling mechanisms triggered by
VISTA.COMP in both mouse and human T-cells.
4.6 Conclusions
The striking activity of VISTA.COMP in suppressing T-cell responses both in-vitro and in-vivo,
combined with the observation of exacerbated autoimmune diseases upon genetic deletion of
VISTA, suggest a potential utility of targeting the VISTA inhibitory pathway using a high-avidity
VISTA IgV-like domain containing scaffold to clinically suppress undesired immune responses.
116
117
Chapter 5: Conclusions and Future Directions
118
Conclusion and Future Directions
5.1 Thesis Conclusions
The overarching goal of this thesis was to derive functional ligands which agonize or antagonize
immune checkpoint receptors, and to explore the potential therapeutic relevance of such ligands
using in-vivo models human disease.
Chapters 2 & 3 cover projects which involved the extension of aptamer technology to immune
checkpoint targets. As DNA aptamers have several advantages relative to the protein-based
therapeutics, such as low cost and reduced immunogenicity, aptamer-based therapeutics targeting
validated immune checkpoint components (ligands and/or receptors) may represent attractive
alternatives to mAbs currently used in the clinic (Keefe et al., 2010). Chapter 2 provides a proof-
of-principle that one can derive DNA aptamers which bind specifically to murine PD-1 and block
PD-1:PD-L1 signaling to enhance anti-tumor immunity. Importantly, treatment with a PEGylated
form of the lead aptamer (PEG-MP7) suppresses the growth of murine syngeneic colon carcinoma
MC38 cells in C57Bl/6 mice as efficiently as a PD-1 blocking mAb. Chapter 3 is an account of
our success in deriving novel DNA aptamers to another immune inhibitory receptor, CD200R1. In
this case however, we identified aptamers which acted as agonists, capable of stimulating
CD200R1 signaling to suppress inflammatory responses both in-vitro and in-vivo. Specifically, we
demonstrated that PEGylated anti-CD200R1 agonistic aptamers could prolong allograft survival
in a model of transplant rejection, and reduce airway inflammation in a model of HDM-induced
allergy. To the best of our knowledge, these results represent the first report of an agonistic aptamer
towards an immune inhibitory receptor. Another important advance detailed in Chapter 3, was the
successful incorporation of NGS analysis into SELEX screens, which led to the identification of
cross-species CD200R1-binding aptamers. Altogether, findings from these two chapters confirm
that aptamers directed at IgV-like domain containing checkpoint receptors can potently modulate
(augment or dampen) disease-related immunological responses, by acting as either agonists or
antagonists.
Chapter 4 similarly involved the design of a novel ligand targeting an IgV-like domain containing
checkpoint molecule, namely the VISTA-receptor. VISTA is a newly discovered checkpoint
molecule which binds to an uncharacterized receptor on T-cells to suppress their activation (Wang
et al. 2011). Several studies have implicated this pathway as a key regulator of T-cell mediated
119
immune responses (Lines, Pantazi, et al., 2014; Nowak et al., 2017; Wang et al., 2014). However,
the currently unknown nature of the VISTA-receptor(s) precludes the ability to target this
checkpoint receptor with mAbs or aptamers. Thus, we constructed a multivalent form of VISTA
by fusing its extracellular IgV-like domain to a short, pentamer-forming α-helical peptide derived
from the cartilage oligomeric matrix protein (COMP). This fusion protein, termed VISTA.COMP,
was expressed as a stable pentamer and potently suppressed T-cell activation. Importantly,
VISTA.COMP inhibits T-cell activation in-vitro as a soluble entity, without the requirement of
immobilization on a solid surface as is the case with a dimeric VISTA-Fc chimera. In-vivo,
VISTA.COMP treatment was found to suppress inflammatory responses in models of transplant
rejection and acute inflammatory hepatitis, supporting the hypothesis that agonizing the
VISTA:VISTA-receptor represents a new strategy for therapeutically modulating T-cell mediated
inflammatory diseases.
5.2 Work in Progress and Future Directions
5.2.1 Continued Derivation of Aptamers to Immune Checkpoint Molecules
Work by our laboratory and others, has led to the derivation of RNA or DNA aptamers targeting
several immune checkpoints including PD-1, CD200R1, CTLA-4 and TIM-3 (Hervas-Stubbs et
al., 2016; Prodeus et al., 2015; Prodeus et al., 2014; Santulli-Marotto et al., 2003). Importantly,
these studies have consistently found aptamers to be as effective as characterized mAbs to the
same target, supporting the use of aptamers as useful immune modulators. Moving forward, there
are a growing number of immune co-stimulatory and inhibitory receptors which represent
attractive targets for further aptamer discovery (Le Mercier et al., 2015). Furthermore, our proof-
of-principle research with murine PD-1 targeting aptamers suggests that an equivalent aptamer
targeting human PD-1 is of therapeutic relevance. To this end, our laboratory is currently
performing SELEX screens on several targets including human PD-1 and VISTA. In the case of
VISTA, our SELEX screens will be utilizing NGS in an attempt to identify cross-species
mouse/human VISTA-blocking aptamers using the same successful SELEX strategy that yielded
CD200R1 aptamer agonists. The primary goal of this screen is to identify antagonistic VISTA
aptamers with a view to characterize their potential to provoke anti-tumor immune responses.
Notably, VISTA is expressed by myeloid-derived suppressor cells within the tumor
microenvironment (TME) of most human cancers where it is postulated to contribute to immune
120
evasion by suppressing the function of infiltrating anti-tumor T-cells, suggesting that VISTA is a
valid target for cancer immunotherapy (Deng, Le Mercier, Kuta, & Noelle, 2016; Lines, Sempere,
et al., 2014). Of interest, a recent report found that VISTA expression is significantly upregulated
in prostate cancers which are resistant to CTLA-4 blockade (Gao et al., 2017). Antagonistic anti-
VISTA mAbs have shown positive results in several pre-clinical syngeneic tumor models, both as
a monotherapy and in combination with anti-PD-1 mAbs, leading to the recent initiation of a Phase
I trial of a humanized anti-VISTA mAb in solid tumors (Le Mercier et al., 2014). Thus, SELEX-
screens which identify cross-species VISTA-blocking aptamers may lead to the derivation of
checkpoint inhibitors with rapid bench-to-bedside potential.
5.2.2 Pharmacokinetics of DNA aptamers
The biggest limitation to the clinical translation of checkpoint targeting aptamers is the poor
pharmacokinetic properties associated with their systemic use as was described in Section 1.6.4.
Specifically, the t1/2 of most PEGylated aptamers has been reported to range from 12-48 hours,
which is far inferior to the t1/2 of 12-20 days seen with checkpoint targeting humanized mAbs
(Healy et al., 2004). It is important to note that most of the aptamer pharmacokinetic studies
reported in the literature have focused on the t1/2 and biodistribution of protected RNA aptamers.
We have not yet determined the t1/2 of any aptamer used in this thesis. However, it is clear that
PEGylation of M49 was critical to its ability to prolong allograft survival (Figure 3-8). Future
research is warranted to determine the PK properties of the DNA aptamers discovered in this thesis,
particularly aptamer CCS13, the mouse/human CD200R1 agonist which carries clinical relevance.
For this purpose, we have synthesized a CCS13-derivative that contains a 5’-termini 20 kDa linear
polyethylene glycol group as well as a metal chelator, namely diethylene triamine pentaacetic acid
(DTPA) at its 3’ end. Future research will involve labelling this aptamer-derivative with gamma-
emitting radionucleotides such as 111In or 99mTc to carry out imaging and biodistribution studies in
mice using PET/SPECT/CT. Furthermore, radiolabeled chelator-aptamer conjugates can be used
to determine the importance of mass (20kDa vs 40kDa vs 80kDa) and presentation (linear vs
branched PEG) of PEG polymers on their circulation half-life and biodistribution. For instance, a
recent report found that conjugation of a protected anti-IL17A RNA aptamer to a highly-branched
PEG group (2x2 arm, 80kDa PEG) extended the half-life to ~4.5 days in cynomolgus monkeys, a
number far superior to that seen with linear PEG groups (Haruta et al., 2017). These critical
experiments will provide insight into the PK and biodistribution properties of differing DNA
121
aptamer formulations; findings which could have critical implications on the clinical translation of
immunomodulatory aptamers.
5.2.3 Local Delivery of CD200R1 Aptamers to Treat Allergic Disorders
While the poor PK properties of unmodified aptamers currently limit their potential systemic use,
the local delivery of aptamers to effected tissues may be appropriate in certain indications. For
example, the only FDA-approved aptamer Pegatinib (a PEGylation anti-VEGF protected RNA
aptamers), used to treat age related macular degeneration, is delivered to the vitreous humor by
direct intraocular injection to inhibit local angiogenesis (Ng et al., 2006). The results presented in
Chapter 3 demonstrated that the systemic (intravenous) injection of a PEGylated CD200R1
agonistic aptamer (PEG-CCS13) could suppress airway hyper-responsiveness in a mouse model
of HDM-induced allergy. Current research is now ongoing to determine if intranasal
administration of this aptamer can suppress local inflammation and lung remodeling in a mouse
model of chronic HDM-induced allergic asthma. Preliminary results thus far have indicated a
therapeutic advantage in this local delivery route, in terms of decreased methacholine-induced
airway hyper-responsiveness after the HDM-challenge of sensitized mice receiving intratracheal
injections of PEG-CCS13 when compared to control animals. Importantly, this local delivery
approach bypasses the limitations associated with the systemic use of oligonucleotide aptamers
and in doing so provides a clinically effective modality for the treatment of these indications.
