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Engineering chimeric antigen receptor-directed immune cells for enhanced antitumor efficacy in solid tumors
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Engineering chimeric antigen receptor-directed immune cells for enhanced antitumor efficacy in solid tumors
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i
Engineering Chimeric Antigen Receptor-Directed Immune Cells
for Enhanced Antitumor Efficacy in Solid Tumors
By
Yun Qu
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfullment of
the Requirements for the Degree
DOCTOR OF PHILOSOPHY
(CHEMICAL ENGINEERING)
May 2021
Copyright 2021 Yun Qu
ii
Dedication
This dissertation is dedicated to my grandparents, Suidian Qu and Cuiqi Jiang,
who exemplify the spirits of lifelong, true scholars despite all hardships
and inspire me to pursue a career in exploratory research.
iii
Acknowledgement
I would like to express my great gratitude to my advisor, Dr. Pin Wang, for his guidance
and support over the past five years. I have always felt very fortunate to be mentored by Dr. Pin
Wang over the transitory period of my life that is marked with challenges, risks and
opportunities. Dr. Wang is a role model for me. His dedication to scientific research and
translational efforts has inspired and encouraged me to always strive for excellence and have
unwavering courage in face of challenges. Meanwhile, I would like to thank my committee
members: Dr. Stacey Finley, Dr. Noah Malmstadt, Dr. Nicholas Graham, Dr. Paulo
Branicio for their valuable advice and insightful comments regarding my research.
Everlasting memories have been created at Room 515 of Ronald Tutor Hall where I share
with my fellow labmates. Dr. Xianhui Chen taught me the essential skills for biological
research and has supported me tremendously in all my research endeavors. Dr. Natnaree
Siriwon taught me hand-to-hand the techniques of animal studies that have greatly benefited me
for my following studies and her research aspirations encouraged me to pursue further scientific
novelty. Collaborative work with Dr. Elizabeth Siegler has made me truly understand and
appreciate the essence of teamwork and I thank her for guiding me through my first complete
project as well as always encouraging original thinking and creativity from me. I would like to
express special thanks to my collaborator of later projects, Zachary Dunn, for his aspirations,
tenacity and unparalleled scientific curiosity that encourage me to continue my research efforts
during the difficult times of the COVID-19 pandemic with more advanced study designs and
even greater efficiency. I also want to thank Dr. Si Li, Dr. Yu Jeong Kim, Dr. Xiaoyang
Zhang, Dr. John Mac, Dr. Paul Bryson, Dr. Xiaolu Han, Dr. Jennifer Rohrs, Gunce Cinay,
Melanie MacMullan, Chumeng Cheng, Jiangyue Liu, Meng Wang, Siyi Guo, Shuai Yang,
iv
and Fangheng Hu. All my labmates have been great comfort and support to me, especially when
I was experiencing difficulties in research as well as enduring personal hardships. I am very
honored to have shared this journey with everyone at Wang lab and befriended some most
wonderful people in my life.
My family and friends, despite the physical distances between them and me, have
provided me with their unconditional love and support. I want to thank everyone of them for
sharing with me different perspectives about life and bringing into my life tremendous amount of
joy and laughter. Last but not least, I reserve my deepest gratitude to my parents, Jun Qu and
Ping Chen, my most important source of love, strength, and motivation.
v
Table of Contents
Dedication ....................................................................................................................................... ii
Acknowledgement ......................................................................................................................... iii
List of Figures .............................................................................................................................. viii
Abstract .......................................................................................................................................... ix
Chapter 1. Introduction ................................................................................................................... 1
1.1 Adoptive cell therapy: current landscape ...................................................................... 2
1.2 CAR-engineered immune cell therapy —challenges and strategies .............................. 6
1.3 Challenges of CAR-engineered immune cell therapy in solid tumor ......................... 11
1.4 Focus of dissertation ................................................................................................... 15
Chapter 2. Engineering CAR-Expressing Natural Killer Cells with Cytokine Signaling and
Synthetic Switch for an Off-the-Shelf Cell-Based Cancer Immunotherapy ................................. 16
2.1 Abstract ....................................................................................................................... 17
2.2 Introduction ................................................................................................................. 18
2.3 Materials and methods ................................................................................................ 21
2.3.1 Cell culture ................................................................................................... 21
2.3.2 Viral vector production ................................................................................ 21
2.3.3 Transduction of NK92 and SKOV3 cells .................................................... 22
2.3.4 CAR detection on NK cell surface............................................................... 22
2.3.5 Intracellular cytokine staining...................................................................... 23
2.3.6 Cytotoxicity assay ........................................................................................ 23
2.3.7 Chemical inducer of dimerization sensitivity assay..................................... 24
2.3.8 Xenograft tumor model ................................................................................ 24
2.3.9 Ex vivo NK cell staining .............................................................................. 24
2.3.10 Statistical analysis ...................................................................................... 25
2.4 Results ......................................................................................................................... 26
2.4.1 Generation of anti-mesothelin CAR-NK92 cell lines with retroviral vector 26
2.4.2 Enhanced in vitro functions of engineered CAR-NK92 cells ...................... 27
2.4.3 CID induced apoptosis of iCAS9 transduced NK92 cells ........................... 29
2.4.4 Membrane bound IL15/IL15Rα complex enhanced antitumor efficacy of
anti-mesothelin CAR-NK in vivo.......................................................................... 30
vi
2.4.5 Membrane bound IL15/IL15Rα complex enhanced cell proliferation in vivo
............................................................................................................................... 31
2.4.6 CID AP20187 induced apoptosis of iCAS9-transduced CAR-NK92 cells in
vivo ........................................................................................................................ 32
2.5 Discussion ................................................................................................................... 34
Chapter 3. Engineering CAR-T Cells to Overexpress Adenosine Deaminase 1 (ADA) for
Enhanced Anti-tumor Efficacy in Solid Tumor ............................................................................ 35
3.1 Abstract ....................................................................................................................... 36
3.2 Introduction ................................................................................................................. 37
3.3 Results ......................................................................................................................... 40
3.3.1 Design, generation, and validation of CAR T cells engineered to secrete
ADA ...................................................................................................................... 40
3.3.2 Incorporation of ADA, aADA, or cADA into CAR T cells does not
comprise in vitro effector functions ...................................................................... 44
3.3.3 ADA overexpression reduces CAR T cell exhaustion, Treg induction, and
adenosine susceptibility in vitro............................................................................ 45
3.3.4 ADA overexpression and cADA expression enhance CAR T cell antitumor
activity and improve overall survival in a xenograft solid tumor model .............. 47
3.3.5 ADA overexpression and cADA expression enhances intratumoral CAR T
cell expansion........................................................................................................ 48
3.3.6 ADA overexpression reduces tumor burden in a syngeneic solid tumor
model..................................................................................................................... 49
3.4 Discussion ................................................................................................................... 52
3.4.1 Interpreting experimental results ................................................................. 52
3.4.2 Design advantages and disadvantages ......................................................... 53
3.4.3 Clinical relevancy and translational value ................................................... 55
3.5 Materials and methods ................................................................................................ 57
3.5.1 Antibodies .................................................................................................... 57
3.5.2 Cell lines and cell culture ............................................................................. 57
3.5.3 Mice ............................................................................................................. 58
3.5.4 Plasmid design ............................................................................................. 58
3.5.5 ELISA and Western blotting ........................................................................ 58
3.5.6 aADA and cADA isolation and characterization ......................................... 59
3.5.7 Tumor model and animal studies schematics .............................................. 60
3.5.8 Statistical analysis ........................................................................................ 61
vii
Chapter 4. Engineering CAR-T Cells to Secrete 4-1BB Ligand Crosslinked to PD-1 Checkpoint
Inhibitor for Enhanced Anti-tumor Efficacy in Solid Tumor ....................................................... 62
4.1 Abstract ....................................................................................................................... 63
4.2 Introduction ................................................................................................................. 64
4.3 Results ......................................................................................................................... 67
4.3.1 Design of the CAR and protein characterization ......................................... 67
4.3.2 In vitro functional analysis........................................................................... 68
4.3.3 Secreted fusion protein protects parental CAR T cells from exhaustion ..... 70
4.3.4 Phenotype analysis of CAR T cells ............................................................. 71
4.3.5 In vivo antitumor efficacy of CAR T cells ................................................... 72
4.3.6 Ex vivo analysis of CAR T cells in xenograft mouse model........................ 73
4.4 Discussion ................................................................................................................... 75
4.4.1 Potential mechanisms behind the improved efficacy................................... 75
4.4.2 Limitations, future outlook and translational value ..................................... 77
4.5 Materials and methods ................................................................................................ 79
4.5.1 Antibodies .................................................................................................... 79
4.5.2 Cell lines and cell culture ............................................................................. 79
4.5.3 Mice ............................................................................................................. 80
4.5.4 Plasmid design ............................................................................................. 80
4.5.5 Protein isolation and characterization .......................................................... 80
4.5.6 Tumor model and animal studies schematics .............................................. 82
4.5.7 Statistical analysis ........................................................................................ 82
References ..................................................................................................................................... 83
viii
List of Figures
Figure 1-1. Novel CAR designs to manage tumor escape and mitigate toxicities. ........................ 8
Figure 1-2. Immunosuppressive factors in the solid tumor microenvironment ............................ 12
Figure 2-1. Anti-mesothelin CAR designs and expression levels ................................................ 26
Figure 2-2. Enhanced in vitro functions of engineered CAR-NK92 cells .................................... 28
Figure 2-3. CID induced apoptosis of iCAS9 transduced NK92 cells. ........................................ 29
Figure 2-4. Tumor growth control by anti-mesothelin CAR-NK ................................................. 30
Figure 2-5. Enhanced cell proliferation of anti-mesothelin CAR-NK in vivo .............................. 31
Figure 2-6. CID drug induction of iCAS9-transduced cells apoptosis in vivo ............................. 32
Figure 3-1. ADA injections enhance CAR T cell therapy tumor inhibition but does not improve
overall survival.............................................................................................................................. 40
Figure 3-2. Design and characterization of CAR and ADA expression in transduced PBMCs ... 41
Figure 3-3. Binding kinetics of purified aADA and cADA .......................................................... 42
Figure 3-4. ADA overexpression does not compromise CAR T cell in vitro cytotoxicity, effector
cytokine production, and proliferation. ......................................................................................... 43
Figure 3-5. ADA overexpression prevents CAR T cell exhaustion and reduces Treg
differentiation ................................................................................................................................ 45
Figure 3-6. The overexpression of ADA and cADA increases overall survival........................... 46
Figure 3-7. ADA overexpressing CAR T cells exhibit enhanced in vivo expansion ................... 47
Figure 3-8. Mouse ADA expression reduces the growth rate of CT26 subcutaneous tumors. ..... 48
Figure 4-1. Schematics of CAR construct and fusion protein ...................................................... 65
Figure 4-2. Secreted protein characterization and binding kinetics .............................................. 66
Figure 4-3. In vitro analysis of CAR-T cells ................................................................................ 67
Figure 4-4. Secreted fusion protein protects parental CAR T cells from exhaustion ................... 68
Figure 4-5. Phenotype analysis of CAR T cells ............................................................................ 70
Figure 4-6. Antitumor efficacy of CAR19.aPD1-41BBL T cells in xenograft mouse model ...... 71
Figure 4-7. Ex vivo analysis of CAR T cells in xenograft mouse model ...................................... 73
ix
Abstract
This dissertation is a compilation of three projects aiming at engineering chimeric antigen
receptor-directed immune cells for enhanced antitumor efficacy in solid tumors. Project one
Engineering CAR-expressing Natural Killer Cells with Cytokine Signaling and Safety Switch
demonstrated the capacity at which the off-the-shelf candidate NK92 cells can be engineered
with synthetic biology tools for enhanced tumor targeting capability, better proliferative
potential, and treatment regulation. Project two and Project three focus on armoring CAR-
directed T cells with self-secreted immune-modulating compounds to protect CAR-T cells from
select immunosuppressive mechanisms in the solid tumor microenvironment. In Project two
Engineering CAR-T Cells to Overexpress Adenosine Deaminase 1 (ADA) for Enhanced Anti-
tumor Efficacy in Solid Tumor, CAR-T cells were engineered to overexpress ADA1 or express
protein modified-ADA1 to target adenosine accumulation in the tumor microenvironment and
help ameliorate its suppressive effect on T cells. In Project three Engineering CAR-T Cells to
Secrete 4-1BB Ligand Crosslinked to PD-1 Checkpoint Inhibitor for Enhanced Anti-tumor
Efficacy in Solid Tumor, CAR-T cells were engineered to overexpress a fusion protein consisting
of single-chain 4-1BB ligand crosslinked to anti-PD-1 scFv to simultaneously engage the
protective effect of anti-PD-1 scFv against PD-1 checkpoint and the costimulatory effect of 4-
1BB ligand interacting with 4-1BB upregulated on activated T cells.
1
Chapter 1. Introduction
2
1.1 Adoptive cell therapy: current landscape
Adoptive cellular therapy utilizes the body’s native weapons—immune cells—to fight
against cancer and has demonstrated its potency in a number of malignancies such as metastatic
melanoma, leukemia, B cell lymphoma, advanced cervical carcinoma, etc.
1-3
Adoptive cellular
therapy, or adoptive cellular transfer (ACT), is often considered an “living” treatment since
immune cells are isolated from the body, expanded and/or engineered in cultures, and then
reinfused into the patient with capabilities to continue to proliferate in vivo and exhibit antitumor
effector functions.
4
There are several types of ACT currently under investigations both in
preclinical and clinical settings including tumor infiltrating lymphocytes (TILs), T cell receptor-
engineered T cells (TCR-T), chimeric antigen receptor-engineered T cells (CAR-T), and natural
killers cells engineered to express CAR (CAR-NK).
1
TILs therapy was first explored in 1980s by Dr. Steven Rosenberg and others when they
isolated tumor infiltrating lymphocytes from murine tumors. They showed that the cells had
greater in vivo antitumor efficacy for treating mice with various tumor metastases when infused
with T cell growth factor IL-2 compared to lymphokine-activated killer cells.
5
Currently, the
therapy uses TILs isolated from the patient’s resected tumor or biopsies and expands the cell ex
vivo. After the patient is given a lymphodepleting regimen usually in the form of chemotherapy,
expanded TILs are then adoptive-transferred into the patient with subsequent IL-2 infusions to
support the in vivo expansion of TILs. One advantage of TILs therapy is that cells already
recognize a broad range of antigens and neoantigens present in the specific tumor.
4
For
metastatic melanoma, TILs have shown consistent antitumor reactivity and its clinical benefit
correlates with the high mutational load and high neoantigen rates of the tumor.
7
Although TILs
have shown great potential for treatment of metastatic melanoma and there is currently a phase
3
III clinical trial comparing ipilimumab to TILs therapy for advanced melanoma, the production
and reactivity of TILs for other solid tumor types is highly variable.
1
Efforts have been attempted
to improve the expansion methods for TILs, but there remain the consequences of loss of critical
costimulatory molecules and loss of proliferative capacity after TIL expansion.
6
The
heterogenous nature of the TILs therapy made it difficult to study the mechanism behind its
antitumor efficacy. Further studies are needed to determine if certain subsets of TILs mediate the
tumor regression while others are dispensable and expansion methods can be tailored for the
effective subsets, or if a mixture or a balance between different subsets (i.e. effector memory T
cells vs. central memory T cells) are required for optimal antitumor efficacy. Hopefully these
studies as well as other future insights into cancer immunotherapy can shed light into whether
the clinical response of TILs in metastatic melanoma could be similarly achieved in other cancer
types.
The development of genetic engineering tools made it possible to create new anti-tumor
treatment modalities. TCRs and CARs are both engineered, tumor-specific receptors that can be
introduced into immune cells and equip them with cancer directing capabilities.
1,4
Antigen
binding to TCR or CAR triggers downstream signaling and activates the immune cells to target
the antigen-expressing cells. In contrast to TILs therapy for which cells are isolated from tumor
resections and are thus limited in number, TCR-T and CAR-engineered immune cells typically
use peripheral blood mononuclear cells as sources. PBMCs are self-renewable cell populations
with great expansion capacity.
8
In autologous engineered-T cell therapy, the patient’s peripheral
blood is collected. T cells are isolated from PBMCs, activated, and engineered to express TCRs
or CARs on cell surface.
1
4
T cell receptors are made of heterodimers consisting of alpha and beta peptide chains that
can recognize polypeptide fragments presented by MHC class I molecules on cancer cells.
9
Endogenous TCRs have low affinity to antigen peptides and are degenerate. Engineered TCRs
used in TCR-T therapy are artificially designed for high affinity to antigen peptides and
specificity to cancer cells.
10
In order to construct the high-affinity TCR, antigen peptides only
present on cancer cells and not on normal tissues are identified as targets. Usually a TCR phage
display library is created to screen for TCRs with high affinity and specificity. TCR-T therapy
has the advantage of recognizing a broad range of antigens including cell surface antigens,
subcellular antigens, neoantigens from mutation because of the MHC class I presentation, but is
also limited by it since MHC is downregulated in some cancer cells as a method of immune
escape.
11
Preclinical studies have shown the potential of TCR-T therapy for both hematological
malignancies (i.e. targeting HA-1, WT1, CMV) and solid tumors (i.e. targeting MAGE, MART-
1, NY-ESO-1, etc.).
10
Chimeric antigen receptors are recombinant receptors that combine an antigen-
recognizing extracellular moiety, a hinge, a transmembrane domain, and intracellular T cell
activating and co-stimulatory domains.
12
The extracellular domain can take the form of single-
chain variable fragment (scFv) of an antigen-specific antibody, or antigen-binding Fabs fragment
selected from libraries, or novel designs such as antibody mimetics.
12-14
The intracellular domain
is comprised of a CD3ζ chain as the TCR signaling domain and an additional co-signaling
domain, mainly CD28 and 4-1BB (CD137) or others, to deliver co-stimulatory signals. Unlike
TCRs, CARs recognize antigen targets in an MHC-independent manner and recognize
carbohydrate and glycolipid antigens in addition to protein and peptides targets, but their
limitation is that they are only able to recognize surface antigens due to the artificial antigen-
5
recognizing CAR modality. CAR-T therapies targeting CD19, an optimal target in B cell
malignancies, have achieved great success in clinical settings and a few products approved by
FDA are now available to benefit subsets of patients with leukemia and lymphoma. Ongoing
studies are evaluating its antitumor efficacy for other targets like BCMA, CD20, CD22 in
hematological malignancies and mesothelin, EGFR, GPC3, HER2, etc. in solid tumors.
10
Natural killer (NK) cells are lymphocytes of the innate immune system and exhibit direct
cytotoxicity against malignant cells. They have the potential to be allogenic therapeutics because
they do not require human leukocyte antigen (HLA) matching and almost never induce graft
versus host disease (GvHD).
16
NK cells also can be derived from a number of sources including
peripheral blood, umbilical cord blood, human embryonic stem cells, and human induced
pluripotent stem cells. CAR-NK therapy for both hematological malignancies and solid tumors
are currently undergoing preclinical and clinical investigations.
16
Target selection remains one of the biggest challenges for TCR-T, CAR-T, CAR-NK
therapies. The number of tumor-specific antigens (TSA) as targets is limited, and novel methods
are being utilized to discover neoantigens that are tumor specific and thus targetable by TCR or
CAR.
2,17
Targeting tumor associated antigens (TAA) that overexpress on tumor cells and express
at a lower level on normal tissues may result in severe side effects of “on-target, off-tumor”
toxicities,
10
which can potentially be addressed by improved cell engineering designs.
13
In general, adoptive cellular therapy provides more possibilities for patients because of its
precision medicine nature. Combinations of ACT with other therapeutic modalities such as
immune checkpoint inhibitors are of great clinical potential in order for the treatments to achieve
better clinical outcomes.
6
1.2 CAR-engineered immune cell therapy—challenges and strategies
Great efforts have been devoted into developing better CAR designs and addressing
issues that include T cell activation and persistence, tumor escape, CAR-induced toxicities,
tumor immunosuppression, etc.
13
Design of CAR signaling in the intracellular domain has upgraded through several
generations to produce T cell products with better activation and persistence. The first generation
of CAR-T comprised of only the CD3ζ intracellular domain for T cell activation and target cell
lysis signaling.
13
However, its in vivo efficacy was very limited due to insufficient cytokine
secretion and proliferation.
12
Adding a costimulatory domain (i.e. 4-1BB, CD28, OX40, etc.) in
the intracellular domain gave rise to the second generation of CAR-T cells that has shown more
sustained activation of T cell and greatly enhanced proliferation and antitumor effects. Choice of
co-stimulatory domains affects the in vivo proliferation and effector function of CAR-T cells.
18
CD28 and 4-1BB are the two most commonly used costimulatory domain. CD28 co-stimulated
CAR-T cells are associated with faster tumor burden clearance and robust antitumor activity at
lower effector-to-target ratios,
19
while CAR-T cells expressing 4-1BB exhibit much longer
persistence ex vivo and in vivo than CAR-T cells expressing CD28.
18
It has been shown that
CD28 co-stimulation promotes effector memory differentiation and upregulates glycolytic
metabolism while 4-1BB co-stimulation promotes central memory differentiation and
upregulates oxidative metabolism and mitochondrial biogenesis. In the clinical setting, CD19
CAR-T cells co-stimulated by 4-1BB also showed better safety profile than CD19 CAR-T cells
co-stimulated by CD28 for B cell non-Hodgkin’s lymphoma, as CD28 co-stimulated CD19
CAR-T cells induced more severe cytokine release syndrome and immune effector cell-
associated neurotoxicity.
20
Third generation of CAR-T cells have also been developed that
7
include two costimulatory domains, in the hope that the CAR-T cells would be able to overcome
the limitations of each costimulatory domain alone. A clinical study investigating the use of
CD28 CD19 CAR.T cells versus CD28-4-1BB CD19 CAR.T cells for B cell non-Hodgkin’s
lymphoma showed that the third generation CAR-T greatly enhanced CAR-T expansion and may
be of particular value when treating low disease burden in patients when compared to second
generation CAR-T stimulated by CD28.
21
In addition to supplying CAR-T cells with costimulatory signaling, transgenic expression
of cytokines or constitutively active cytokine receptors is another strategy to enhance cell
proliferation and persistence in vivo.
22
CAR-engineered immune cells are simultaneously
encoded to secret a cytokine such as IL-2, IL-7, IL-15, IL-12, IL-23 etc., or express membrane-
bound cytokines such as mbIL15, or express constitutively active cytokine receptor such as IL-7
receptor α (C7R).
Tumor escape is another challenge commonly encountered in CAR-engineered immune
therapy. Antigen recognition by CAR-T cells is mediated through the binding of scFv or
antibody mimetics in the extracellular domain to surface antigens on tumor cells. Most previous
CAR-T designs target a single tumor associated antigen (TAA). Loss or decreased expression
level during or after CAR-T therapy and the growth of antigen-negative tumor cells result in
tumor escape and largely limit the success of CAR-directed immune cell therapy.
23
This
highlights the importance of targeting more than one antigen for therapeutic benefits. One
strategy called “dual CARs” enables cells to express two CARs simultaneously and recognition
of one of the two antigen targets will activate the cells to attack and kill (Figure 1-1, A).
24
Another strategy, called tandem CAR, involves linking two scFvs together that target different
antigens (Figure 1-1, BC).
25
Studies have shown that, with optimization in linker and spacer
8
design, binding of either scFv structure to its cognate antigen is sufficient to achieve full T cell
activation.
25-26
Interestingly, it has been noticed that when dual or tandem CAR-T cells recognize
both antigens at the same time, enhanced T cell function is observed.
Figure 1-1. Novel CAR designs to manage tumor escape and mitigate toxicities. Figure adapted from Guedan S,
Calderon H, Posey J, Maus MV. Engineering and Design of Chimeric Antigen Receptors. Molecular therapy
Methods & clinical development. 2019;12:145-156. doi:10.1016/j.omtm.2018.12.009
The next generation CARs also have witnessed new strategies to mitigate toxicities
associated with CAR-engineered immune cell therapy. As mentioned above, tumor-specific
antigens are the best targets for CAR, but there are very few of them. Most antigens currently
9
targeted by CAR-directed immune cell therapy also express on normal tissues. Even when they
are expressed at very low levels, CAR targeting can result in severe side effects.
27
Strategies to
mitigate such dangerous toxicities utilize alternative ways to tune CAR signals. In the
combinatorial approach, T cells were engineered to express two different CARs, one with CD3ζ
signaling in intracellular domain and the other with a costimulatory intracellular domain (Figure
1-1, C).
28
Binding of either CAR sends suboptimal signal to T cells, thus limiting the CAR
activation and function at normal tissues expressing one antigen. Another strategy called
“synthetic Notch” also utilizes two CARs (Figure 1-1, D).
29
Activation of the synNotch receptor
through binding of one CAR to its antigen sends transcription signal and induce the formation of
another CAR. For both combinatorial strategy and synthetic Notch strategy, T cells are fully
activated only at the tumor site where both antigens are present. These strategies successfully
alleviate “on-target, off-tumor” toxicities, but their efficacies may be greatly defeated when
antigen escape for one antigen occurs and CAR-T cells are no longer able to be fully activated.
Other strategies for managing CAR toxicity at normal tissue involves fragmented CAR
signaling design that requires heterodimerizing drugs at tumor site to trigger CAR activation
(Figure 1-1, F) and simultaneously expressing inhibitory CAR that recognize antigens on normal
tissues.
30-31
The emergence of universal CARs allows versatility of switching CAR targets and aims
to address CAR-related toxicity and antigen escape at the same time.
32-35
Universal CARs are
composed of an antibody-based molecule that recognize a tumor antigen of choice and is linked
to a switch, and a universal CAR that does not have antigen specificity and can be turned on by
the antibody switch (Figure 1-1, G). For therapeutic efficacy, both the universal CAR and the
antibody switch recognizing one tumor antigen are given. Binding of the switch and the
10
universal CAR unleashes the CAR-T functions, and the intensity can be adjusted with differing
levels of antibody switch to mitigate potential side effects. If antigen escape happens when
universal CAR-T cells are still present in circulation, injecting another antibody switch that
recognize a different antigen recruits the universal CAR-T cells again for cytotoxic functions.
The development of synthetic biology tools has truly benefited the novel strategies for
CAR designs that promise more efficacious and safe therapeutic modalities. Hopefully, more
clinical evidence showing the potency of these novel strategies will be available soon.
11
1.3 Challenges of CAR-engineered immune cell therapy in solid tumor
Solid tumor microenvironment remains one of the biggest challenges to replicate the
success of CAR-T or CAR-NK therapies for hematological malignancies in the realm of solid
tumors. A solid tumor is composed of not only tumor cells but also stromal cells, inflammatory
cells, blood vessels, and extracellular matrices (ECM), creating both a physical barrier difficult
for adoptive cells to home to and penetrate as well as an immunosuppressive tumor
microenvironment (TME) inhibiting proliferation and function of antitumor effector cells.