5.2.4 Identification of the VISTA Receptor
The study presented in Chapter 4, along with work from others, clearly define the VISTA:VISTA-
receptor axis as a critical pathway for the regulation of T-cell mediated immune responses (Nowak
et al., 2017). Modulation of immunity by either promoting or inhibiting this checkpoint pathway
is now proposed as a therapeutic strategy to treat autoimmune disorders and cancer respectively
(Ceeraz et al., 2014; Lines, Sempere, et al., 2014). However, there is still much that remains
unknown about the specific biology of this pathway. Primarily, the presently unknown identity of
the VISTA-receptor severely limits our understanding of this pathway in human health and
disease. Accordingly, ongoing research in our laboratory is aimed at identifying the putative
VISTA-receptor(s), with a view to characterize its identify, function, and temporal expression
patterns on various tissue and cell types. We have confirmed that VISTA does not bind to the
structurally related PD-1 and PD-L1, and does not bind to itself in a homotypic fashion as is seen
122
with the N-terminal IgV-like domain of CEACAM-5 (Appendix 7). Several approaches to identify
this receptor are underway, including the use of the high avidity VISTA.COMP, as bait in IP/MS-
based proteomic assays. In the event that the putative receptor is elucidated, future work will
revolve around characterizing its role in immune regulation. Such experiments would include, for
example, the phenotypic characterization of VISTA-receptor knockout mice. In the long term,
mAbs or aptamers targeting this checkpoint receptor should be investigated for their potential as
anti-cancer therapies.
In addition to acting as a checkpoint ligand, several studies have shown that VISTA is also
expressed by T-cells, where it functions as a receptor to transduce immune-inhibitory signals upon
binding to a previously uncharacterized ligand. A recent poster by J. Wang et al. (R&D Systems),
suggested that IGSF11 (VSIG3) is a ligand for VISTA, which functions to inhibit T-cell function.
Experimentally, we have since confirmed that VSIG3 does bind to VISTA (Figure 5-1A,B;
unpublished data). SPR-based binding assays show that IGSF11 binds to VISTA-Fc with an
avidity constant of 1.3 µM, and to VISTA.COMP with an increased avidity of 0.024 µM (Figure
5-1C,D). Importantly, the physiological relevance of this interaction is not yet understood. Of note,
VSIG3 does not have an expression pattern consistent with other immune checkpoint molecules,
as it is pre-dominantly expressed in the brain where it binds to postsynaptic scaffolding protein
(PSD-95) and AMPA glutamate receptors (AMPARs) and functions as a synaptic adhesion model
(Jang et al., 2016). IGSF11 is also expressed in the testes and ovaries, but has not been detected in
immune tissues such as the spleen and lymph nodes. Current work is ongoing to determine if
IGSF11 is inducibly-expressed on myeloid, lymphoid, or cancer cells, with a view to characterize
if an immunological role for this ligand exists. In particular, we are focused on identifying if
IGSF11 is also expressed on T-cells, and if so, determine if it is the unknown receptor responsible
for transducing VISTA immune-inhibitory signals.
5.2.5 VISTA-mediated Signaling Events in T-cells
In addition to characterizing of the putative VISTA binding partner(s), current work is ongoing to
define the genome wide changes in T-cells induced by VISTA:VISTA-receptor inhibitory
signaling. To accomplish this, we have recently performed RNA-sequencing experiments to
identify genes which are differentially expressed in an activated clonal T-cell line upon treatment
with VISTA.COMP relative to the control COMP-only or PD-L1.COMP constructs. Preliminary
123
data analysis showed that of 10,593 genes sequenced, 19 were differentially regulated (adjusted
P-value < 0.05 and fold change > 1.5) by VISTA.COMP relative to COMP (Figure 5-2A). Of these
genes, 6 were found to be upregulated, and 13 downregulated (Figure 5-2B). As expected, a
number pro-inflammatory cytokine and chemokines and receptors normally expressed by T-cell
activation were downregulated by VISTA.COMP (Figure 5-3C). Further investigations into the
remaining differentially-expressed genes are warranted, in particular focusing on hits that were
identified to be differentially expressed by VISTA.COMP treatment, but not PD-L1.COMP, as
these genes are more likely to be responsible for effecting VISTA inhibitory signaling, and not a
result of general suppression of T-cell activation. One such gene encodes the protein PIK3IP1, a
negative regulator of PI3K-signalling a common pathway involved in the transduction of signals
from the TCR, CD28, and IL2-receptor (Okkenhaug & Vanhaesebroeck, 2003). Notably,
transfection of T-cell lines with PIK3IP1 suppresses T-cell activation and IL-2 secretion, pointing
to an interesting causal relationship of VISTA-mediated PIK3IP1 upregulation and T-cell
suppression (DeFrances, Debelius, Cheng, & Kane, 2012). Future key studies will focus on
validating PIK3IP1 as a VISTA-induced gene, and on interrogating the role played, if any, by this
pathway in imparting the inhibitory effects of the VISTA:VISTA-receptor pathway.
124
Buf
fer
IgG-F
c
IGSF11
-Fc
0
10
20
30
40
50
Binding to VISTA-Fc
Bin
din
g S
tabili
ty (
RU
)
Buf
fer
mVIS
TA-F
c
hVIS
TA-F
c0
50
100
150
Binding to hIGSF-11
A B
C D
VISTA-Fc > IGSF11-Fc
KD = 1.3 µM
VISTA.COMP > IGSF11-Fc
KD = 0.024 µM
Concentration (M)
Bin
din
g R
espo
nse
(R
U)
Concentration (M)
Bin
din
g R
espo
nse
(R
U)
Figure 5-1. VISTA binds to IGSF11. The direct interaction of recombinant VISTA-Fc and
IGSF11-Fc was confirmed by Surface Plasmon Resonance (SPR) (A) A negative control Fc (IgG-
Fc) or recombinant human IGSF11-Fc were injected over a CM5 chip containing immobilized
human VISTA-Fc. IGSF11-Fc, but not IgG-Fc, readily bound to immobilized VISTA-Fc.
Binding Stability (RU) was measured by subtracting the binding response from of the VISTA-Fc
containing flow cell with that of an empty reference flow cell. (B) Reverse orientation of
experiment as in A. hVISTA-Fc readily binds to immobilized hIGSF-11. Interestingly, mVISTA-
Fc does not bind to hIGSF-11 showing that the interaction is species dependent. (C) SPR-based
steady state affinity assay of VISTA-Fc binding to immobilized IGSF11. Binding curves were
created by plotting the steady-state binding response (RU) as a function of VISTA-Fc
concentration, yielding an avidity constant of 1.2µM. (D) Steady-state binding experiment of
VISTA.COMP binding to IGSF-11 show an increased avidity constant of 0.024µM.
125
A
COMP VISTA.
COMP
IL2ra
Ifng
Cd69
IL2
Mif
Ccl3 Ccl4
Pdcd1
IL10
Ccl1
B C
Figure 5-2 Differential expression of genes in a T-cell line treated with VISTA.COMP. (A)
RNA sequencing (Ion Torrent HiSeq 2500) was performed on anti-CD3 activated 2.10 T-cells
treated with VISTA.COMP, or COMP (negative control) (n=3). Gene counts across these
samples were normalized and compared using the DeSeq2 algorithm (Illumina BaseSpace
Software) identifying 19 differentially-expressed genes as defined by fold change (FC) > 1.5 and
adjusted P-value < 0.05. (B) Volcano plot of RNA-sequencing data showing fold changes and
adjusted P-values of genes differentially regulated upon VISTA.COMP treatment. The
horizontal line (P=0.05) indicates significance, and vertical lines are placed at a fold change of
± 1.5. Genes labelled with blue dots were not significantly changed. Genes labelled with red and
grey dots were found to be significantly changed, with grey dots representing significance and
an absolute fold change of >1.5. (C) Heat map showing Z-scores of selected differentially
expressed genes across replicate samples (columns). Selected genes include pro-inflammatory
chemokines, cytokines, or cell surface receptors which are preferentially expressed by activated
T-cells. With the exception of CD69, these genes are found to be downregulated by
VISTA.COMP.
126
5.3 Concluding Remarks
Over the past decade there has been an immense amount of research focused on the discovery and
characterization of immune regulatory checkpoint receptors. Excitingly, this research has led to
direct benefits to society in the form of advanced anti-cancer therapies with never-before-seen
efficacy. With countless clinical trials ongoing, it is likely that the full potential of checkpoint
inhibitors targeting PD-1 and CTLA-4 are yet to be realized. Further research in the field has also
led to the recent discovery of checkpoint molecules such as VISTA, whose inhibition may also
prove to have clinical utility. In addition to blocking these pathways to mobilize anti-tumor
responses, it is also now clear that enhancing checkpoint-mediated immune-inhibitory signaling
may prove useful to patients suffering from autoimmune and inflammatory disorders. The work
presented in this thesis has focused on these checkpoint molecules, with a view to advance our
understanding on the best methods to therapeutically target these receptors by developing novel
nucleic acid and protein-based targeting agents. Collectively, the primary research presented
within should serve as a stepping-stone to advance our knowledge of checkpoint receptors and
how to target them with effective therapeutic formulations.
127
128
References
Alam, K. K., Chang, J. L., & Burke, D. H. (2015). FASTAptamer: A Bioinformatic Toolkit for
High-throughput Sequence Analysis of Combinatorial Selections. Mol Ther Nucleic
Acids, 4, e230. doi:10.1038/mtna.2015.4
Auchincloss, H., & Turka, L. A. (2011). CTLA-4: not all costimulation is stimulatory. J
Immunol, 187(7), 3457-3458. doi:10.4049/jimmunol.1102316
Azuma, T., Zhu, G., Xu, H., Rietz, A. C., Drake, C. G., Matteson, E. L., & Chen, L. (2009).
Potential role of decoy B7-H4 in the pathogenesis of rheumatoid arthritis: a mouse model
informed by clinical data. PLoS Med, 6(10), e1000166.
doi:10.1371/journal.pmed.1000166
Berezhnoy, A., Stewart, C. A., Mcnamara Ii, J. O., Thiel, W., Giangrande, P., Trinchieri, G., &
Gilboa, E. (2012). Isolation and Optimization of Murine IL-10 Receptor Blocking
Oligonucleotide Aptamers Using High-throughput Sequencing. Mol Ther, 20(6), 1242-
1250. doi:10.1038/mt.2012.18
Bertrand, A., Kostine, M., Barnetche, T., Truchetet, M. E., & Schaeverbeke, T. (2015). Immune
related adverse events associated with anti-CTLA-4 antibodies: systematic review and
meta-analysis. BMC Med, 13, 211. doi:10.1186/s12916-015-0455-8
Blind, M., & Blank, M. (2015). Aptamer Selection Technology and Recent Advances. Mol Ther
Nucleic Acids, 4, e223. doi:10.1038/mtna.2014.74
Boudakov, I., Liu, J., Fan, N., Gulay, P., Wong, K., & Gorczynski, R. M. (2007). Mice lacking
CD200R1 show absence of suppression of lipopolysaccharide-induced tumor necrosis
factor-alpha and mixed leukocyte culture responses by CD200. Transplantation, 84(2),
251-257. doi:10.1097/01.tp.0000269795.04592.cc
Broderick, C., Hoek, R. M., Forrester, J. V., Liversidge, J., Sedgwick, J. D., & Dick, A. D.
(2002). Constitutive retinal CD200 expression regulates resident microglia and activation
state of inflammatory cells during experimental autoimmune uveoretinitis. Am J Pathol,
161(5), 1669-1677. doi:10.1016/S0002-9440(10)64444-6
Callahan, M. K., & Wolchok, J. D. (2013). At the bedside: CTLA-4- and PD-1-blocking
antibodies in cancer immunotherapy. J Leukoc Biol, 94(1), 41-53.
doi:10.1189/jlb.1212631
Ceeraz, S., Nowak, E. C., Burns, C. M., & Noelle, R. J. (2014). Immune checkpoint receptors in
regulating immune reactivity in rheumatic disease. Arthritis Res Ther, 16(5), 469.
Ceeraz, S., Nowak, E. C., & Noelle, R. J. (2013). B7 family checkpoint regulators in immune
regulation and disease. Trends Immunol, 34(11), 556-563. doi:10.1016/j.it.2013.07.003
129
Ceeraz, S., Sergent, P. A., Plummer, S. F., Schned, A. R., Pechenick, D., Burns, C. M., & Noelle,
R. J. (2016). VISTA deficiency accelerates the development of fatal murine lupus
nephritis. Arthritis Rheumatol. doi:10.1002/art.40020
Chames, P., Van Regenmortel, M., Weiss, E., & Baty, D. (2009). Therapeutic antibodies:
successes, limitations and hopes for the future. Br J Pharmacol, 157(2), 220-233.
doi:10.1111/j.1476-5381.2009.00190.x
Chattopadhyay, K., Lazar-Molnar, E., Yan, Q., Rubinstein, R., Zhan, C., Vigdorovich, V., . . .
Almo, S. C. (2009). Sequence, structure, function, immunity: structural genomics of
costimulation. Immunol Rev, 229(1), 356-386. doi:10.1111/j.1600-065X.2009.00778.x
Chen, L., & Flies, D. B. (2013). Molecular mechanisms of T cell co-stimulation and co-
inhibition. Nat Rev Immunol, 13(4), 227-242. doi:10.1038/nri3405
Cheng, X., Veverka, V., Radhakrishnan, A., Waters, L. C., Muskett, F. W., Morgan, S. H., . . .
Davis, S. J. (2013). Structure and interactions of the human programmed cell death 1
receptor. J Biol Chem, 288(17), 11771-11785. doi:10.1074/jbc.M112.448126
Cherwinski, H. M., Murphy, C. A., Joyce, B. L., Bigler, M. E., Song, Y. S., Zurawski, S. M., . . .
Phillips, J. H. (2005). The CD200 receptor is a novel and potent regulator of murine and
human mast cell function. J Immunol, 174(3), 1348-1356.
Cho, C. H., Kammerer, R. A., Lee, H. J., Steinmetz, M. O., Ryu, Y. S., Lee, S. H., . . . Koh, G.
Y. (2004). COMP-Ang1: a designed angiopoietin-1 variant with nonleaky angiogenic
activity. Proc Natl Acad Sci U S A, 101(15), 5547-5552. doi:10.1073/pnas.0307574101
Collins, M., Ling, V., & Carreno, B. M. (2005). The B7 family of immune-regulatory ligands.
Genome Biol, 6(6), 223. doi:10.1186/gb-2005-6-6-223
Copland, D. A., Calder, C. J., Raveney, B. J., Nicholson, L. B., Phillips, J., Cherwinski, H., . . .
Dick, A. D. (2007). Monoclonal antibody-mediated CD200 receptor signaling suppresses
macrophage activation and tissue damage in experimental autoimmune uveoretinitis. Am
J Pathol, 171(2), 580-588. doi:10.2353/ajpath.2007.070272
Czajkowsky, D. M., Hu, J., Shao, Z., & Pleass, R. J. (2012). Fc-fusion proteins: new
developments and future perspectives. EMBO Mol Med, 4(10), 1015-1028.
doi:10.1002/emmm.201201379
DeFrances, M. C., Debelius, D. R., Cheng, J., & Kane, L. P. (2012). Inhibition of T-cell
activation by PIK3IP1. Eur J Immunol, 42(10), 2754-2759. doi:10.1002/eji.201141653
Delves, P. J., & Roitt, I. M. (2000a). The immune system. First of two parts. N Engl J Med,
343(1), 37-49. doi:10.1056/NEJM200007063430107
Delves, P. J., & Roitt, I. M. (2000b). The immune system. Second of two parts. N Engl J Med,
343(2), 108-117. doi:10.1056/NEJM200007133430207
130
Deng, J., Le Mercier, I., Kuta, A., & Noelle, R. J. (2016). A New VISTA on combination therapy
for negative checkpoint regulator blockade. J Immunother Cancer, 4, 86.
doi:10.1186/s40425-016-0190-5
Dollins, C. M., Nair, S., Boczkowski, D., Lee, J., Layzer, J. M., Gilboa, E., & Sullenger, B. A.
(2008). Assembling OX40 aptamers on a molecular scaffold to create a receptor-
activating aptamer. Chem Biol, 15(7), 675-682. doi:10.1016/j.chembiol.2008.05.016
Dollins, C. M., Nair, S., & Sullenger, B. A. (2008). Aptamers in immunotherapy. Hum Gene
Ther, 19(5), 443-450. doi:10.1089/hum.2008.045
Drake, C. G., Jaffee, E., & Pardoll, D. M. (2006). Mechanisms of immune evasion by tumors.
Adv Immunol, 90, 51-81. doi:10.1016/S0065-2776(06)90002-9
Dyke, C. K., Steinhubl, S. R., Kleiman, N. S., Cannon, R. O., Aberle, L. G., Lin, M., . . .
Rusconi, C. P. (2006). First-in-human experience of an antidote-controlled anticoagulant
using RNA aptamer technology: a phase 1a pharmacodynamic evaluation of a drug-
antidote pair for the controlled regulation of factor IXa activity. Circulation, 114(23),
2490-2497. doi:10.1161/CIRCULATIONAHA.106.668434
Efimov, V. P., Lustig, A., & Engel, J. (1994). The thrombospondin-like chains of cartilage
oligomeric matrix protein are assembled by a five-stranded alpha-helical bundle between
residues 20 and 83. FEBS Lett, 341(1), 54-58.