36
Solid tumors that do not attract immune cells are often considered “cold tumors”; they are
either completely ignored by the immune surveillance system, or only have a T cell infiltrate in
their periphery.
37
Engineering T cells to express chemokine receptors that respond to chemokines
secreted by tumor cells has been shown as a way to enhance the ability of tumor-specific T cells
to home to tumor sites and result in therapeutic benefits in preclinical models.
38,39
Using protein-
degrading enzymes to target the protective proteins of ECM (such as heparin sulfate
proteoglycans) has shown promise in preclinical studies. CAR T cells expressing heparinase
have demonstrated improved tumor infiltration and antitumor efficacy in vivo.
40
Efforts to turn
“cold tumors” into immune-responsive “hot tumors”
may be a prerequisite for CAR directed
immune cell therapy to truly have therapeutic potentials for solid tumor types previously
considered “cold tumors”.
In the cases opposite to “cold tumors”, when TILs or TCR/CAR directed effector cells
are successful in homing to the tumor, their functions are largely inhibited by a number of
mechanisms existing in the TME (Figure 1-2). Cell populations including tumor-associated
macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), regulatory T cells, and
cancer-associated fibroblasts (CAFs) employ suppressive mechanisms via (1) secretion of
12
immunosuppressive cytokines such as IL-10 and transforming growth factor (TGF)-β, (2)
expression of inhibitory receptors such as CTLA-4 and PD-L1, and (3) metabolic pathways that
deplete nutrition for T cell function or upregulate suppressive metabolites.
22,41
Depending on
specific tumor type, the cancer cells also employ at least some of the mechanisms listed above to
mediate immune suppression.
Figure 1-2. Immunosuppressive factors in the solid tumor microenvironment. Adapted from Wagner J, Wickman E,
DeRenzo C, Gottschalk S. CAR T Cell Therapy for Solid Tumors: Bright Future or Dark Reality? Molecular
therapy. 2020;28(11):2320-2339. doi:10.1016/j.ymthe.2020.09.015
Immunosuppressive cytokines playing major roles in the TME include transforming
growth factor (TGF)-β, vascular endothelial growth factor (VEGF), interleukin-10 (IL-10), etc.
TGF-β inhibits T cell priming and infiltration and suppresses the cytotoxic functions of effector
cell.
42-43
IL-10 inhibits major histocompatibility complex II expression on antigen presenting
cells, suppresses M1 cytokine secretion and inducible nitric oxide synthase, and induces T cell
anergy.
44
VEGF accumulation inhibits dendritic cell maturation, enhances PD1/PD-L1
expression and IL-10 secretion.
45
Strategies to deal with the accumulation of immunosuppressive
13
cytokines involve direct inhibition of the suppressive signaling and overexpression of a negative
form of the respective receptor on T cells.
46
Immune inhibitory receptors constitute an important part of the tumor microenvironment.
For example, immune checkpoint PD-1 is upregulated on activated T cells. Binding of PD-1 and
its inhibitory ligand PD-L1 expressed by tumor cells can induce inhibition and apoptosis of T
cells.
41
Another immune checkpoint CTLA-4 expressed by antigen presenting cells binds to
CD28 competitively with CD80 and CD86, resulting in inhibition of the costimulatory signaling
through CD28 and thus inhibiting T cell activation and expansion. Protecting immune
checkpoints with neutralizing antibodies or soluble ligands has achieved great success in
preclinical and clinical settings. Pembrolizumab, a humanized antibody targeting PD-1, is widely
used to treat melanoma, lung cancer, head and neck cancer, Hodgkin lymphoma and stomach
cancer that overexpress PD-L1. The significance of immune checkpoint blockade for opening up
a whole realm of new possibilities of cancer immunotherapy was awarded the Nobel Prize in
Physiology or Medicine in 2018.
Metabolic pathways are also involved in inhibiting functions of intratumoral effector
cells. Overexpression of CD39 and CD73 converts extracellular ATP to adenosine, the
accumulation of which in the TME inhibits T cell proliferation and activation.
47
Accumulation of
prostaglandin E2, especially promoted by tumor hypoxia, inhibits effector cell function and
recruit MDSCs.
48-50
Upregulation of arginase secreted by myeloid cells degrades L-arginine
needed for cytotoxic iNOS production.
51
The convoluted inhibitory networks in TME make it crucial to understand the dominant
inhibitory mechanism or multiple mechanisms in each solid tumor type before one could decide
on the therapeutic schematics that would have the biggest potential. Due to the complexity of
14
immunosuppressive tumor microenvironment, combination strategies would be very necessary to
tackle with multiple inhibitory mechanisms simultaneously in order to unleash the full potential
of adoptive cell therapy in the battleground.
15
1.4 Focus of dissertation
Projects discussed in this dissertation focus on armoring CAR-directed immune cells to
enhance their persistence and function in the solid tumor microenvironment and achieve better
therapeutic efficacy, especially through secreting proteins with immune-modulating effects.
Engineering CAR-directed immune cells to secret functional proteins has the nature of
combination strategy and facilitates local delivery of the functional proteins in the solid tumor
microenvironment. In the clinical setting, it will have the potential advantages of reduced
systemic toxicity as well as cost-effectiveness compared to delivering several therapeutic
modalities separately. Furthermore, the strategies investigated in this dissertation are
transferrable to other adoptive cellular therapies that face similar roadblocks in solid tumor
microenvironment and may help them with enhanced therapeutic efficacy.
16
Chapter 2. Engineering CAR-Expressing Natural Killer Cells with
Cytokine Signaling and Synthetic Switch for an Off-the-Shelf Cell-
Based Cancer Immunotherapy
Portions of this chapter are adapted from: Qu Y, Siegler E, Cheng C, et al. Engineering CAR-
expressing natural killer cells with cytokine signaling and synthetic switch for an off-the-shelf
cell-based cancer immunotherapy. MRS communications. 2019;9(2):433-440.
doi:10.1557/mrc.2019.31
17
2.1 Abstract
Immune cells genetically engineered with chimeric antigen receptor (CAR) have been
shown to eliminate cancer cells, but their clinical efficacy in solid tumor has been limited partly
due to the lack of persistence in the immunosuppressive tumor microenvironment (TME). The
high cost and logistical burden of autologous adoptive cell therapy call for alternative off-the-
shelf CAR therapies. In this study, we synthetically engineered a 3
rd
generation anti-mesothelin
CAR, a membrane-bound IL-15/IL-15Rα complex, and an inducible caspase 9 “kill switch” to
express in NK92 cells for tumor-specific targeting effect, immuno-stimulatory signaling, and
safety control, and tested this allogeneic cell product in a preclinical ovarian cancer model.
18
2.2 Introduction
Chimeric antigen receptor (CAR)-engineered immune cells have shown great potential in
cancer immunotherapy, as CARs are recombinant receptors that can specifically recognize tumor
associated antigens on cancer cells and dramatically improve the efficacy of tumor-targeted
immune cells.
52
Among the candidates of immune cells to express CARs for adoptive cellular
immunotherapy, the well-characterized cytotoxic natural killer (NK) cell line, NK-92 cell line, is
a promising one due to its lack of immunogenicity and availability as an “off-the-shelf”
product.
12, 53
The standardized NK92 cell line is thus an ideal target for a synthetic biology
approach which equips cells with CARs and other engineered structures to resolve issues on
limited antitumor efficacy and potential toxicities.
Although CAR-engineered immune cells have been highly successful in treating
hematological malignancies, they have shown limited efficacy in solid tumors partly due to the
immunosuppressive tumor microenvironment (TME).
46
NK cells typically rely on interleukin
(IL)-2 for growth and cytotoxic function, so during animal studies or clinical trials, they are often
co-administered with exogenous IL-2, which may cause acute toxicity in high doses.
54
Alternatively, NK92 cells transduced with lentiviral vectors encoding IL-15 resulted in stable
expression of the cytokine and continued proliferation and cytotoxic capabilities without
exogenous IL-2.
55, 56
Moreover, the simultaneously expressed receptor subunit IL-15Rα can form
a stable complex with IL-15 on cell surfaces and enables autocrine stimulation of NK cells by
membrane-bound IL-15 in cis presentation.
57
This membrane-bound IL-15/IL-15Rα complex has
shown to contribute to the long survival of CD8 memory T cells, as well as persistence of T cells
engineered to co-express CAR and the complex in a tethered form.
58-59
We hypothesized that
19
NK92 cells engineered to co-express CAR and the membrane-bound IL-15/IL-15Rα complex
would also survive and proliferate without exogenous IL-2.
For both preclinical animal studies and clinical trials using NK92 cells to treat cancer, the
cells are typically irradiated prior to infusion to prevent engrafting cells from in vivo
tumorigenicity.
54, 60
As a result, the infused NK92 cells experience limited expansion without
long-term persistence. For NK92 cells engineered to express membrane-bound IL-15/IL-15Rα
complex, irradiation prior to infusion would greatly defeat the purpose of the complex to
improve NK92 proliferation and persistence in vivo. One potential alternative to irradiation is to
express an inducible suicide gene on infused cells as a safety switch. Transduced T cells selected
for high expression of the inducible caspase 9 (iCAS9) suicide gene have been shown to respond
well to the chemical inducer of dimerization (CID) AP20187 and resulted in dose-dependent
levels of apoptosis.
61-62
The iCAS9 suicide gene, when transduced into the CAR-NK cells with
membrane-bound IL-15/IL-15Rα complex, serves as a safety mechanism other than irradiation
without affecting the proliferation and persistence of infused NK92 cells. This suicide switch is
also a very useful tool for physicians to regulate the therapy when complications like “on-target,
off-tumor” toxicity or cytokine storms occur.
63
Mesothelin is a cell-surface molecule overexpressed in many carcinomas and is an
attractive target for CAR-engineered immune therapies.
64
A third-generation anti-mesothelin
CAR consisting of a single-chain variable fragment domain targeting mesothelin and two co-
stimulatory domains, CD28 and CD137 (4-1BB), transduced into T cells has shown to eradicate
large, established tumors overexpressing mesothelin antigen.
65
Therefore, we utilized synthetic biology tools and engineered NK92 cells to express anti-
mesothelin CAR and membrane-bound IL-15/IL-15Rα complex for specific killing and better
20
proliferation in an ovarian cancer mouse model. We further transduced the engineered CAR-NK
cells with the iCAS9 suicide gene as a safety switch to eliminate infused CAR-NK cells in case
of adverse side effects or post-treatment, in order to mitigate the risk of tumorigenicity from the
infused cells.
21
2.3 Materials and methods
2.3.1 Cell culture
SKOV3 (ATCC HTB-77) tumor cell lines were cultured in at 37°C and 5% CO2 in high-
glucose DMEM (Corning) media supplemented with 10% FBS(Sigma-Aldrich), 1% 100X pen-
strep(Corning), and 2mM L-glutamine(Corning). NK92 cells (Dr. Jihane Khalife, Children’s
Hospital Los Angeles; ATCC CRL-2407) were maintained in MEM-α (Gibco) supplemented
with 10% FBS, 10% HI horse serum(Gibco), 1% 100X NEAA(LONZA), 1% 100X pen-strep,
1mM sodium pyruvate(Corning), 0.1 mM 2-β mercaptoethanol, 0.2 mM myo-inositol, and 2.5
µM folic acid(all from Sigma-Aldrich). Mesothelin-expressing SKOV3 (SKOV.meso) cells were
generated by transducing SKOV3 cancer cells with lentiviral vectors containing mesothelin
cDNA and sorting mesothelin
positive cells with fluorescence-activated cell sorting.
2.3.2 Viral vector production
The CAR construct consists of a murine-derived scFv from the antigen-binding domain
of mesothelin antibody SS1, the CD8 hinge and CD28 transmembrane regions, and two
costimulatory domains CD28&4-1BB, and CD3ζ cytoplasmic regions in the retroviral MP71
vector. The membrane-bound IL-15/IL-15Rα complex was included in one of the CAR
constructs following a 2A linker. The iCAS9 suicide switch with GFP marker, pMSCV-F-del
Casp9.IRES.GFP, was purchased from Addgene. The plasmid for mesothelin consisted of human
mesothelin cDNA cloned into a lentiviral FUW backbone. The plasmids, together with envelope
and packaging plasmids, were used to transfect HEK 293T cells in 30mL plates using calcium
phosphate precipitation method to generate lentiviral or retroviral vectors. Fresh media (high
glucose DMEM supplemented with 10% FBS and 1% 100X pen-strep was replenished for the
22
cells four hours after initial transfection. Supernatants were harvested and filtered (0.45µm) 48
hours later and used fresh for subsequent transduction.
2.3.3 Transduction of NK92 and SKOV3 cells
NK92 cells were transduced with anti-mesothelin CAR and iCAS9 retroviral vectors.
Non-tissue culture-treated 12-well plates were coated overnight with 25 μg RetroNectin per well
(Clontech Laboratories). Retroviral vectors were subsequently spin-loaded onto the plates by
centrifuging at 2000 x g for two hours at 32°C. NK92 cells were resuspended at a concentration
of 5x10
5
/mL with fresh media complete with 200 U/mL human IL-2 and added to the vector-
coated plates. The plates were centrifuged at 600 x g for 30 min at 32°C and incubated overnight
at 37°C and 5% CO2. CAR
positive or GFP positive NK92 cells were sorted using fluorescence
activated cell sorting.