Ellington, A. D., & Szostak, J. W. (1990). In-vitro selection of RNA molecules that bind specific
ligands. Nature, 346(6287), 818-822. doi:10.1038/346818a0
Ellington, A. D., & Szostak, J. W. (1992). Selection in-vitro of single-stranded DNA molecules
that fold into specific ligand-binding structures. Nature, 355(6363), 850-852.
doi:10.1038/355850a0
Eulberg, D., & Klussmann, S. (2003). Spiegelmers: biostable aptamers. Chembiochem, 4(10),
979-983. doi:10.1002/cbic.200300663
Flies, D. B., Han, X., Higuchi, T., Zheng, L., Sun, J., Ye, J. J., & Chen, L. (2014). Coinhibitory
receptor PD-1H preferentially suppresses CD4⁺ T cell-mediated immunity. J Clin Invest,
124(5), 1966-1975. doi:10.1172/JCI74589
Flies, D. B., Higuchi, T., & Chen, L. (2015). Mechanistic Assessment of PD-1H Coinhibitory
Receptor-Induced T Cell Tolerance to Allogeneic Antigens. J Immunol, 194(11), 5294-
5304. doi:10.4049/jimmunol.1402648
Francisco, L. M., Sage, P. T., & Sharpe, A. H. (2010). The PD-1 pathway in tolerance and
autoimmunity. Immunol Rev, 236, 219-242. doi:10.1111/j.1600-065X.2010.00923.x
Francisco, L. M., Salinas, V. H., Brown, K. E., Vanguri, V. K., Freeman, G. J., Kuchroo, V. K.,
& Sharpe, A. H. (2009). PD-L1 regulates the development, maintenance, and function of
induced regulatory T cells. J Exp Med, 206(13), 3015-3029. doi:10.1084/jem.20090847
131
Freeman, G. J., Long, A. J., Iwai, Y., Bourque, K., Chernova, T., Nishimura, H., . . . Honjo, T.
(2000). Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family
member leads to negative regulation of lymphocyte activation. J Exp Med, 192(7), 1027-
1034.
Ganson, N. J., Povsic, T. J., Sullenger, B. A., Alexander, J. H., Zelenkofske, S. L., Sailstad, J.
M., . . . Hershfield, M. S. (2016). Pre-existing anti-polyethylene glycol antibody linked to
first-exposure allergic reactions to pegnivacogin, a PEGylated RNA aptamer. J Allergy
Clin Immunol, 137(5), 1610-1613.e1617. doi:10.1016/j.jaci.2015.10.034
Gao, J., Ward, J. F., Pettaway, C. A., Shi, L. Z., Subudhi, S. K., Vence, L. M., . . . Sharma, P.
(2017). VISTA is an inhibitory immune checkpoint that is increased after ipilimumab
therapy in patients with prostate cancer. Nat Med, 23(5), 551-555. doi:10.1038/nm.4308
Gilboa, E., McNamara, J., & Pastor, F. (2013). Use of oligonucleotide aptamer ligands to
modulate the function of immune receptors. Clin Cancer Res, 19(5), 1054-1062.
doi:10.1158/1078-0432.CCR-12-2067
Gorczynski, Cattral, M. S., Chen, Z., Hu, J., Lei, J., Min, W. P., . . . Ni, J. (1999). An
immunoadhesin incorporating the molecule OX-2 is a potent immunosuppressant that
prolongs allo- and xenograft survival. J Immunol, 163(3), 1654-1660.
Gorczynski, Chen, Z., He, W., Khatri, I., Sun, Y., Yu, K., & Boudakov, I. (2009). Expression of
a CD200 transgene is necessary for induction but not maintenance of tolerance to cardiac
and skin allografts. J Immunol, 183(3), 1560-1568. doi:10.4049/jimmunol.0900200
Gorczynski, Chen, Z., Lee, L., Yu, K., & Hu, J. (2002). Anti-CD200R ameliorates collagen-
induced arthritis in mice. Clin Immunol, 104(3), 256-264.
Gorczynski, Chen, Z., Yu, K., & Hu, J. (2001). CD200 immunoadhesin suppresses collagen-
induced arthritis in mice. Clin Immunol, 101(3), 328-334. doi:10.1006/clim.2001.5117
Gorczynski, Hu, J., Chen, Z., Kai, Y., & Lei, J. (2002). A CD200-FC immunoadhesin prolongs
rat islet xenograft survival in mice. Transplantation, 73(12), 1948-1953.
Gorczynski, R., Chen, Z., Khatri, I., & Yu, K. (2013). sCD200 present in mice receiving cardiac
and skin allografts causes immunosuppression in-vitro and induces Tregs.
Transplantation, 95(3), 442-447. doi:10.1097/TP.0b013e3182754c30
Gorczynski, R. M., Hu, J., Chen, Z., Kai, Y., & Lei, J. (2002). A CD200-FC immunoadhesin
prolongs rat islet xenograft survival in mice. Transplantation, 73(12), 1948-1953.
Greenfield, E. A., Nguyen, K. A., & Kuchroo, V. K. (1998). CD28/B7 costimulation: a review.
Crit Rev Immunol, 18(5), 389-418.
Gupta, S., Hirota, M., Waugh, S. M., Murakami, I., Suzuki, T., Muraguchi, M., . . . Schneider, D.
J. (2014). Chemically modified DNA aptamers bind interleukin-6 with high affinity and
inhibit signaling by blocking its interaction with interleukin-6 receptor. J Biol Chem,
289(12), 8706-8719. doi:10.1074/jbc.M113.532580
132
Hamid, O., Robert, C., Daud, A., Hodi, F. S., Hwu, W. J., Kefford, R., . . . Ribas, A. (2013).
Safety and tumor responses with lambrolizumab (anti-PD-1) in melanoma. N Engl J Med,
369(2), 134-144. doi:10.1056/NEJMoa1305133
Haraoui, B., & Bykerk, V. (2007). Etanercept in the treatment of rheumatoid arthritis. Ther Clin
Risk Manag, 3(1), 99-105.
Harding, F. A., Stickler, M. M., Razo, J., & DuBridge, R. B. (2010). The immunogenicity of
humanized and fully human antibodies: residual immunogenicity resides in the CDR
regions. MAbs, 2(3), 256-265.
Haruta, K., Otaki, N., Nagamine, M., Kayo, T., Sasaki, A., Hiramoto, S., . . . Yamazaki, H.
(2017). A Novel PEGylation Method for Improving the Pharmacokinetic Properties of
Anti-Interleukin-17A RNA Aptamers. Nucleic Acid Ther, 27(1), 36-44.
doi:10.1089/nat.2016.0627
Hatherley, D., Cherwinski, H. M., Moshref, M., & Barclay, A. N. (2005). Recombinant CD200
protein does not bind activating proteins closely related to CD200 receptor. J Immunol,
175(4), 2469-2474.
Haughn, L., Gratton, S., Caron, L., Sékaly, R. P., Veillette, A., & Julius, M. (1992). Association
of tyrosine kinase p56lck with CD4 inhibits the induction of growth through the alpha
beta T-cell receptor. Nature, 358(6384), 328-331. doi:10.1038/358328a0
Healy, J. M., Lewis, S. D., Kurz, M., Boomer, R. M., Thompson, K. M., Wilson, C., &
McCauley, T. G. (2004). Pharmacokinetics and biodistribution of novel aptamer
compositions. Pharm Res, 21(12), 2234-2246.
Hervas-Stubbs, S., Soldevilla, M. M., Villanueva, H., Mancheño, U., Bendandi, M., & Pastor, F.
(2016). Identification of TIM3 2'-fluoro oligonucleotide aptamer by HT-SELEX for
cancer immunotherapy. Oncotarget, 7(4), 4522-4530. doi:10.18632/oncotarget.6608
Hirano, F., Kaneko, K., Tamura, H., Dong, H., Wang, S., Ichikawa, M., . . . Chen, L. (2005).
Blockade of B7-H1 and PD-1 by monoclonal antibodies potentiates cancer therapeutic
immunity. Cancer Res, 65(3), 1089-1096.
Hirota, M., Murakami, I., Ishikawa, Y., Suzuki, T., Sumida, S., Ibaragi, S., . . . Schneider, D. J.
(2016). Chemically Modified Interleukin-6 Aptamer Inhibits Development of Collagen-
Induced Arthritis in Cynomolgus Monkeys. Nucleic Acid Ther, 26(1), 10-19.
doi:10.1089/nat.2015.0567
Hodi, F. S., Chesney, J., Pavlick, A. C., Robert, C., Grossmann, K. F., McDermott, D. F., . . .
Postow, M. A. (2016). Combined nivolumab and ipilimumab versus ipilimumab alone in
patients with advanced melanoma: 2-year overall survival outcomes in a multicentre,
randomised, controlled, phase 2 trial. Lancet Oncol, 17(11), 1558-1568.
doi:10.1016/S1470-2045(16)30366-7
133
Hodi, F. S., O'Day, S. J., McDermott, D. F., Weber, R. W., Sosman, J. A., Haanen, J. B., . . .
Urba, W. J. (2010). Improved survival with ipilimumab in patients with metastatic
melanoma. N Engl J Med, 363(8), 711-723. doi:10.1056/NEJMoa1003466
Hoek, R. M., Ruuls, S. R., Murphy, C. A., Wright, G. J., Goddard, R., Zurawski, S. M., . . .
Sedgwick, J. D. (2000). Down-regulation of the macrophage lineage through interaction
with OX2 (CD200). Science, 290(5497), 1768-1771.