SKOV3 cells were similarly transduced by lentiviral vectors to highly express human
mesothelin on cell surface. 2 mL of freshly made viral supernatant was added to 1 million
SKOV3 cells in a tissue-culture 12 well plate. The plate was then centrifuged at 1050 x g for 90
minutes at 25°C for spinoculation and then resuspended in fresh media. The transduced cells
were sorted using fluorescence activated cell sorting for 100% positive mesothelin expression.
2.3.4 CAR detection on NK cell surface
After transduction and cell sorting, 1×10
5
NK92 cells were incubated with recombinant
human mesothelin-Fc chimera (R&D Systems) at a volume ratio of 1:50 (2 µg/mL) in PBS at
4°C for 30 minutes and rinsed with PBS. The cells were subsequently incubated with PE-labeled
goat anti-human Fc (Jackson ImmunoResearch) at a volume ratio of 1:150 in PBS at 4°C for 10
23
minutes. The cells were then rinsed, and analyzed through flow cytometry. Nontransduced NK92
cells were used in parallel as a negative control.
2.3.5 Intracellular cytokine staining
1×10
5
NK cells per well were co-incubated with target cells in 96-well round-bottom
plates at a 1:1 ratio for six hours at 37°C. 10 µg/ml of Brefeldin-A (Sigma) was added into the
co-culture to inhibit protein transport. At the end of the incubation, cells were permeabilized
using the CytoFix/CytoPerm kit (BD Biosciences) and stained for CD45 and IFN-γ using Pacific
Blue-conjugated anti-human CD45 (Biolegend) and PE-conjugated anti-human IFN-γ
(Biolegend). NK cells not co-cultured with target cells were used in parallel as a negative
control. Results were analyzed through flow cytometry. The data was collected in triplicates and
presented as the mean ± SEM.
2.3.6 Cytotoxicity assay
2x10
4
target cells were labeled with 5 µM carboxyfluorescein succinimidyl ester (CFSE,
Life Technologies) as previously described,
66
and co-incubated with NK cells at various ratios in
96-well round-bottom plates for 24 hours at 37°C. The cells were then trypsinized and incubated
in 7-AAD (Life Technologies) in PBS (1:1000 dilution) for 10 minutes at room temperature and
analyzed via flow cytometry. Percentages of killed cells were calculated as [CFSE
+
7-AAD
+
cells
/ (CFSE
+
7-AAD
-
+ CFSE
+
7-AAD
+
)] cells, with live/dead gates based on control wells of target
cells only to account for spontaneous cell death. The cytotoxicity assay results were determined
in triplicates and presented as the mean ± SEM.
24
2.3.7 Chemical inducer of dimerization sensitivity assay
CID AP20187 (B/B Homodimerizer, Takara) was diluted in complete NK cell media at a
concentration of 10 nM. 5x10
4
NK cells were incubated in media or 10 nM CID for 24 hours and
analyzed for viability. Number of viable cells were counted under a light microscope via trypan
blue exclusion method. The percentage of viable cells in culture was also detected using
AnnexinV/7-AAD staining(BD Biosciences) per the manufacturer’s instructions and analyzed
through flow cytometry. The assay were set up in triplicates and the data were presented as mean
± SEM.
2.3.8 Xenograft tumor model
All animal experiments were conducted according to the animal protocol approved by the
University of Southern California Institutional Animal Care and Use Committee (IACUC).
Eight-week-old female NOD.Cg-Prkdc
scid
IL2Rγ
tm1Wj1
/SZ (NSG) mice (Jackson Laboratories)
were inoculated subcutaneously with 3.5×10
6
SKOV.meso cells, and treatment began when
tumors grew to 50-70mm
3
. The mice were injected intravenously through the tail vein with
10×10
6
NK92 cells three times over a period of ten days. Tumor growth and body weight of the
remaining mice were closely monitored until the end of the experiment. Tumor volume was
recorded three times a week. A fine caliper was used to measure the length and width of the
subcutaneous tumor. The volume of the tumor was estimated by the calculation ½ × (length) ×
(width). The mice were euthanized when the tumor reached 1000mm
3
or became ulcerated.
2.3.9 Ex vivo NK cell staining
One week after the last NK cell treatment, mice were sacrificed for ex vivo analysis.
Each tissue sample was harvested into a single cell suspension through a 70 µm strainer, washed
25
with PBS, centrifuged, and resuspended in 3 mL TAC buffer for 10 minutes at room temperature
for blood lysis. The samples were then washed and resuspended with PBS for staining and
analysis. Fluorescent-conjugated anti-CD45 antibody (Biolegend) was used to stain the samples
in 100 µL PBS at a 1:100 volume ratio for 15 minutes at 4°C in a 96-well plate. Samples were
analyzed using flow cytometry in triplicates and presented as the mean ± SEM.
2.3.10 Statistical analysis
Statistical analysis was performed in Microsoft Excel and GraphPad Prism 6. One-way
ANOVA with Tukey’s multiple comparison was used to assess the data between groups. P
values of < 0.05 were considered significant. Significance of findings was defined as: ns = not
significant, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
26
2.4 Results
2.4.1 Generation of anti-mesothelin CAR-NK92 cell lines with retroviral vector
Figure 2-1. Anti-mesothelin CAR designs and expression levels. (a) Schematic representation of CAR constructs.
(b) CAR expression over time in the absence of exogenous IL-2. (c) CAR expression of sorted cell populations. (d)
Expansion of NK cells with or without exogenous IL-2.
Anti-mesothelin CARs with or without membrane-bound IL-15/IL-15Rα complex were
transduced efficiently in NK92 cells using retroviral vectors encoding a third-generation anti-
mesothelin CAR, with membrane-bound IL-15/IL-15Rα complex as NK.αmeso.mbIL15, or
without the complex as NK.αmeso.mbIL15. The schematics of the inserted transgene are shown
in Figure 2-1A. Genetically engineering CAR-NK cells with membrane-bound IL-15/IL-15Rα
complex allowed CAR-NK cells to expand in absence of exogenous IL-2 in vitro. Unsorted
transduced NK.αmeso and NK.αmeso.mbIL15 cells were cultured in complete media without IL-
2 supplementation over 10 days. NK.αmeso cells lost CAR expression and were dead by day 5,
while NK.αmeso.mbIL15 increased the purity of the CAR-expressing population over time in
27
absence of IL-2 (Figure 2-1B). Although CAR
+
NK92 cells were sorted later using flow
cytometry for subsequent studies, this demonstrates that starving unsorted cells of IL-2 can serve
to enrich the transduced cell population. After selection for CAR positive NK92 cells via
fluorescence activated cell sorting, the cells displayed high CAR expression (Figure 2-1C).
Nontransduced NK92 cells were used as a negative control for the staining. Previous research
indicates that the membrane-bound IL-15/IL-15Rα complex acts in cis instead of trans, behaving
as an autocrine stimulation by binding to the β and γ IL-15R subunits expressed on the
transduced cells instead of those on surrounding immune cells.
57
This data supports that finding,
as CAR
+
populations selectively proliferated, not the entire cell population—cells were
stimulated by the membrane-bound IL-15/IL-15Rα complex only if the cells already expressed
that transgene. It was further demonstrated that NK.αmeso.mbIL15 cells could survive and
proliferate in absence of exogenous IL-2 by observing the expansion over 10 days of sorted
CAR-NK cells in IL-2-free media (Figure 2-1D). All cells expanded well in media with IL-2
supplementation, but only the NK.αmeso.mbIL15 cells expanded without IL-2—nontransduced
NK92 cells and NK.αmeso cells died by day 5 in media without IL-2 (p < 0.001).
NK.αmeso.mbIL15 cells also expanded significantly more than nontransduced NK92 cells when
supplemented with IL-2 (p < 0.01).
2.4.2 Enhanced in vitro functions of engineered CAR-NK92 cells
CAR-NK cells with membrane-bound IL-15/IL-15Rα complex secreted cytokines and
exhibited cytotoxicity to mesothelin-expressing target cells. In vitro functionality of the CAR-
NK cell lines was assessed by performing interferon-γ (IFN-γ) release and cytotoxicity assays.
SKOV3 cells induced minimal IFN-γ production in NK cell lines, with or without anti-
mesothelin CAR, suggesting low mesothelin expression on the cell surface of SKOV3 cells.
28
SKOV.meso cells transduced to overexpress mesothelin were used as the desired target cells,
while SKOV3 cells were used as negative control. In the IFN-γ assay, nontransduced NK92 cells
did not release IFN-γ in response to SKOV.meso cells, demonstrating the necessity of an anti-
mesothelin CAR to generate an immune response. NK.αmeso cells had an increased IFN-γ
+
population when coincubated with SKOV.meso cells, but NK.αmeso.mbIL15 cells had
significantly higher percentages of IFN-γ
+
cells after exposure to SKOV.meso cells compared
with both nontransduced (p < 0.001) and NK.αmeso cells (p < 0.01). This data is in accordance
with previous work showing that IL-15 aids in greater production of IFN-γ in NK cells.
67-69
Cytotoxicity assays were performed using the above-mentioned target and effector cells in
different ratios. NK.αmeso.mbIL15 cells killed lower percentages of SKOV.meso target cells
compared to NK.αmeso cells, perhaps due to slightly lower CAR expression on the cell surface.
Both CAR-NK cell lines were significantly more cytotoxic to SKOV.meso cells compared to
nontransduced NK92 cells at all effector-to-target ratios (Figure 2-2B).
Figure 2-2. Enhanced in vitro functions of engineered CAR-NK92 cells. (A) Interferon-γ secretion by NK cells
coincubated with mesothelin+ or mesothelin− target cells. (B) Cytotoxicity of NK cells coincubated with
mesothelin+ or mesothelin− target cells. Significance stars compare non-transduced NK cells to NK.αmeso and non-
transduced NK cells to NK.αmeso.mbIL15 (*P < 0.05, **P < 0.01, ***P < 0.001).
29
2.4.3 CID induced apoptosis of iCAS9 transduced NK92 cells
The iCAS9 suicide gene harnesses technology from genetic engineering and small
molecule drugs. This kill switch was efficiently transduced in NK92 cells (NK92 without CAR,
NK.αmeso, and NK.αmeso.mbIL15) using the commercially available retroviral vector
described above in Materials and methods. All cell lines were sorted using fluorescence activated
cell sorting for >99% expression levels and transgene expressions of comparable intensities
across all groups were confirmed (Figure 2-3A). Uniform transgene expression across all groups
is important, as higher iCAS9 expression leads to greater sensitivity to the CID AP20187 used
when triggering apoptosis.
62
Figure 2-3. CID induced apoptosis of iCAS9 transduced NK92 cells. (a)iCAS9 expression of sorted NK cell lines.
(b) Viability of NK cells, analyzed by both trypan blue staining and7-AAD/AnnexinV flow cytometry, after
incubation in either plain media or 10 nM CID for 24 h.
30
NK92 cells expressing iCAS9 were highly sensitive to low concentrations of the CID
AP20187, an otherwise nontoxic small molecule dimerizer. To test the capability of the suicide
transgene to induce apoptosis in cells after exposure to the AP20187, two methods were used to
observe the viable cell populations in culture after addition of CID AP20187: 1) trypan blue
exclusion method with cell counting under optical microscope using a hemocytometer and 2)
AnnexinV/7-AAD staining and analysis with flow cytometry. Six cell lines were tested—
nontransduced NK92, NK.αmeso, and NK.αmeso.mbIL15 cells, each either with or without
iCAS9 transgene. Only the groups of cells expressing iCAS9 showed markedly decreased
viability after CID exposure (p < 0.001) in both trypan blue exclusion and AnnexinV/7-AAD
staining (Figure 2-3B). These experiments demonstrate that, if CAR-NK therapy needs to be
stopped, either due to unwanted toxicity or simply at the end of treatment, CID can be
administered and quickly trigger apoptosis only in cells expressing the iCAS9 transgene.
2.4.4 Membrane bound IL15/IL15Rα complex enhanced antitumor efficacy of anti-
mesothelin CAR-NK in vivo
31
Figure 2-4. Tumor growth control by anti-mesothelin CAR-NK. (A) Animal study schedule. (B) Tumor growth
curve for mice receiving different treatments. (C) Comparison between tumor sizes across groups at day 9 of last
treatment and day 20.
To evaluate the antitumor efficacy of NK.αmeso.mbIL15 cells in vivo, 10 million cells
were injected intravenously into NSG mice with established SKOV.meso tumors twice a week
for two weeks. The data demonstrate that NK.αmeso.mbIL15 cells slowed tumor growth and
exhibited better tumor growth control than NK.αmeso cells (Figure 2-4). The treatment with
NK.αmeso cells did not show better antitumor efficacy than that of NK92 or PBS only.
2.4.5 Membrane bound IL15/IL15Rα complex enhanced cell proliferation in vivo
Figure 2-5. Enhanced cell proliferation of anti-mesothelin CAR-NK in vivo. (A) Animal study schedule. (B)
Percentage of NK cells in mouse peripheral blood over time. (C) Percentages of NK cells in bone marrow. (D)
Representative FACS plots of NK cells in mouse bone marrow 7 days after the final NK injection.
Infused NK.αmeso.mbIL15 cells proliferated more in vivo compared with NK.αmeso
cells. Tumor-bearing mice were separated into two groups, with one group receiving
NK.αmeso.mbIL15 cells and the other group NK.αmeso cells on days 0, 5, and 9. The mice were
tail bled three times a week following the first NK injection to monitor its expansion in
32
peripheral blood. By day 12, the group receiving NK.αmeso.mbIL15 cells had significantly
higher NK cell populations in tail blood (Figure 2-5B). One week after the last injection of NK
cells, the mice were euthanized, and organs were harvested and analyzed for NK cell
populations. Interestingly, the group receiving NK.αmeso.mbIL15 cells treatment exhibited
significantly higher populations of NK cells homing to the bone marrow, while NK.αmeso cells
had few NK cells in the bone marrow. Representative flow cytometry plots are shown in Figure
2-5D, and quantitative data are displayed in Figure 2-5C. NK92 cells used for adoptive cell
therapy has not been reported in literature to be able to home to bone marrow, making NK92
cells expressing membrane-bound IL-15/IL-15Rα a potential therapy for malignancies which
heavily metastasize to the bone marrow.