Hoffmann, S., Hoos, J., Klussmann, S., & Vonhoff, S. (2011). RNA aptamers and spiegelmers:
synthesis, purification, and post-synthetic PEG conjugation. Curr Protoc Nucleic Acid
Chem, Chapter 4, Unit 4.46.41-30. doi:10.1002/0471142700.nc0446s46
Ishiguro, A., Akiyama, T., Adachi, H., Inoue, J., & Nakamura, Y. (2011). Therapeutic potential
of anti-interleukin-17A aptamer: suppression of interleukin-17A signaling and
attenuation of autoimmunity in two mouse models. Arthritis Rheum, 63(2), 455-466.
doi:10.1002/art.30108
Janeway, C. (2005). Immunobiology : the immune system in health and disease (6th ed.). New
York: Garland Science.
Jang, S., Oh, D., Lee, Y., Hosy, E., Shin, H., van Riesen, C., . . . Kim, E. (2016). Synaptic
adhesion molecule IgSF11 regulates synaptic transmission and plasticity. Nat Neurosci,
19(1), 84-93. doi:10.1038/nn.4176
Jenmalm, M. C., Cherwinski, H., Bowman, E. P., Phillips, J. H., & Sedgwick, J. D. (2006).
Regulation of myeloid cell function through the CD200 receptor. J Immunol, 176(1),
191-199.
Joller, N., Hafler, J. P., Brynedal, B., Kassam, N., Spoerl, S., Levin, S. D., . . . Kuchroo, V. K.
(2011). Cutting edge: TIGIT has T cell-intrinsic inhibitory functions. J Immunol, 186(3),
1338-1342. doi:10.4049/jimmunol.1003081
Juneja, V. R., McGuire, K. A., Manguso, R. T., LaFleur, M. W., Collins, N., Haining, W. N., . . .
Sharpe, A. H. (2017). PD-L1 on tumor cells is sufficient for immune evasion in
immunogenic tumors and inhibits CD8 T cell cytotoxicity. J Exp Med, 214(4), 895-904.
doi:10.1084/jem.20160801
Keefe, A. D., Pai, S., & Ellington, A. (2010). Aptamers as therapeutics. Nat Rev Drug Discov,
9(7), 537-550. doi:10.1038/nrd3141
Khatri , I. B., Brent Lamptey , Adriana Taseva , Karrie Wong , Anna Podnos , Reginald M.
Gorczynski. (2012). Structural and functional consequences of switching carboxy
terminal domains in mouse CD200 receptors. In. Open Journal of ImmunologyOpen
Journal of Immunology.
Krieg, A. M. (2002). CpG motifs in bacterial DNA and their immune effects. Annu Rev
Immunol, 20, 709-760. doi:10.1146/annurev.immunol.20.100301.064842
134
Kroner, A., Schwab, N., Ip, C. W., Ortler, S., Göbel, K., Nave, K. A., . . . Wiendl, H. (2009).
Accelerated course of experimental autoimmune encephalomyelitis in PD-1-deficient
central nervous system myelin mutants. Am J Pathol, 174(6), 2290-2299.
doi:10.2353/ajpath.2009.081012
Krug, A., Rothenfusser, S., Hornung, V., Jahrsdörfer, B., Blackwell, S., Ballas, Z. K., . . .
Hartmann, G. (2001). Identification of CpG oligonucleotide sequences with high
induction of IFN-alpha/beta in plasmacytoid dendritic cells. Eur J Immunol, 31(7), 2154-
2163. doi:10.1002/1521-4141(200107)31:7<2154::AID-IMMU2154>3.0.CO;2-U
Larkin, J., Chiarion-Sileni, V., Gonzalez, R., Grob, J. J., Cowey, C. L., Lao, C. D., . . . Wolchok,
J. D. (2015). Combined Nivolumab and Ipilimumab or Monotherapy in Untreated
Melanoma. N Engl J Med, 373(1), 23-34. doi:10.1056/NEJMoa1504030
Latchman, Y., Wood, C. R., Chernova, T., Chaudhary, D., Borde, M., Chernova, I., . . . Freeman,
G. J. (2001). PD-L2 is a second ligand for PD-1 and inhibits T cell activation. Nat
Immunol, 2(3), 261-268. doi:10.1038/85330
Lauzon-Joset, J. F., Langlois, A., Lai, L. J., Santerre, K., Lee-Gosselin, A., Bossé, Y., . . .
Bissonnette, E. Y. (2015). Lung CD200 Receptor Activation Abrogates Airway
Hyperresponsiveness in Experimental Asthma. Am J Respir Cell Mol Biol, 53(2), 276-
284. doi:10.1165/rcmb.2014-0229OC
Le, D. T., Uram, J. N., Wang, H., Bartlett, B. R., Kemberling, H., Eyring, A. D., . . . Diaz, L. A.
(2015). PD-1 Blockade in Tumors with Mismatch-Repair Deficiency. N Engl J Med,
372(26), 2509-2520. doi:10.1056/NEJMoa1500596
Le Mercier, I., Chen, W., Lines, J. L., Day, M., Li, J., Sergent, P., . . . Wang, L. (2014). VISTA
Regulates the Development of Protective Antitumor Immunity. Cancer Res, 74(7), 1933-
1944. doi:10.1158/0008-5472.CAN-13-1506
Le Mercier, I., Lines, J. L., & Noelle, R. J. (2015). Beyond CTLA-4 and PD-1, the Generation Z
of Negative Checkpoint Regulators. Front Immunol, 6, 418.
doi:10.3389/fimmu.2015.00418
Leach, D. R., Krummel, M. F., & Allison, J. P. (1996). Enhancement of antitumor immunity by
CTLA-4 blockade. Science, 271(5256), 1734-1736.
Levay, A., Brenneman, R., Hoinka, J., Sant, D., Cardone, M., Trinchieri, G., . . . Berezhnoy, A.
(2015). Identifying high-affinity aptamer ligands with defined cross-reactivity using high-
throughput guided systematic evolution of ligands by exponential enrichment. Nucleic
Acids Res, 43(12), e82. doi:10.1093/nar/gkv534
Li, N., Xu, W., Yuan, Y., Ayithan, N., Imai, Y., Wu, X., . . . Wang, L. (2017). Immune-
checkpoint protein VISTA critically regulates the IL-23/IL-17 inflammatory axis. Sci
Rep, 7(1), 1485. doi:10.1038/s41598-017-01411-1
Lin, D. Y., Tanaka, Y., Iwasaki, M., Gittis, A. G., Su, H. P., Mikami, B., . . . Garboczi, D. N.
(2008). The PD-1/PD-L1 complex resembles the antigen-binding Fv domains of
135
antibodies and T cell receptors. Proc Natl Acad Sci U S A, 105(8), 3011-3016.
doi:10.1073/pnas.0712278105
Lines, J. L., Pantazi, E., Mak, J., Sempere, L. F., Wang, L., O'Connell, S., . . . Noelle, R. (2014).
VISTA is an immune checkpoint molecule for human T cells. Cancer Res, 74(7), 1924-
1932. doi:10.1158/0008-5472.CAN-13-1504
Lines, J. L., Sempere, L. F., Broughton, T., Wang, L., & Noelle, R. (2014). VISTA is a novel
broad-spectrum negative checkpoint regulator for cancer immunotherapy. Cancer
Immunol Res, 2(6), 510-517. doi:10.1158/2326-6066.CIR-14-0072
Liu, Y., Bando, Y., Vargas-Lowy, D., Elyaman, W., Khoury, S. J., Huang, T., . . . Chitnis, T.
(2010). CD200R1 agonist attenuates mechanisms of chronic disease in a murine model of
multiple sclerosis. J Neurosci, 30(6), 2025-2038. doi:10.1523/JNEUROSCI.4272-
09.2010
Luo, X. G., Zhang, J. J., Zhang, C. D., Liu, R., Zheng, L., Wang, X. J., . . . Ding, J. Q. (2010).
Altered regulation of CD200 receptor in monocyte-derived macrophages from
individuals with Parkinson's disease. Neurochem Res, 35(4), 540-547.
doi:10.1007/s11064-009-0094-6
Lázár-Molnár, E., Yan, Q., Cao, E., Ramagopal, U., Nathenson, S. G., & Almo, S. C. (2008).
Crystal structure of the complex between programmed death-1 (PD-1) and its ligand PD-
L2. Proc Natl Acad Sci U S A, 105(30), 10483-10488. doi:10.1073/pnas.0804453105
Mahoney, K. M., Rennert, P. D., & Freeman, G. J. (2015). Combination cancer immunotherapy
and new immunomodulatory targets. Nat Rev Drug Discov, 14(8), 561-584.
doi:10.1038/nrd4591
McNamara, J. O., Kolonias, D., Pastor, F., Mittler, R. S., Chen, L., Giangrande, P. H., . . .
Gilboa, E. (2008). Multivalent 4-1BB binding aptamers costimulate CD8+ T cells and
inhibit tumor growth in mice. J Clin Invest, 118(1), 376-386. doi:10.1172/JCI33365
Meyer, C., Berg, K., Eydeler-Haeder, K., Lorenzen, I., Grötzinger, J., Rose-John, S., & Hahn, U.
(2014). Stabilized Interleukin-6 receptor binding RNA aptamers. RNA Biol, 11(1), 57-65.
doi:10.4161/rna.27447
Mihrshahi, R., Barclay, A. N., & Brown, M. H. (2009). Essential roles for Dok2 and RasGAP in
CD200 receptor-mediated regulation of human myeloid cells. J Immunol, 183(8), 4879-
4886. doi:10.4049/jimmunol.0901531
Mittal, D., Gubin, M. M., Schreiber, R. D., & Smyth, M. J. (2014). New insights into cancer
immunoediting and its three component phases--elimination, equilibrium and escape.