70
2.4.6 CID AP20187 induced apoptosis of iCAS9-transduced CAR-NK92 cells in vivo
Figure 2-6. CID drug induction of iCAS9-transduced cells apoptosis in vivo. (A) Animal study schematic. (B) Fold-
change of NK cells in mouse peripheral blood comparing before and after CID or PBS administration. (C)
Percentages of NK cells in mouse bone marrow after CID or PBS administration.
33
AP20187 injected into the mice induced apoptosis of NK cells expressing the iCAS9
transgene. Tumor-bearing mice received 3 injections of 10 million NK.αmeso.mbIL15 cells as
described above. On day 13, one third of the mice received 50 µg free CID through
intraperitoneal injection; one third received the same amount of CID encapsulated in a
previously reported drug-delivery liposomal nanoparticle through intravenous injection;
71
and
one third did not receive CID treatment. The mice were tail bled on days 7, 12, and 16 and were
analyzed for NK cell populations in tail blood. Mice without any CID treatment had higher NK
cell populations in tail blood compared with mice receiving CID injections through either route
(Figure 2-6B). On day 16, the mice were euthanized, and their organs were analyzed for NK cell
populations. Mice treated with CID had minimal NK cell populations in the bone marrow, while
mice without CID treatment retained high levels of NK cell populations that homed to the bone
marrow (Figure 2-6C). Overall, the addition of an engineered cytokine and suicide switch allows
for the optimization of biological functions in CAR-NK cells.
34
2.5 Discussion
CAR-engineered immune cells have been successful in treating a variety of
hematological cancers, but their clinical success in treating solid tumors is lacking. CARs are a
clinically relevant application of synthetic biology, and their modular design offers the potential
to target a variety of diseases simply by switching out the antigen-binding domain. CARs can be
further improved by genetically modifying human immune cells with additional engineered
receptors. This research presents two more synthetic molecules in addition to the anti-mesothelin
CAR: a membrane-bound cytokine, IL-15/IL-15Rα, and a suicide switch, iCAS9, for immune
stimulation and safety mechanisms. Instead of autologous T cells, which are used in most CAR
studies, the NK92 cell line has proven effective in executing CAR-mediated antitumor functions
in preclinical settings and provides a renewable, off-the-shelf source of engineered cells. A
homogenous, allogenic cellular product has the potential for more precise modulation of
biological outputs than autologous cells, which vary widely in function from patient to patient.
There are many more applications of synthetic biology for CARs beyond the scope of this
research letter, including logic gates and synthetic notch receptors.
26,72-73
CARs require fine-
tuning through molecular engineering to have the precision and persistence needed to eradicate
solid tumors, and additional artificial receptors modulating the immune environment and cell
response are key to more effective CARs in the future.
35
Chapter 3. Engineering CAR-T Cells to Overexpress Adenosine
Deaminase 1 (ADA) for Enhanced Anti-tumor Efficacy in Solid
Tumor
Portions of this chapter are adapted from: Qu Y, Dunn Z, Chen X, et al. Adenosine Deaminase 1
Overexpression Enhances the Antitumor Efficacy of CAR-engineered T Cells. (manuscript
submitted to Human Gene Therapy, under review)
36
3.1 Abstract
Chimeric antigen receptor (CAR) T cell therapy has spawned unprecedented responses in
certain leukemias and lymphomas but has yet to achieve similar success in combating solid
tumors. A substantial body of work indicates that the accumulation of adenosine in the solid
tumor microenvironment (TME) plays a crucial role in abrogating immunotherapies. Adenosine
deaminase 1 (ADA) catabolizes adenosine into inosine and is indispensable for a functional
immune system. We have, for the first time, engineered CAR T cells to overexpress ADA. To
potentially improve the pharmacokinetic profile of ADA, we have modified the overexpressed
ADA in two ways, through the incorporation of an (1) albumin-binding domain or (2) collagen-
binding domain. ADA and modified ADA were successfully expressed by CAR T cells and
augmented CAR T cell exhaustion resistance. In a preclinical engineered ovarian carcinoma
xenograft model, ADA and collagen-binding ADA overexpression significantly enhanced CAR
T cell expansion, tumor tissue infiltration, tumor growth control, and overall survival, whereas
albumin-binding ADA overexpression did not. Furthermore, in a syngeneic colon cancer solid
tumor model, the overexpression of mouse ADA by cancer cells significantly reduced tumor
burden. The overexpression of ADA for enhanced cell therapy is a safe, straightforward,
reproducible genetic modification that can be utilized in current CAR T cell constructs to result
in an armored CAR T product with superior therapeutic potential.
37
3.2 Introduction
Despite revolutionizing the treatment of B cell malignancies,
74-77
CAR T cells have had
disappointing efficacy against solid tumor.
78-80
A formidable roadblock to the use of CAR-T cell
in solid tumor treatment is the immunosuppressive tumor microenvironment (TME).
81-82
Among
the variety of cellular populations, soluble factors, and structural proteins that contribute to a pro-
tumor microenvironment,
83-85
the past decade has witnessed a focus on the ubiquitous purine
nucleoside adenosine.
86-88
Originally identified as a molecule essential for preventing excessive
tissue damage during inflammation,
89
adenosine has since been discovered to reach micromolar
concentrations in the TME,
90-91
especially due to an increase in the expression of adenosine
generating ectonucleotidases CD39 and CD73.
92-93
In contrast to the source of its generation, the
immuno-stimulatory ATP, adenosine is a potent anti-inflammatory signal, suppressing the
function of anti-tumor immune cells while simultaneously enhancing the activity of suppressor
cells.
94
Four adenosine receptors (ARs) A1R, A2AR, A2BR and A3R bind adenosine,
95
and the
predominant AR subtype present on T lymphocytes is A2AR.
96-97
A2AR stimulation by adenosine
dampens TCR activation through cyclic adenosine monophosphate-protein kinase A (cAMP-
PKA) signaling, leading to severe inhibition of T cell effector function and pro-inflammatory
cytokine production.
98-100
Previous studies have addressed the problem of adenosine accumulation in tumor
microenvironment by inhibiting the enzymatic activities of CD39 and CD73 to reduce adenosine
production,
101-102
or by blocking A2AR with antagonists to prevent the activation of A2AR and
downstream signaling pathways.
103-104
Both strategies ameliorate the immunosuppressive effect
of adenosine on antitumor T cells, more specifically CD8+ CAR T cells, and restores their
antitumor efficacy.
105-106
38
Adenosine deaminase is an enzyme involved in purine metabolism. It catalyzes the
irreversible degradation of adenosine into inosine and is essential for a functional immune
system by preventing the buildup of toxic metabolites.
107-108
One isoform, adenosine deaminase
1(ADA), is widely expressed in most cells in the body, particularly lymphocytes and
macrophages, and also has a costimulatory role for T cells.
109
Overexpression of ADA on CAR T
cells is a potential strategy to reduce the adenosine accumulation in solid tumor
microenvironment and ameliorate the hypofunction of CAR T cells caused by adenosinergic
signaling.
ADA has a short half-life in circulation, which is a common challenge shared by other
protein drugs.
110
One strategy to improve the retention time of protein drugs is to modify proteins
with albumin binding peptides.
111
Albumin is an abundant 67 kDa serum protein with an
exceptionally long half-life (19 days in humans) due to its interactions with Fc natal receptor,
and proteins bound to albumin exhibit increased in vivo persistence. Rather than encumbering a
protein with albumin, a short peptide that binds albumin can take advantage of the unique serum
longevity of albumin while minimizing the risk to protein functionality.
112
Another strategy to improve retention time of ADA in vivo takes advantage of the
extracellular matrix of solid tumor. Cancerous extracellular matrix remodeling can result in
increased concentrations of collagen and uncontrolled, unorganized tumor growth causes
hyperpermeable tumor vasculature.
113
This leakiness causes tumor associated collagen to be
exposed to molecules in the blood preferentially to other tissues.
114-115
The von Willebrand factor
(VWF) is a hemostasis that tightly binds collagen I and III to initiate thrombosis upon blood
vessel injury, and within VWF the A3 domain is responsible for collagen binding.
116
The
conjugation of VWF A3 to immune checkpoint inhibitors and fusion to IL-2 has been
39
demonstrated to enhance the targeted delivery of the immunomodulatory agents to the TME.
117
ADA linked with the collagen binding domain VWF A3 may preferentially accumulate in TME
for a localized adenosine-degrading function.
In this study, we transduced CAR T cells to overexpress ADA in a single bicistronic
vector with an anti-CD19 CAR. To improve the pharmacokinetic profile of overexpressed ADA,
we proposed two ways to modify it through the incorporation of (1) albumin-binding domain or
(2) collagen-binding domain. We proved that CAR T cells overexpressing ADA/modified ADA
exhibited enhanced exhaustion resistance and better functions in adenosine-rich environment.
CAR T cells overexpressing ADA and ADA with collagen binding domain outperformed
parental CAR T cells in an engineered ovarian carcinoma xenograft solid tumor model. We
further validated ADA overexpression as a potential cancer treatment in a proof of concept
syngeneic colon cancer solid tumor model, in which the overexpression of mouse ADA by
cancer cells resulted in reduced tumor burden compared to control cancer cells.
40
3.3 Results
3.3.1 Design, generation, and validation of CAR T cells engineered to secrete ADA
Figure 3-1. ADA injections enhance CAR T cell therapy tumor inhibition but does not improve overall survival. A)
Schematic representation of experiment procedure for tumor challenge, CAR T cell injection, and ADA treatment.
NSG mice were subcutaneously challenged with 3.5x10^6 SKOV3.CD19 tumor cells. Once the tumors reached a
size of 50-80 mm^3 (day 0), 3x10^6 CAR19 T cells were adoptively transferred via intravenous injection. The
CAR19 + ADA group received 5U bovine ADA through intraperitoneal injection daily from Day 0 to Day 12. B)
Tumor growth curve for mice treated with CAR19 or CAR19 + ADA injection. Data were presented as mean tumor
volume 土 SEM at indicated time points (n= 5). C) Overall survival of mice bearing SKOV3.CD19 tumors after the
indicated treatment. Survival curve was plotted using the Kaplan-Meier method and compared using the log-rank
(Mantel-Cox) test (n = 5).
A preliminary combination study in which commercial bovine ADA was injected
systemically into tumor-bearing mice in conjunction with CAR T cells has shown that ADA
injections initially enhanced tumor growth inhibition, but this did not culminate in an improved
overall survival (Fig. 1A-C). The results indicate that sustained administration of ADA may be
necessary to engender long-term benefits. Therefore, we engineered CAR T cells to secrete ADA
constitutively to circumvent the challenges associated with continuous exogenous delivery of
41
ADA to the TME. Retroviral vector constructs used for our anti-CD19 CAR (CAR19) T cells,
anti-CD19 CAR with ADA secretion (CAR19.ADA), anti-CD19 CAR secreting ADA with
albumin binding domain (CAR19.aADA), and anti-CD19 CAR secreting ADA with collagen
binding domain (CAR19.cADA) are depicted in Figure 3-2A.
Figure 3-2. Design and characterization of CAR and ADA expression in transduced PBMCs. A) Schematic
representations of parental anti-CD19 CAR T cells (CAR19) and ADA overexpressing anti-CD19 CAR T cell
(CAR19.ADA, CAR19.aADA, CAR19.cADA) constructs. B) Expression of the four CAR constructs. T cells were
stained with biotinylated goat anti-mouse antibodies followed by APC conjugated streptavidin to detect CAR
expression on the cell surface. C-D) Overexpression of ADA in the supernatant from 48-hour T cell only culture or
coculture with SKOV3.CD19 at a 1:1 effector T cell: tumor cell ratio was analyzed by ELISA. E) Western blot
analysis of ADA. T cells were cultured in the presence of Brefeldin A followed by cell lysis and protein
quantification using standard BCA assay. 20ug total protein was then used for anti-ADA immunoblotting. F)
Characterization of modified ADA.
All constructs were efficiently transduced into and expressed by T cells and CAR
expression levels are shown in Figure 3-2B. Following successful CAR transduction, we
normalized CAR expression levels among groups and assessed cell culture supernatants and cell
42
lysates for ADA and modified ADA. A significantly higher concentration of ADA in the cell
culture supernatants of ADA engineered CAR T cells was observed by ELISA (Fig. 3-2C, 3-2D),
both when T cells were cultured alone and co-cocultured with antigen presenting tumor cells. We
further validated the presence and size of CAR T cell expressed aADA and cADA by
immunoblot following protein secretion inhibited T cell culture (Fig. 3-2E). Cell lysates were
stained for ADA and, as expected, endogenous ADA expression was identified for T cell groups,
and the aADA and cADA were identified at their respective molecular weights, 44 kDa and 62
kDa. Intracellular ADA and ecto-ADA flow cytometry analysis did not reveal differences in
ADA expression levels (data not shown), likely due to the endogenous production and transport
of ADA. To produce the necessary quantities of purified protein for binding analysis, we stably
transduced HEK-293T cells to secrete his-tagged aADA or cADA. Following protein isolation
and quantification, purified aADA and cADA were evaluated for target binding, specific activity,
and size (Fig. 3-2F, Fig. 3-3A-D). The modified ADAs of the predicted molecular weight bound
their targets with nanomolar affinities similar to those previously reported for SA21 and VWF
A3 fusion proteins,
39,44
and retained the catalytic function of ADA.