Curr Opin Immunol, 27, 16-25. doi:10.1016/j.coi.2014.01.004
Nelson, A. L., Dhimolea, E., & Reichert, J. M. (2010). Development trends for human
monoclonal antibody therapeutics. Nat Rev Drug Discov, 9(10), 767-774.
doi:10.1038/nrd3229
136
Ng, E. W., Shima, D. T., Calias, P., Cunningham, E. T., Guyer, D. R., & Adamis, A. P. (2006).
Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease. Nat Rev Drug
Discov, 5(2), 123-132. doi:10.1038/nrd1955
Nishimura, H., Nose, M., Hiai, H., Minato, N., & Honjo, T. (1999). Development of lupus-like
autoimmune diseases by disruption of the PD-1 gene encoding an ITIM motif-carrying
immunoreceptor. Immunity, 11(2), 141-151.
Nowak, E. C., Lines, J. L., Varn, F. S., Deng, J., Sarde, A., Mabaera, R., . . . Noelle, R. J. (2017).
Immunoregulatory functions of VISTA. Immunol Rev, 276(1), 66-79.
doi:10.1111/imr.12525
Okkenhaug, K., & Vanhaesebroeck, B. (2003). PI3K in lymphocyte development, differentiation
and activation. Nat Rev Immunol, 3(4), 317-330. doi:10.1038/nri1056
Orava, E. W., Abdul-Wahid, A., Huang, E. H., Mallick, A. I., & Gariépy, J. (2013). Blocking the
attachment of cancer cells in vivo with DNA aptamers displaying anti-adhesive
properties against the carcinoembryonic antigen. Mol Oncol, 7(4), 799-811.
doi:10.1016/j.molonc.2013.03.005
Orava, E. W., Cicmil, N., & Gariépy, J. (2010). Delivering cargoes into cancer cells using DNA
aptamers targeting internalized surface portals. Biochim Biophys Acta, 1798(12), 2190-
2200. doi:10.1016/j.bbamem.2010.02.004
Orava, E. W., Jarvik, N., Shek, Y. L., Sidhu, S. S., & Gariépy, J. (2013). A short DNA aptamer
that recognizes TNFα and blocks its activity in-vitro. ACS Chem Biol, 8(1), 170-178.
doi:10.1021/cb3003557
Pardoll, D. M. (2012). The blockade of immune checkpoints in cancer immunotherapy. Nat Rev
Cancer, 12(4), 252-264. doi:10.1038/nrc3239
Pastor, F., Kolonias, D., McNamara Ii, J. O., & Gilboa, E. (2011). Targeting 4-1BB
Costimulation to Disseminated Tumor Lesions With Bi-specific Oligonucleotide
Aptamers. Mol Ther, 19(10), 1878-1886. doi:10.1038/mt.2011.145
Pastor, F., Soldevilla, M. M., Villanueva, H., Kolonias, D., Inoges, S., de Cerio, A. L., . . .
Bendandi, M. (2013). CD28 aptamers as powerful immune response modulators. Mol
Ther Nucleic Acids, 2, e98. doi:10.1038/mtna.2013.26
Pawar, R. D., Goilav, B., Xia, Y., Herlitz, L., Doerner, J., Chalmers, S., . . . Putterman, C.
(2015). B7x/B7-H4 modulates the adaptive immune response and ameliorates renal
injury in antibody-mediated nephritis. Clin Exp Immunol, 179(2), 329-343.
doi:10.1111/cei.12452
Podojil, J. R., Liu, L. N., Marshall, S. A., Chiang, M. Y., Goings, G. E., Chen, L., . . . Miller, S.
D. (2013). B7-H4Ig inhibits mouse and human T-cell function and treats EAE via IL-
10/Treg-dependent mechanisms. J Autoimmun, 44, 71-81. doi:10.1016/j.jaut.2013.04.001
137
Podojil, J. R., & Miller, S. D. (2013). Targeting the B7 family of co-stimulatory molecules:
successes and challenges. BioDrugs, 27(1), 1-13. doi:10.1007/s40259-012-0001-6
Pratico, E. D., Sullenger, B. A., & Nair, S. K. (2013). Identification and characterization of an
agonistic aptamer against the T cell costimulatory receptor, OX40. Nucleic Acid Ther,
23(1), 35-43. doi:10.1089/nat.2012.0388
Prodeus, A., Abdul-Wahid, A., Fischer, N. W., Huang, E. H., Cydzik, M., & Gariépy, J. (2015).
Targeting the PD-1/PD-L1 Immune Evasion Axis With DNA Aptamers as a Novel
Therapeutic Strategy for the Treatment of Disseminated Cancers. Mol Ther Nucleic
Acids, 4, e237. doi:10.1038/mtna.2015.11
Prodeus, A., Cydzik, M., Abdul-Wahid, A., Huang, E., Khatri, I., Gorczynski, R., & Gariépy, J.
(2014). Agonistic CD200R1 DNA Aptamers Are Potent Immunosuppressants That
Prolong Allogeneic Skin Graft Survival. Mol Ther Nucleic Acids, 3, e190.
doi:10.1038/mtna.2014.41
Raptopoulou, A. P., Bertsias, G., Makrygiannakis, D., Verginis, P., Kritikos, I., Tzardi, M., . . .
Boumpas, D. T. (2010). The programmed death 1/programmed death ligand 1 inhibitory
pathway is up-regulated in rheumatoid synovium and regulates peripheral T cell
responses in human and murine arthritis. Arthritis Rheum, 62(7), 1870-1880.
doi:10.1002/art.27500
Riella, L. V., Paterson, A. M., Sharpe, A. H., & Chandraker, A. (2012). Role of the PD-1
pathway in the immune response. Am J Transplant, 12(10), 2575-2587.
doi:10.1111/j.1600-6143.2012.04224.x
Robert, C., Schachter, J., Long, G. V., Arance, A., Grob, J. J., Mortier, L., . . . investigators, K.-.
(2015). Pembrolizumab versus Ipilimumab in Advanced Melanoma. N Engl J Med,
372(26), 2521-2532. doi:10.1056/NEJMoa1503093
Rohloff, J. C., Gelinas, A. D., Jarvis, T. C., Ochsner, U. A., Schneider, D. J., Gold, L., & Janjic,
N. (2014). Nucleic Acid Ligands With Protein-like Side Chains: Modified Aptamers and
Their Use as Diagnostic and Therapeutic Agents. Mol Ther Nucleic Acids, 3, e201.
doi:10.1038/mtna.2014.49
Rozali, E. N., Hato, S. V., Robinson, B. W., Lake, R. A., & Lesterhuis, W. J. (2012).
Programmed death ligand 2 in cancer-induced immune suppression. Clin Dev Immunol,
2012, 656340. doi:10.1155/2012/656340
Rusconi, C. P., Roberts, J. D., Pitoc, G. A., Nimjee, S. M., White, R. R., Quick, G., . . .
Sullenger, B. A. (2004). Antidote-mediated control of an anticoagulant aptamer in vivo.
Nat Biotechnol, 22(11), 1423-1428. doi:10.1038/nbt1023
Salehi, S., Wang, X., Juvet, S., Scott, J. A., & Chow, C. W. (2017). Syk Regulates Neutrophilic
Airway Hyper-Responsiveness in a Chronic Mouse Model of Allergic Airways
Inflammation. PLoS One, 12(1), e0163614. doi:10.1371/journal.pone.0163614
138
Sanmamed, M. F., & Chen, L. (2014). Inducible expression of B7-H1 (PD-L1) and its selective
role in tumor site immune modulation. Cancer J, 20(4), 256-261.
doi:10.1097/PPO.0000000000000061
Santulli-Marotto, S., Nair, S. K., Rusconi, C., Sullenger, B., & Gilboa, E. (2003). Multivalent
RNA aptamers that inhibit CTLA-4 and enhance tumor immunity. Cancer Res, 63(21),
7483-7489.
Schrand, B., Berezhnoy, A., Brenneman, R., Williams, A., Levay, A., & Gilboa, E. (2015).
Reducing toxicity of 4-1BB costimulation: targeting 4-1BB ligands to the tumor stroma
with bi-specific aptamer conjugates. Oncoimmunology, 4(3), e970918.
doi:10.4161/21624011.2014.970918
Schreiber, R. D., Old, L. J., & Smyth, M. J. (2011). Cancer immunoediting: integrating
immunity's roles in cancer suppression and promotion. Science, 331(6024), 1565-1570.
doi:10.1126/science.1203486
Sica, G. L., Choi, I. H., Zhu, G., Tamada, K., Wang, S. D., Tamura, H., . . . Chen, L. (2003). B7-
H4, a molecule of the B7 family, negatively regulates T cell immunity. Immunity, 18(6),
849-861.
Simelyte, E., Criado, G., Essex, D., Uger, R. A., Feldmann, M., & Williams, R. O. (2008).
CD200-Fc, a novel antiarthritic biologic agent that targets proinflammatory cytokine
expression in the joints of mice with collagen-induced arthritis. Arthritis Rheum, 58(4),
1038-1043. doi:10.1002/art.23378
Song, K. M., Lee, S., & Ban, C. (2012). Aptamers and their biological applications. Sensors
(Basel), 12(1), 612-631. doi:10.3390/s120100612
Strand, V., Balsa, A., Al-Saleh, J., Barile-Fabris, L., Horiuchi, T., Takeuchi, T., . . . Marshall, L.
(2017). Immunogenicity of Biologics in Chronic Inflammatory Diseases: A Systematic
Review. BioDrugs. doi:10.1007/s40259-017-0231-8
Sullenger, B. A., White, R. R., & Rusconi, C. P. (2003). Therapeutic aptamers and antidotes: a
novel approach to safer drug design. Ernst Schering Res Found Workshop(43), 217-223.
Terawaki, S., Chikuma, S., Shibayama, S., Hayashi, T., Yoshida, T., Okazaki, T., & Honjo, T.
(2011). IFN-α directly promotes programmed cell death-1 transcription and limits the
duration of T cell-mediated immunity. J Immunol, 186(5), 2772-2779.
doi:10.4049/jimmunol.1003208
Thompson, C. B., & Allison, J. P. (1997). The emerging role of CTLA-4 as an immune
attenuator. Immunity, 7(4), 445-450.
Tiegs, G., Hentschel, J., & Wendel, A. (1992). A T cell-dependent experimental liver injury in
mice inducible by concanavalin A. J Clin Invest, 90(1), 196-203. doi:10.1172/JCI115836
Tivol, E. A., Borriello, F., Schweitzer, A. N., Lynch, W. P., Bluestone, J. A., & Sharpe, A. H.
(1995). Loss of CTLA-4 leads to massive lymphoproliferation and fatal multiorgan tissue
139
destruction, revealing a critical negative regulatory role of CTLA-4. Immunity, 3(5), 541-
547.
Topalian, S. L., Hodi, F. S., Brahmer, J. R., Gettinger, S. N., Smith, D. C., McDermott, D. F., . . .
Sznol, M. (2012). Safety, activity, and immune correlates of anti-PD-1 antibody in
cancer. N Engl J Med, 366(26), 2443-2454. doi:10.1056/NEJMoa1200690
Tuerk, C., & Gold, L. (1990). Systematic evolution of ligands by exponential enrichment: RNA
ligands to bacteriophage T4 DNA polymerase. Science, 249(4968), 505-510.
van der Merwe, P. A., Bodian, D. L., Daenke, S., Linsley, P., & Davis, S. J. (1997). CD80 (B7-1)
binds both CD28 and CTLA-4 with a low affinity and very fast kinetics. J Exp Med,
185(3), 393-403.
van der Vlist, M., Kuball, J., Radstake, T. R., & Meyaard, L. (2016). Immune checkpoints and
rheumatic diseases: what can cancer immunotherapy teach us? Nat Rev Rheumatol,
12(10), 593-604. doi:10.1038/nrrheum.2016.131
Vater, A., & Klussmann, S. (2003). Toward third-generation aptamers: Spiegelmers and their
therapeutic prospects. Curr Opin Drug Discov Devel, 6(2), 253-261.
Vesely, M. D., & Schreiber, R. D. (2013). Cancer immunoediting: antigens, mechanisms, and
implications to cancer immunotherapy. Ann N Y Acad Sci, 1284, 1-5.
doi:10.1111/nyas.12105
Wakeman, C. A., Winkler, W. C., & Dann, C. E. (2007). Structural features of metabolite-
sensing riboswitches. Trends Biochem Sci, 32(9), 415-424.
doi:10.1016/j.tibs.2007.08.005
Wang, Le Mercier, I., Putra, J., Chen, W., Liu, J., Schenk, A. D., . . . Noelle, R. J. (2014).
Disruption of the immune-checkpoint VISTA gene imparts a proinflammatory phenotype
with predisposition to the development of autoimmunity. Proc Natl Acad Sci U S A,
111(41), 14846-14851. doi:10.1073/pnas.1407447111
Wang, L., Rubinstein, R., Lines, J. L., Wasiuk, A., Ahonen, C., Guo, Y., . . . Noelle, R. J. (2011).
VISTA, a novel mouse Ig superfamily ligand that negatively regulates T cell responses. J
Exp Med, 208(3), 577-592. doi:10.1084/jem.20100619
Wang, X., Hao, J., Metzger, D. L., Mui, A., Ao, Z., Akhoundsadegh, N., . . . Warnock, G. L.
(2011). Early treatment of NOD mice with B7-H4 reduces the incidence of autoimmune
diabetes. Diabetes, 60(12), 3246-3255. doi:10.2337/db11-0375
Wang, X. J., Ye, M., Zhang, Y. H., & Chen, S. D. (2007). CD200-CD200R regulation of
microglia activation in the pathogenesis of Parkinson's disease. J Neuroimmune
Pharmacol, 2(3), 259-264. doi:10.1007/s11481-007-9075-1
Ward, J. P., Gubin, M. M., & Schreiber, R. D. (2016). The Role of Neoantigens in Naturally
Occurring and Therapeutically Induced Immune Responses to Cancer. Adv Immunol,
130, 25-74. doi:10.1016/bs.ai.2016.01.001
140
Webber, J., Stone, T. C., Katilius, E., Smith, B. C., Gordon, B., Mason, M. D., . . . Clayton, A.
(2014). Proteomics analysis of cancer exosomes using a novel modified aptamer-based
array (SOMAscan™) platform. Mol Cell Proteomics, 13(4), 1050-1064.
doi:10.1074/mcp.M113.032136
Wei, J., Loke, P., Zang, X., & Allison, J. P. (2011). Tissue-specific expression of B7x protects
from CD4 T cell-mediated autoimmunity. J Exp Med, 208(8), 1683-1694.
doi:10.1084/jem.20100639
White, R. R., Sullenger, B. A., & Rusconi, C. P. (2000). Developing aptamers into therapeutics.
J Clin Invest, 106(8), 929-934. doi:10.1172/JCI11325
Wright, G. J., Cherwinski, H., Foster-Cuevas, M., Brooke, G., Puklavec, M. J., Bigler, M., . . .
Barclay, A. N. (2003). Characterization of the CD200 receptor family in mice and
humans and their interactions with CD200. J Immunol, 171(6), 3034-3046.
Wright, G. J., Puklavec, M. J., Willis, A. C., Hoek, R. M., Sedgwick, J. D., Brown, M. H., &
Barclay, A. N. (2000). Lymphoid/neuronal cell surface OX2 glycoprotein recognizes a
novel receptor on macrophages implicated in the control of their function. Immunity,
13(2), 233-242.
Wu, D., Katilius, E., Olivas, E., Dumont Milutinovic, M., & Walt, D. R. (2016). Incorporation of
Slow Off-Rate Modified Aptamers Reagents in Single Molecule Array Assays for
Cytokine Detection with Ultrahigh Sensitivity. Anal Chem, 88(17), 8385-8389.
doi:10.1021/acs.analchem.6b02451
Yaddanapudi, K., Mitchell, R. A., & Eaton, J. W. (2013). Cancer vaccines: Looking to the
future. Oncoimmunology, 2(3), e23403. doi:10.4161/onci.23403
Yokosuka, T., Takamatsu, M., Kobayashi-Imanishi, W., Hashimoto-Tane, A., Azuma, M., &
Saito, T. (2012). Programmed cell death 1 forms negative costimulatory microclusters
that directly inhibit T cell receptor signaling by recruiting phosphatase SHP2. J Exp Med,
209(6), 1201-1217. doi:10.1084/jem.20112741
Yoon, K. W., Byun, S., Kwon, E., Hwang, S. Y., Chu, K., Hiraki, M., . . . Lee, S. W. (2015).
Control of signaling-mediated clearance of apoptotic cells by the tumor suppressor p53.
Science, 349(6247), 1261669. doi:10.1126/science.1261669
Yu, K., Chen, Z., & Gorczynski, R. (2013). Effect of CD200 and CD200R1 expression within
tissue grafts on increased graft survival in allogeneic recipients. Immunol Lett, 149(1-2),
1-8. doi:10.1016/j.imlet.2012.11.004
Zak, K. M., Kitel, R., Przetocka, S., Golik, P., Guzik, K., Musielak, B., . . . Holak, T. A. (2015).
Structure of the Complex of Human Programmed Death 1, PD-1, and Its Ligand PD-L1.
Structure, 23(12), 2341-2348. doi:10.1016/j.str.2015.09.010
Zhang, S., Cherwinski, H., Sedgwick, J. D., & Phillips, J. H. (2004). Molecular mechanisms of
CD200 inhibition of mast cell activation. J Immunol, 173(11), 6786-6793.
141
Zhang, S., & Phillips, J. H. (2006). Identification of tyrosine residues crucial for CD200R-
mediated inhibition of mast cell activation. J Leukoc Biol, 79(2), 363-368.
doi:10.1189/jlb.0705398
Zhang, S., Wang, X. J., Tian, L. P., Pan, J., Lu, G. Q., Zhang, Y. J., . . . Chen, S. D. (2011).
CD200-CD200R dysfunction exacerbates microglial activation and dopaminergic
neurodegeneration in a rat model of Parkinson's disease. J Neuroinflammation, 8, 154.
doi:10.1186/1742-2094-8-154
Zhou, J., & Rossi, J. (2017). Aptamers as targeted therapeutics: current potential and challenges.