43
Figure 3-3. Binding kinetics of purified aADA and cADA. His-tagged aADA and cADA proteins were purified and
quantified using BCA assay. Kd values of aADA against human serum albumin (A) and mouse serum albumin (B),
and cADA against collagen III (C) were measured by ELISA at different concentrations of purified protein. D)
Purified proteins were loaded to SDS-PAGE and detected with chemiluminescent western blot detection.
44
3.3.2 Incorporation of ADA, aADA, or cADA into CAR T cells does not comprise in vitro
effector functions
Figure 3-4. ADA overexpression does not compromise CAR T cell in vitro cytotoxicity, effector cytokine
production, and proliferation. A) Cytotoxicity of the four CAR groups against target cells. CAR T cells were
cocultured for 16 hours with SKOV3-CD19 cells at 0.2:1, 1:1, 5:1, and 10:1 effector-to-target ratios, and
cytotoxicity against SKOV3-CD19 was measured. Nontransduced (NT) T cells were used as a control. B) The four
groups of CAR T cells were cocultured for 24, 48, 72, and 96 hours with SKOV3-CD19 cells at 1:1 effector-to-
target ratios, and Ki67 expression of CD3+ cells was measured. Nontransduced (NT) T cells were used as a control.
C) T cells were plated 6hrs before SKOV3-CD19 cells were added at 1:1 effector-to-target ratios and brefeldin A
was supplemented into the coculture. 6hrs after coculture, IFN-γ expression of CD3+CD8+ cells was measured
through intracellular cytokine staining, representative FACS scatterplots gated on CD3+CD8+ cells shown.
Nontransduced (NT) T cells were used as a control.
ADA overexpressing CAR T cells exhibited comparable cytotoxicity, proliferation, and
IFN-γ expression level to parental CD19 CAR T cells (Fig. 3-4). The cytolytic function of the
CAR T cells was assessed in 16-hour cytotoxicity assays, in which CAR T cells and
SKOV3.CD19 cells were cocultured at effector to target cell ratios of 0.2:1, 1:1, 5:1, 10:1 (Fig.
3-4A). The cytotoxicity assays revealed commensurate cytotoxicity among the groups. ADA has
45
been reported to increase the proliferation of T cells,
118,119
but this phenomenon was not observed
in our Ki67 staining proliferation studies nor CFSE staining (Fig. 3-4B), possibly due to higher
concentrations of ADA achieved via exogenous ADA supplementation in previous studies than
through ADA overexpression from our modified CAR T cells. Intracellular expression level of
IFN-γ was also comparable among CAR expressing groups (Fig. 3-4C). Thus, ADA
overexpression does not hinder in vitro effector functions, but potential costimulatory effects
were not observed.
3.3.3 ADA overexpression reduces CAR T cell exhaustion, Treg induction, and adenosine
susceptibility in vitro
To assess the effect of ADA overexpression on protecting CAR T cells from effector
inhibition, the parental and modified CAR T cells were cocultured with SKOV3.CD19 target
cells and then analyzed by flow cytometry for the expression of established T cell exhaustion
markers PD-1, LAG-3, or TIM-3 and transcription factor FOXP3 for T regulatory phenotype
determination. In addition, coinhibitory receptor PD-L1 upregulation was assessed, as PD-L1
engagement on T cells was recently shown to promote self-tolerance and suppression of
neighboring effector T cells in cancer.
120
Baseline pre-coculture exhaustion marker expression
and phenotype proportions were similar across groups (data not shown). ADA overexpressing
CAR T cells displayed reduced PD-1 and PD-L1 expression levels in CD8+ cells when
compared to parental CAR T cells (Figure 3-5AB), whereas there was no significant difference
in LAG-3 and TIM-3 expression (data not shown). Furthermore, unmodified CAR T cells had a
pronounced higher percentage of CD4+FOXP3+ T regulatory cells (Fig. 3-5CD). The reduction
in expression of two critical T cell inhibitory receptors as well as Treg differentiation suggests
46
that ADA overexpression can augment CAR T cell resistance to immunosuppressive signaling in
the TME.
We also examined the functionality of the CAR T cell groups in adenosine supplemented
assays (Fig. 3-5EF). After 12-hour cocultures in media containing 50 uM adenosine or 0 uM
control wells, cytotoxicity and PD-1 expression were assessed. ADA overexpressing CAR T
cells were resistant to adenosine-mediated cytotoxicity inhibition, whereas parental CAR T cells
experienced a 20% reduction in cytotoxicity. The PD-1 exhaustion study results were replicated
in the presence of 50 uM adenosine, with ADA overexpressing groups having less PD-1 positive
CD8+ T cells compared to parental CAR T cells.
Figure 3-5. ADA overexpression prevents CAR T cell exhaustion and reduces Treg differentiation. A-B) CAR T
cells were cocultured for 24 hours with SKOV3-CD19 cells at a 1:1 effector-to-target ratio and stained for the
expression of exhaustion markers. Nontransduced (NT) T cells were used as a control. C-D) CAR T cells were
cocultured for 24 hours with SKOV3-CD19 cells at a 1:1 effector-to-target ratio and stained for the expression of
FOXP3. Nontransduced (NT) T cells were used as a control. Representative FACS scatterplots gated on CD3+CD4+
cells (D) and summarized in (C). The four CAR Groups were cocultured with target cells at a ratio of 1:1 for 12 hrs
in the presence of 0 or 50 uM adenosine. Data presented as percentage of 0 uM adenosine killing in the 50 uM
adenosine samples. F) CAR T cells were cocultured for 12 hours with SKOV3-CD19 cells at a 1:1 effector-to-target
ratio in the presence of 50 uM adenosine and stained for the expression of PD-1 expression.
47
3.3.4 ADA overexpression and cADA expression enhance CAR T cell antitumor activity
and improve overall survival in a xenograft solid tumor model
Figure 3-6. The overexpression of ADA and cADA increases overall survival. A) Schematic representation of
experiment procedure for tumor challenge and adoptive cellular transfer. B) Tumor growth curve for mice treated
with NT or CAR T cells. Data were presented as mean tumor volume ± SEM at indicated time points (n=7 for all
groups). C) Mouse survival curves for the different treatment groups were calculated using the Kaplan–Meier
method (n=7). D) The percentage of human CD45+ T cells in the blood of SKOV3.CD19 bearing mice that were
treated with NT or CAR T cells was analyzed by flow cytometry at day 12. E) Ratio CD45+CD4+FOXP3+ T cells
in peripheral blood. F) Relative concentration of inosine in peripheral serum compared to inosine concentration in
NT treated mouse serum (n=3 mice per group).
Following the promising in vitro assessment of ADA overexpressing CAR T cells, we
evaluated the therapeutic potency of our treatments in an immunocompromised mouse model
with xenograft ovarian cancer. Once subcutaneous SKOV3.CD19 tumors reached a volume of
50-80 mm
3
, 3 million CAR+ T cells were administered for treatment and tumor growths were
monitored (Fig. 3-6A). All NT group mice reached endpoint by day 15, and by day 18 the
48
CAR19 and CAR19.aADA groups had reached their median overall survival, achieving an
increase in median OS of 6.7% and 20% respectively when compared to the control mice (Fig. 3-
6C). The CAR19.ADA and CAR19.cADA treated mice had significantly reduced tumor burden
by day 13 when compared to all other groups, and experienced prolonged overall survival, both
with an approximately doubled median OS than that of the control mice.
On day 12 post treatment, 3 mice from each group were tail bled for circulating T cell
analysis and serum inosine concentration measurements. CAR19.ADA and CAR19.cADA T
cells were more prevalent in the blood (Fig. 3-6D). ADA overexpression also resulted in a
decreased proportion of peripheral T regulatory cells (Fig. 3-6E). Serum inosine levels were
analyzed using an Inosine Assay Kit (Sigma), which revealed an increase in inosine
concentration contingent on ADA overexpression (Fig. 3-6F).
3.3.5 ADA overexpression and cADA expression enhances intratumoral CAR T cell
expansion
49
Figure 3-7. ADA overexpressing CAR T cells exhibit enhanced in vivo expansion. A) Schematic representation of
experiment procedure for tumor challenge and adoptive cellular transfer. NSG mice were subcutaneously challenged
with 3.5x10
6
SKOV3.CD19 tumor cells. Once the tumors reached a size of 50-80 mm
3
(day 0), 3x10
6
CAR+ T cells
were adoptively transferred via intravenous injection. B) The percentage of human CD45+ T cells in the tumor,
blood, spleen, and bone marrow of SKOV3.CD19 bearing mice that were treated with NT or CAR T cells was
analyzed by flow cytometry at day 10 (n=3 per group). C) CD8+:CD4+ ratios of T cells in the tumor, blood, spleen,
and bone marrow. CAR19.ADA.CBD cells exhibit a higher CD8+:CD4+ T cell ratio at the local tumor site. D) PD-1
expression in TILs. E) Ratio of CD45+CD8+:CD45+CD4+FOXP3+ cells in the tumor tissue (n=3 mice per group).
To study whether the enhanced antitumor efficacy observed in CAR19.ADA and
CAR19.cADA groups are correlated with intratumoral CAR T cells, we performed another
animal study following a similar schematic (Fig. 3-7A). Once the subcutaneous SKOV3.CD19
tumors reached a volume of 50-80 mm
3
, 3 million CAR+ T cells were administered. On day 10
the mice were sacrificed and ex vivo analysis was performed. When compared to unmodified
CAR T group, CAR19.ADA and CAR19.cADA groups displayed superior in vivo expansion as
shown by increased percentages of CD45+ T cells in the tumor, blood, spleen, and bone marrow
(Fig. 3-7B). Cells harvested from the organs were further characterized for phenotype, PD-1
expression, and pCREB expression. Notably, only the CAR19.cADA TILs displayed an
increased CD8+:CD4+ ratio (Fig. 3-7C). TILs from all the ADA overexpressing groups
displayed lower PD-1 expression compared to parental CAR T cells (Fig. 3-7D), whereas the
intensities for pCREB were equivalent between groups (data not shown). Although the
percentage of T regulatory in the CD4+ helper T cell populations in the tumor and spleen were
commensurate amongst the parental and modified CAR T cell groups (data not shown), the
increase in CD8+ TILs for the cADA overexpressing treatment resulted in a significantly higher
CD8+ : Treg ratio in the TME (Fig. 3-7E).
3.3.6 ADA overexpression reduces tumor burden in a syngeneic solid tumor model
Adenosine precipitates tumor tolerance and promotion by binding to ARs on a variety of
immune and stromal cellular populations.
94,101
While immunocompromised preclinical models
50
enable the study of human T cells and cancer cells, corroborating our ADA overexpression in a
syngeneic model is necessary to address the multifaceted role adenosine plays in the TME. In a
straightforward ADA gene therapy proof-of-concept study, we transduced CT26 murine colon
carcinoma cells to express GFP (CT26.GFP) or GFP and mADA (CT26.GFP.mADA) and sorted
for GFP+ cells of comparable intensity (Fig. 3-8A). We then verified mADA overexpression
(Fig. 3-8B) and commensurate growth rates for the two engineered CT26 cells lines (Fig. 3-8C).
We then challenged BALB/c mice with subcutaneously inoculated CT26.GFP or
CT26.GFP.mADA. Regardless of the starting tumor inoculum (1M or 3M cancer cells),
CT26.GFP.mADA groups exhibited significantly smaller tumor burden by day 25 when
compared to CT26.GFP groups (Fig. 3-8D). Weight of the mice were also monitored and no
toxicities related to mADA overexpression were observed (Fig. 3-8E).
51
Figure 3-8. Mouse ADA expression reduces the growth rate of CT26 subcutaneous tumors. A) CT26 cell were
stably transduced with lentiviral vectors expressing FUGW or FUGW.mADA, and sorted with flow cytometry for
comparable GFP levels. B) Overexpression of mADA in cell culture supernatant was measured by ELISA. C) In
vitro growth kinetics of CT26.GFP and CT26.GFP.mADA cells. D) BALB/c mice were injected subcutaneously
with 1x10
6
or 3x10
6
CT26.GFP or CT26.GFP.mADA cancer cells and monitored for tumor growth (n = 5 mice per
group) and weight (E).
52
3.4 Discussion
3.4.1 Interpreting experimental results
In this study, we engineered CAR T cells to overexpress adenosine deaminase 1 or
express protein-modified ADA for enhanced pharmacokinetic profiles and demonstrated that the
overexpression increased the amount of ADA secreted by the CAR19.ADA, CAR19.aADA, and
CAR19.cADA T cells. The sizes, binding affinities, and enzymatic activities were confirmed for
ADA with albumin binding domain and ADA with collagen binding domain. We found that
ADA overexpression was not toxic to CAR T cells, nor did it impede in vitro effector functions,
as the CAR groups had commensurate cytotoxicity, proliferation, and IFN-γ production.
Discrepancies surfaced when performing exhaustion and adenosine-supplemented studies. The
upregulation of co-inhibitory receptors and promotion of Treg differentiation are well-
documented consequences of A2AR stimulation.