Nat Rev Drug Discov, 16(3), 181-202. doi:10.1038/nrd.2016.199
Zuker, M. (2003). Mfold web server for nucleic acid folding and hybridization prediction.
Nucleic Acids Res, 31(13), 3406-3415.
142
Appendices
143
Primer Sequence
IFNα Fwd CTACTGGCCAACCTGCTCTC
IFNα Rvs CCTTCTTGATCTGCTGGGCA
IL-12 Fwd TTATGTTGTAGAGGTGGACTGG
IL-12 Rvs CCTTTGTGGCAGGTGTACTG
Appendix 1. Primer sequences used for IFNα and IL-12 RT-PCR in RAW264.7 mouse
macrophage cells.
144
MP7
PEG MP7 NHS
Column: xBridge C18 OST (10x50mm) Phase A: 0.1M TEAA + 5% CH
3CN
Phase B: 0.1M TEAA + 90% CH3CN
Gradient: 0-100%B over 60min Flow Rate: 2.2 mL/min
Appendix 2. HPLC Purification of PEGylated anti-PD-1 aptamer MP7. The PEGylation
reaction products were prepared in 0.1M TEAA and purified by reverse phase HPLC using a C18
OST semi-preparative column. The bound PEGylated aptamers were eluted using a linear
gradient going from 5-90% CH3CN over a 60-minute period. Absorbance (UV260nm) was used
to track elution of free nucleic acid and nucleic acid conjugates. The second peak at 22 minutes
corresponds to unreacted aptamer and the last peak (38 minutes) corresponds to PEGylated
aptamer.
145
10
15
20 25
30
40
50
60
85 100
120
150
200
70
1. Media
2. CD200-Fc (+DTT) 3. CD200-Fc (-DTT)
1 2 3
Appendix 3. Purification of CD200-Fc used in Chapter 3. A plasmid encoding the CD200
extracellular domain linked to the Fc-region of mIgG2a which was mutated for reduced
complement and FcR binding was used to express CD200-Fc. Recombinant protein was
expressed by transient transfection using Expi293 cells, and purified from the culture media by
Protein A chromatography. Expression and homogeneity was confirmed by SDS-PAGE analysis
of the purified fractions in both reduced (+DTT) and non-reduced (-DTT) conditions. CD200-Fc
is a homodimer of 150kDa.
146
Ab
so
rban
ce (
mA
U)
100
200
300
400
500
7.93 PEG-Aptamer
9.66 NH2-Aptamer
50
100
150
200
250
300
5.0 10.0 15.0 20.0 25.0 ml
7.93 PEG-Aptamer
Volume
Appendix 4. Purification of PEGylated CD200R1 aptamers with size exclusion
chromatography. DNA aptamers were synthesized with a 5’ amine with a C6 spacer and reacted
with an excess of monofunctional NHS-PEG overnight. FPLC size exclusion chromatography
using a Superdex75 column was used to purify the crude reaction (top) to yield purified full
length PEGylated aptamers (bottom). Absorbance (UV260nm) was used to track elution of free
nucleic acid and nucleic acid conjugates. The first peak at 7.93mL corresponds to PEG-Aptamer,
and 9.66mL peak corresponds to unreacted aptamer.
147
Aptamer Sequence (variable region only)
CCS1 ATAGGGAGCATTACGTAACCATCTC
CCS.2 AAATCCCCGCTGAATTACCACTTTA
CCS.4 GGGTCGGCACGGCGGAGGATGCGGGA
CCS.5 CCCCAGGAAGAACCTACTCACTGAT
CCS.8 CCCCTCCGAGTGATATGTAATCCTA
CCS.10 TGGTGGTGATTTTTGGTGGCTAAT
CCS.11 ATTGATTCCGGGTACTGTATTCTAC
CCS.13 CACCGCTCTTATGCCACCATTTTCA
Appendix 5. Sequences of cross-species anti-CD200R1 aptamers. At cycle 15 enriched SELEX
pools towards both human and mouse CD200R1 were sequences by IonTorrent NGS. The top 50
highest abundance sequences in each dataset were cross-compared to that of the opposite species,
leading to the identification of 8 aptamers with 100% identity across the two pools. Sequences
shown are of the internal variable region only. Full sequence includes the 5’ and 3’ constant primer
regions.
148
> VISTA-Ig
METDTLLLWVLLLWVPGSTGFKVTTPYSLYVCPEGQNATLTCRILGPVSKGHDVTIYKT
WYLSSRGEVQMCKEHRPIRNFTLQHLQHHGSHLKANASHDQPQKHGLELASDHHGNFS
ITLRNVTPRDSGLYCCLVIELKNHHPEQRFYGSMELQVQAGKGSGSTCMASNEQDSDSI
TAIEGRMDPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS
NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS
LSPGK
> VISTA.COMP
METDTLLLWVLLLWVPGSTGEFKVTTPYSLYVCPEGQNATLTCRILGPVSKGHDVTIYK
TWYLSSRGEVQMCKEHRPIRNFTLQHLQHHGSHLKANASHDQPQKHGLELASDHHGNF
SITLRNVTPRDSGLYCCLVIELKNHHPEQRFYGSMELQVQAGKGSGSTCMASNEQDSDS
ITAEFGSGPGPSGTDLAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMECDACG
GSPQPQSENLYFQGGPQPQGGSGSGSGGRHHHHHH
Appendix 6. Amino acid sequences of murine VISTA.COMP and VISTA-Ig used in Chapter 5
149
OD
450 n
m
Appendix 7. VISTA does not bind to itself or structurally related IgV-like domain
containing checkpoint molecules. ELISA based binding experiment assaying if the indicated
analyte binds to the coated ligand over a titrated range of concentrations. Notably, VISTA.COMP
did not bind to PD-1, PD-L1, or itself at any concentration tested. As expected PD-L1.COMP
readily bound PD-1. CEA-N domain was used as a positive control for a homotypic interactions,
showing the CEA-N.COMP binds to a bacterially expressed immobilized CEA-N domain.
150
Appendix: List of Publications
Prodeus, A., Abdul-Wahid, A., Sparkes, A., Fischer, N.W., Cydzik, M., Chiang, N., Alwash, M.,
Ferzoco, A., Vacaresse, N., Julius M., Gorczysnki, R.M., Gariépy J. VISTA.COMP: an
engineered checkpoint receptor agonist that potently suppresses T-cell mediated immune
responses. Accepted for publication in JCI Insight
Prodeus, A., Abdul-Wahid, A., Fischer, N. W., Huang, E. H., Cydzik, M., & Gariépy, J. (2015).
Targeting the PD-1/PD-L1 Immune Evasion Axis With DNA Aptamers as a Novel Therapeutic
Strategy for the Treatment of Disseminated Cancers. Mol Ther Nucleic Acids, 4, e237.
doi:10.1038/mtna.2015.11
Prodeus, A., Cydzik, M., Abdul-Wahid, A., Huang, E., Khatri, I., Gorczynski, R., & Gariépy, J.
(2014). Agonistic CD200R1 DNA Aptamers Are Potent Immunosuppressants That Prolong
Allogeneic Skin Graft Survival. Mol Ther Nucleic Acids, 3, e190. doi:10.1038/mtna.2014.41
Wu FT, Lee CR, Bogdanovic E, Prodeus A, Gariépy J, Kerbel RS. (2015). Vasculotide reduces
endothelial permeability and tumor cell extravasation in the absence of binding to or agonistic
activation of Tie2. EMBO Mol Med. 7(6):770-87. doi: 10.15252/emmm.201404193.
Fischer NW, Prodeus A, Malkin D, Gariépy J. (2016). p53 oligomerization status modulates cell
fate decisions between growth, arrest and apoptosis. Cell Cycle 15(23):3210-3219.
Abdul-Wahid A, Cydzik M, Prodeus A, Alwash M, Stanojcic M, Thompson M, Huang EH,
Shively JE, Gray-Owen SD, Gariépy J. (2016). Induction of antigen-specific TH 9 immunity
accompanied by mast cell activation blocks tumor cell engraftment. Int J Cancer. 15;139(4):841-
53
Cydzik M, Abdul-Wahid A, Park S, Bourdeau A, Bowden K, Prodeus A, Kollara A, Brown TJ,
Ringuette MJ, Gariépy J. (2015). Slow binding kinetics of secreted protein, acidic, rich in
cysteine-VEGF interaction limit VEGF activation of VEGF receptor 2 and attenuate
angiogenesis. FASEB J. 29(8):3493-505. doi: 10.1096/fj.15-271775.
Jennings W, Doshi S, Hota PK, Prodeus A, Black S, Epand RM. (2017). Expression,
Purification, and Properties of a Human Arachidonoyl-Specific Isoform of Diacylglycerol
Kinase. Biochemistry. 56(9):1337-1347. doi: 10.1021/acs.biochem.6b01193.
Prodeus A, Berno B, Topham MK, Epand RM. (2013). The basis of the substrate specificity of
the epsilon isoform of human diacylglycerol kinase is not a consequence of competing
hydrolysis of ATP. Chem Phys Lipids. 166:26-30. doi: 10.1016/j.chemphyslip.2012.11.006.