121-123
After 24-hour co-cultures with antigen-
presenting cancer cells, ADA overexpression prevented CAR T cell exhaustion as shown by a
pronounced decrease in PD-1 and PD-L1 expression, and significantly reduced the induction of
T regulatory cells (Tregs). ADA overexpression also enhanced CAR T cell resistance to
adenosine suppression, as CAR19.ADA, CAR19.aADA, and CAR19.cADA T cells maintained
almost complete cytolytic capabilities (CAR19 experienced a 20% reduction) and reduced PD-1
expression compared to parental CAR T cells in adenosine supplemented cultures.
In our engineered ovarian solid tumor model, ADA overexpression as unmodified ADA
or cADA resulted in superior tumor growth control compared to parental CAR T cells.
CAR19.ADA and CAR19.cADA T cells had robust in vivo expansion, persisting in tumor,
blood, spleen, and bone marrow at significantly higher levels than control CAR T cells.
Peripheral Tregs have been reported to correlate with poor prognosis in many solid tumor
53
types,
124
and we observed a lower prevalence of peripheral Tregs for CAR19.ADA,
CAR19.aADA, and CAR19.cADA T cell groups. Further characterization of the CD45+ T cells
in the tumor revealed that TILs from all three ADA overexpressing CAR variants had diminished
PD-1 expression compared to CAR19 TILs. Only cADA overexpression favored a CD8+
phenotype in the TME, and markedly increased the CD8+ : Treg ratio among TILs. CD8+ : Treg
ratio can be used to predict response to PD-1 inhibition, and indicates a TME that fosters
antitumor immunity.
125
CAR19.ADA and CAR19.cADA T cells significantly prolonged OS,
resulting in approximately double median OS compared to NT and CAR19 control groups.
Interestingly, aADA overexpression did not promote superior expansion nor increase OS.
Although albumin-binding modifications hold promise for improving anticancer therapeutics and
are included in clinically relevant constructs,
126
binding to albumin was shown to reduce the in
vivo efficacy of some drugs.
127-128
We hypothesize that the albumin binding modification
abrogates the benefits of overexpressed ADA because of its binding to albumin. Despite
increased half-life, binding to albumin may prevent ADA from accumulating and being
accessible in the TME where T cells would benefit directly from ADA activity, and further
studies would be required to test this theory. Throughout our in vivo studies we monitored body
weight and witnessed no differences between groups, indicating that the overexpression of ADA
and modified ADA will not exacerbate CAR T cell treatment-associated toxicity.
3.4.2 Design advantages and disadvantages
Previously, researchers have developed pegylated form of adenosine deaminase
isoenzyme ADA2 (PEG-ADA2) for cancer treatment and the administration of PEGADA2
slowed tumor growth in multiple syngeneic models, although survival data was not shown.
129-130
Despite recognition as isoenzymes, ADA and ADA2 differ in structure, cellular localization, and
54
expression. ADA is a 41 kDa monomer expressed in all tissues (highest expression in
lymphocytes) intracellularly or bound to CD26, whereas ADA2 is a 59 kDa protein that forms
homodimers and is secreted primarily by myeloid cells.
131
Although ADA2 has higher serum
stability, we introduced ADA into our CAR constructs rather than ADA2 for several reasons: (i)
ADA is one-hundred fold more catalytically efficient;
129,132
(ii) ADA, through binding cell
membrane receptor CD26, can act as a costimulatory molecule for T cells;
109,118,119
(iii) ADA is
natively expressed by T lymphocytes, and is thus unlikely to cause toxicity upon its
overexpression; and (iv) ADA gene therapy has proven to be effective for treating ADA
deficiency Severe Combined Immunodeficiency (ADA-SCID) and demonstrated a good safety
profile.
133
The therapeutic efficacies of CAR19.ADA and CAR19.cADA groups demonstrated the
advantage of CAR T cells to locally deliver additional ADA to the tumor microenvironment.
Crosslinking ADA with collagen-binding domain utilizes the abundant collagen in extracellular
matrix of tumor structure and could allow more ADA enzymatic activity at tumor site to degrade
adenosine. Compared to systemic delivery of ADA that requires frequent injections and wastes
much injected ADA due to fast clearance from the body, ADA overexpressing CAR T cells
(CAR19.ADA, CAR19.cADA) present advantages of better therapeutic efficacy in terms of
enhanced overall survival and convenience in administration.
Despite the efficacy of ADA overexpression shown in our study, the roles of ADA and its
catalysis product inosine in cancer progression remain controversial. ADA blockade suppressed
the progression of breast cancer in a 4T1 preclinical model,
134
and inosine, an A2AR agonist, can
facilitate immunosuppression.
135
Contrary to these findings, supplementation with inosine
enhanced the anti-tumor efficacy of immune checkpoint inhibitors and ACT in multiple solid
55
tumor models.
136,137
Interestingly, inosine has a unique signaling bias upon A2AR binding,
favoring the ERK1/2 pathway to PKA stimulation, and, relative to adenosine, inosine is
approximately four orders of magnitude less potent at the A2AR (EC50 300uM vs 6 nM),
138,139
which may account for its differing effect on immunotherapies.
We also recognize that our NSG preclinical studies have shortcomings. We chose
SKOV3 as our tumor model for its prevalent adenosine signaling,
92, 140-141
and modified the cell
line to stably express CD19. Studies using ADA overexpressing CAR T cells targeting solid
tumor antigens, such as CEA or HER2, could be conducted to better evaluate the clinical
efficacy of ADA overexpression. Additionally, although the NSG model allows the study of
human T cells and cancer cells, it fails to accurately represent the complex TME found in
patients. Adenosine is a key suppressor of antitumor immunity, not only through its effect on T
cells but also by thwarting the antitumor activity of natural killer cells and polarizing dendritic
cells, macrophages, myeloid derived suppressor cells, endothelial cells, and fibroblasts toward
tolerogenic, pro-tumor phenotypes.
142
To address the dynamic interplay of adenosine in the
TME, we assessed ADA overexpression in a syngeneic solid tumor model. In this proof-of-
concept study, CT26 colon cancer cells expressing GFP or GFP and mADA secretion were
inoculated subcutaneously into BALB/c mice. Without any treatment, tumors with mADA
secretion grew at significantly slower rates in comparison to CT26.GFP tumors in vivo,
suggesting the antitumor effects of overexpressed ADA in immunocompetent mice beyond its
effect on CAR T cell treatment.
3.4.3 Clinical relevancy and translational value
The strategy of ADA overexpression in CAR T cells to enhance CAR T antitumor
efficacy in solid tumor has potential translational value. The first human gene therapy was
56
performed thirty years ago for ADA-deficient patients, in which autologous peripheral T cells
were extracted and transduced with the human adenosine deaminase 1 (ADA), followed by
reinfusion into the patients.
133
This track record proved the clinical safety profile of ADA gene
therapy and allowed its potential use for other diseases. ADA overexpression provides another
option in combatting with adenosine immunosuppression in the tumor microenvironment.
Extensive studies of A2AR inhibition or CD73/CD39 blockade have solidified the adenosinergic
pathway as a targetable checkpoint, but there are potential downsides to current approaches.
A2AR inhibitors fail to prevent immunosuppression caused by other ARs (i.e. adenosine inhibits
dendritic cell activation through A1R signaling).
143
There are alternative adenosine production
pathways,
144
which tumors may exploit to compensate for CD39/CD73 inhibition. T cell intrinsic
modifications, such as the genetic ablation of the A2AR or intracellular expression of PKA
localization altering peptide,
106,145
enhance the CAR T cell product but fail to combat with
adenosine signaling for other immune cell types in the tumor microenvironment. ADA, by
directly targeting adenosine, has the potential to alleviate the widespread immunosuppression of
adenosine irrespective of AR. The genetic addition of the relatively small ADA transgene (~1
kb) can be easily incorporated into other immunotherapies, such as oncolytic viruses and cancer
vaccines, and these other formulations will deepen our understanding of the therapeutic potential
ADA gene therapy for cancer. Spurred on by the success of our ADA overexpressing CAR T
cells and the favorable safety profile of ADA gene therapy, we hope to add ADA gene therapy to
the cancer treatment armamentarium.
57
3.5 Materials and methods
Protocols used for retroviral vector production, T-cell transduction and expansion,
surface immunostaining analysis, and intracellular cytokine staining analysis are based on
protocols in a previous publication.
146
3.5.1 Antibodies
Primary antibodies used in this study include biotinylated goat anti-mouse Fab antibody
(Invitrogen, Carlsbad, CA); PE-anti-CD45, PE-Cy5.5-anti-CD3, FITC-anti-CD4, Pacific Blue
TM
-
anti-CD8, FITC-anti-CD8, PE-anti-IFN-γ, Brilliant Violet 421
TM
-anti-PD-1, PE-anti-PD-L1,
PerCP/Cy5.5-anti-LAG-3, PE-anti-TIM-3 (BioLegend, San Diego, CA), Anti-ADA Antibody
Picoband (BosterBio, Pleasanton, CA), and beta-actin mouse monoclonal antibody, (LI-COR,
Lincoln, NE). The secondary antibodies used were APC-conjugated streptavidin (BioLegend,
San Diego, CA), IRDye
®
680RD Goat anti-Rabbit IgG Secondary Antibody, and IRDye
®
800CW Goat anti-Mouse IgG (LI-COR, Lincoln, NE).
3.5.2 Cell lines and cell culture
SKOV3 and 293T(ATCC) cells were maintained in DMEM with 10% FBS (Gibco), 2
mM L-glutamine, 1% Pen-Strep (Corning, Corning, NY). SKOV3.CD19 cells were generated by
transduction with lentiviral vector encoding the cDNA of human CD19 (hCD19). PBMCs were
cultured in TCM composed of AIM-V medium (Thermo Fisher, Waltham, MA) supplemented
with 5% human AB serum (GemCell, West Sacramento, CA), 10mM HEPES (Gibco, Grand
Island, NY), 1% Pen-Strep (Corning), 1% GlutaMax (Gibco), and 12.25mM N-Acetyl Cysteine
(Sigma-Aldrich, St. Louis, MO).
58
3.5.3 Mice
Female 6-8 weeks old NOD.Cg-Prkdc
scid
IL2Rγ
tm1Wj1
/SZ (NSG) were purchased from The
Jackson Laboratory. All animal studies were performed in accordance with the Animal Care and
Use Committee guidelines of the NIH (Bethesda, MD) and were conducted under protocols
approved by the Animal Care and Use Committee of the USC.
3.5.4 Plasmid design
The retroviral vector encoding anti-CD19 CAR (CAR) was constructed based on the
MP71 retroviral vector kindly provided by Prof. Wolfgang Uckert. The vector encoding anti-
CD19 CAR with adenosine deaminase 1 (ADA, EC. 3.5.4.4) overexpression (ADACAR) was
then generated from the anti-CD19 CAR. The insert for ADACAR consisted of the following
components in frame 5’ to 3’ end: anti-CD19 CAR, P2A linker, human IL-2 leader sequence,
and ADA. For the vectors encoding anti-CD19 CAR with albumin binding ADA (CD19.aADA)
or collagen binding ADA (CD19.cADA), ADA is followed by a GS linker and the albumin
binding peptide SA21 or a collagen binding domain VWF A3 respectively.
39,44
The lentiviral
vectors encoding his-tagged ADA with albumin binding (FUGW.aADA.his) and ADA with
CBD binding (FUGW.cADA.his) were based off the FUGW backbone. A His-tag was added to
the C terminus of the albumin or collagen binding sequence.
3.5.5 ELISA and Western blotting
T cells only and T cells co-cultured with SKOV3.CD19 at a 1:1 effector to tumor ratio
were maintained in TCM for 48 hours, after which the supernatant was measured for ADA using
a human ADA ELISA kit (BosterBio, Pleasanton, CA). CT26.GFP and CT26.GFP.mADA were
59
cultured at cell density of 0.5 M/mL for 48 hrs, after which the supernatant was measured for
mADA using a mouse ADA ELISA kit (Biomatik, Wilmington, Delaware).
For Western blotting, CAR T cells were cultured in media supplemented with brefeldin A
overnight followed by cell lysis and protein quantification using standard BCA assay. 20ug total
protein was used per group for SDSPAGE using 7.5% Mini-PROTEAN precast gel (Bio-rad,
Hercules, CA) together with Chameleon
®
700 Pre-stained Protein Ladder (LI-COR, Lincoln,
NE) and transferred to a nitrocellulose membrane (Thermo Scientific, Waltham, MA) for
Western blot analysis. The membrane was stained with Rabbit anti-ADA antibody (BosterBio,
Pleasanton, CA) at 1:1000 in 5% milk in TBS-T and IRDye
®
680RD Goat anti-Rabbit
IgG Secondary Antibody (LI-COR) at 1:10000, and its chemiluminescence detected with
Odyssey
®
Fc imaging unit (LI-COR).
3.5.6 aADA and cADA isolation and characterization
HEK-293T cells were transduced with FUGW.aADA.his or FUGW.cADA.his lentivirus
for the stable expression of his-tagged aADA or cADA. Following successful transduction and
expansion, the engineered cells were seeded in 10 mL plates in D10. Sixteen hours later the cells
were rinsed twice with PBS and then cultured for 48 hrs in 10 mL serum free media (SFM).
Supernatants were subsequently collected, clarified, and then centrifuged in 10 kDa isolation
columns (Sigma) for 1 hr at 5000xg 4°C. The remaining supernatant was purified for his-tagged
protein using Dynabeads (Thermo) according to the manufacturer’s instructions, and standard
BCA assay (Sigma) was used for quantification of the purified proteins. Specific activity was
measured using an ADA activity kit (Sigma) according to the manufacturer’s instructions.
Binding assays were conducted as previously described for the albumin and collagen binding
domains.
39, 44
Briefly, 96 well plates (Maxisorp) were coated with 10 ng/mL human serum
60
albumin (HSA) (Sigma), mouse serum albumin (MSA) (Sigma), or collagen III (Sigma) in PBS
for 4 hours at 4°C, after which HSA and MSA coated wells were blocked with 1% ovalbumin in
PBS with .05% Tween 20 (PBS-T) and collagen coated wells blocked with 2% BSA in PBS-T
for 1 hour at room temperature (RT). Then, wells were washed with PBS-T and incubated with
increasing concentrations (0, 5, 25, 50, 150, 300 nm in duplicates) of aADA or cADA for 1.5
hours at RT. After three washes with PBS-T, wells were incubated for 1 hour at RT with rabbit
anti-hADA (BosterBio) antibody, followed by washes and 1-hour RT incubation with secondary
HRP-conjugated anti-rabbit antibody. After washes, enzyme concentrations were detected with
tetramethylbenzidine (TMB) substrate by measurement of the absorbance at 450 nm with
subtraction of the absorbance at 570 nm. The apparent Kd values were obtained by nonlinear
regression analysis in Prism software (version 7, GraphPad) assuming one-site–specific binding.
3.5.7 Tumor model and animal studies schematics
6-8 weeks old NSG mice were injected subcutaneously with 3.5 M SKOV3.CD19.
Tumor volume was determined by caliper measurement (LxW
2
/2). Once tumors reached an
average size of 50-80 mm
3
, CAR T cells were injected intravenously at the dose of 3M CAR+
cells per group. Tumor sizes were measured three times a week and mice were euthanized when
they displayed obvious weight loss, ulceration of tumors, or tumor size larger than 750 mm
3
.
Tumor, spleen, and bone marrow tissue were harvested from mice and filtered through 70
μm nylon strainers (BD Falcon, Franklin Lakes, NJ) for single cell suspensions. Blood from
cardiac puncture was immediately transferred to TAC buffer for red blood cell lysis. The filtered
cells and blood samples were washed and incubated with 1% BSA in PBS. Cells were then
stained for fluorescent activated cell sorting (FACS). For peripheral blood analysis, mice were
tail bled. A portion of the collected whole blood was added to TAC buffer for FACS staining,
61
and the remainder was left undisturbed at room temperature for 30 minutes. The coagulated
blood samples were then centrifuged at 1,000 x g for 10 minutes, and the supernatant was
immediately transferred to a clean polypropylene tube for assessment of inosine concentration in
serum using an inosine assay kit (Sigma-Aldrich, St. Louis, MO.)
3.5.8 Statistical analysis
Statistical analysis was performed in GraphPad Prism version 5.01. The differences
among three or more groups were determined with one-way analysis of variance (ANOVA) with
Tukey’s posttest for multiple comparisons. Tumor growth curves were analyzed using two-way
ANOVA with Tukey’s posttest for multiple comparisons. Mice survival curves were evaluated
by the Kaplan-Meier analysis (log-rank test with Bonferroni correction). A P value <0.05 was
considered statistically significant. Significance of findings were defined as: ns = not significant,
P>0.05; * = P<0.05; ** = P<0.01; *** = P<0.001, **** = P<0.0001.
62
Chapter 4. Engineering CAR-T Cells to Secrete 4-1BB Ligand
Crosslinked to PD-1 Checkpoint Inhibitor for Enhanced Anti-tumor
Efficacy in Solid Tumor
63
4.1 Abstract
Chimeric antigen receptor (CAR) T cell therapies have transformed the treatment of
hematological malignancies but have not achieved significance success in treating solid tumors
due to the lack of persistence and function in tumor microenvironment. We previously reported
the augmentation of CD19-28z-CAR T cell therapy in solid tumor through the secretion of anti-
PD-1 scFv (CAR19.aPD1),
147
as shown by enhanced T cell antitumor efficacy, expansion, and
vitality. We have since matured the platform to create a superior CAR T cell product – CAR T
cells secreting single-chain trimeric 4-1BB ligand crosslinked to anti-PD-1 scFv. 4-1BB
signaling promotes cytotoxic T lymphocytes proliferation and survival but targeting 4-1BB with
agonist antibodies in the clinic has been hindered by low antitumor activity and high toxicity.
CAR T cells using 4-1BB endodomain for costimulatory signals have demonstrated milder anti-
tumor response and longer persistence compared to CAR T cells co-stimulated by CD28
endodomain. We have, for the first time, engineered CD28 co-stimulated CAR T cells to secrete
a fusion protein containing the soluble trimeric 4-1BB ligand, which provided additional co-
stimulatory signaling to CAR T cells through soluble 4-1BBL binding to 4-1BB upregulated on
CAR T cells upon activation. The novel CAR T cells exhibited reduced exhaustion, enhanced
persistence and proliferation, and a less differentiated memory status compared to CAR T cells
without additional 4-1BB:4-1BBL costimulation. The novel CAR T cells achieved much better
tumor growth control and significantly prolonged overall survival in an engineered solid tumor
xenograft model.
64
4.2 Introduction
Cancer immunotherapy has recently caused a paradigm shift in cancer treatment.
148
In
contrast to the three traditional pillars of cancer therapy, namely surgery, radiation, and
chemotherapy, which directly target or remove cancer cells, immunotherapy harnesses the power
of the immune system to eradicate cancer. To date, a centerpiece for successful cancer
immunotherapies is engaging T lymphocyte of the immune system and improving its antitumor
efficacy, especially through the use of immune checkpoint inhibitors or by engineering T cells to
express chimeric antigen receptors (CAR).
149-150
CARs are comprised of an antigen-recognizing
extracellular moiety, a hinge, a transmembrane domain, and intracellular T cell activating and
co-stimulatory domains.
12
CAR T cells recognize tumor associated antigen on tumor cells
through the extracellular domain, which triggers intracellular domains to activate the T cells to
attack tumor cells. CAR T cell therapy has achieved remarkable success in hematological
malignancies, but its clinical efficacy in solid tumor requires further improvement.
151
A big
challenge encountered by CAR T cells in both hematological malignancies and solid tumors is its
lack of function and proliferation upon prolonged antigen exposure.
22
The immunosuppressive
tumor microenvironment (TME) only worsens the problem with a multitude of inhibitory
molecules and receptors present in the TME suppressing effector cell functions.
36
Substantial efforts have been attempted at improving CAR designs to generate CAR T
cells with better resistance to exhaustion and hypofunctionality, focusing on (1) providing
additional signals to promote T cell activation or co-stimulation,
152-153
(2) engineering CAR T
cells to transgenicly express cytokines or constitutively active cytokine receptors that support
proliferation,
154-155
(3) silencing or knocking-out molecules that restrict T cell activation,
156
and
(4) modulating transcription factors in CAR T cells.
157-158
Among them, 4-1BB signaling
65
components have been a prominent candidate given the costimulatory role of 4-1BB:4-1BBL in
supporting clonal expansion, survival, cytokine release and effector function of immune cells.
159
Besides the use of 4-1BB endodomain for costimulation in CARs which favors CAR T cell
persistence,
160
transgenic expression of 4-1BB ligand on CD28-costimulated CAR T cells
provided significantly better therapeutic efficacy compared to both 4-1BB-costimulated and
CD28-costimulated CAR T cells.
161
Another method of targeting 4-1BB involve the use of
monoclonal antibody agonists.
Although strong anti-tumor efficacy has been observed in
preclinical models, their clinical development was restricted because of either low efficacy or
severe liver toxicity.
162
Compared to 4-1BB agonist antibodies, 4-1BB ligand has demonstrated
better immunomodulatory activity and safety profile,
163
but the soluble ligand on its own is
inactive and requires crosslinking for T cell co-stimulation activity.
164
We formerly reported the augmentation of antitumor efficacy by CAR T cells engineered
to secrete anti-PD-1 checkpoint inhibitor.
147
The secreted anti-PD-1 scFv protected CAR T cells
from inhibition by PD-1:PD-L1 interaction and significantly enhanced the anti-tumor efficacy of
the CAR19.aPD1 T cells compared to parental CAR T cells, especially in H292 tumor model
where PD-L1 is upregulated. Despite the clinical success of PD-1/PD-L1 blockade in treating a
number of malignancies, PD-1 blockade does not always benefit patients and a number of
mechanisms lead to both primary and acquired resistance to PD-1 blockade.
165
One prominent
cause is loss of intratumoral T cell function characterized by a lack of memory T cells and re-
exhaustion of effectors cells invigorated by PD-1 blockade. Since 4-1BB costimulation favors T
cell expansion, persistence and development of T cell memory,
159
and 4-1BB ligand requires
crosslinking for costimulatory activity,
we hypothesize that crosslinking the current anti-PD-1
scFv with 4-1BB ligand will provide additional benefits to CAR T cells and is potentially of
66
translational value in the management of tumors resistant to PD-1 blockade due to lack of T cell
function. Therefore, we have engineered CAR T cells to secrete a novel immunomodulatory
fusion protein consisting of anti-PD-1 scFv crosslinked to a single-chain format of trimeric 4-
1BB ligand. The single-chain format is based on a previous design that connects three
extracellular domain units with polypeptide linkers.
166
We hypothesized that the fusion protein
would benefit CAR T cell effector function and persistence and help achieve better antitumor
efficacy in solid tumor model.
67
4.3 Results
4.3.1 Design of the CAR and protein characterization
Figure 4-1. Schematics of CAR construct and fusion protein. (A) Schematics of CAR19, CAR19 with anti-PD-1
scFv secretion, CAR19 with anti-PD-1-4-1BBL secretion. (B) Schematic of fusion protein aPD1 scFv crosslinked
with single-chain 4-1BB ligand. (C) CAR expression of transduced T cells for CAR19, CAR19.aPD1,
CAR19.aPD1-41BBL groups.
We previously engineered an anti-PD1 scFv secreting CAR T cell (CAR19.aPD1) from a
traditional CD19 targeting, CD28 co-stimulated second-generation CAR construct. For the new
CAR19.aPD1-41BBL construct, we linked anti-PD-1 scFv with 4-1BBL trimer of a single-chain
format to create the fusion protein aPD-1-4-1BBL (Figure 4-1A). Three units of ectodomain of
the 4-1BB ligand are linked by flexible GS linkers. A schematic of the fusion protein is shown in
Figure 4-1B. Following the successful cloning of the CAR constructs, retroviral vectors
containing the CARs were generated and used to transduced activated human PBMCs. All three
CAR constructs were expressed in T cells (Figure 4-1C). CAR expressions were normalized
with non-transduced T cells for all the in vitro and in vivo studies.
68
The two modified CAR T cells secreted their respective immunomodulatory proteins.
Anti-HA stained immunoblot identified anti-PD-1 scFv at a MW of ~27 kDa, and anti-PD-1-sc4-
1BBL at ~94 kDa (Figure 4-2A). We assessed the binding kinetics of secreted anti-PD-1 scFv
and fusion protein to recombinant human PD-1 (Figure 4-2B) and recombinant human 4-1BB
(Figure 4-2C) using ELISA. Similar to previously reported,
166
fusion of antibody scFv to the
single chain format of 4-1BBL significantly reduces antibody binding affinity to recombinant
human PD-1, EC50 increased from ~2.5nM to ~100nM. The fusion protein had nanomolar
affinity for recombinant human 4-1BB with EC50 ~ 6.25nM.
Figure 4-2. Secreted protein characterization and binding kinetics. (A) Western blotting of purified proteins. (B)
Binding kinetics of secreted proteins to recombinant human PD1. (C) Binding kinetics of secreted aPD1-41BBL
fusion protein to recombinant human 4-1BB.
4.3.2 In vitro functional analysis
For specific cell lysis assay, CAR T cells were cocultured with SKOV3.CD19 cells at
effector to tumor cell (E:T) ratios of 1:1, 3:1, and 5:1 for 16 hrs, followed by flow cytometry
69
analysis. SKOV3.CD19 cells were almost completely lysed for all the CAR T cell variants at all
the tested E:T ratios (Figure 4-3A).
Figure 4-3. In vitro analysis of CAR-T cells. (A) Specific cell lysis results of CAR T cells targeting SKOV3.CD19
cancer cells. (B) Co-expression of number of intracellular cytokines (IFN-gamma, TNF-alpha, IL-2) expressed on
CAR T cells after coculture with SKOV3.CD19 cancer cells. (C) Expression of IFN-gamma, TNF-alpha, IL-2,
Granzyme B on CAR T cells after coculture with SKOV3.CD19 cancer cells. (D) Expression of exhaustion markers
(PD-1, LAG-3, TIM-3) on CAR T cells after coculture with SKOV3.CD19 cancer cells.
Following a 24hr coculture with antigen presenting tumor cells in which Brefeldin A was
added at hour 18 to block protein transport, CAR T cells were stained for intracellular cytokines
expression. For Figure 4-3B, cells were stained simultaneously for IFN-γ, TNF-α, and IL-2 and
percentages of cells expressing all three cytokines, or only two of the three cytokines, or only
one cytokine were plotted for NT and CAR T groups. Interestingly, CAR19.aPD1-41BBL T cells
overall express less effector cytokines following the 24-hr antigen exposure. Figure 4-3C shows
the expression levels for IFN-γ, TNF-α, IL-2 and Granzyme B for NT and CAR T groups. CAR
T cells with protein secretions express less IFN-γ, TNF-α, and Granzyme B compared to parental
70
CAR19 T cells for both CD8+ cell and CD4+ cells. CAR19.aPD1 expresses commensurate IL-2
level compared to parental CAR19 T cells and CAR19.aPD1-41BBL T cells show lower IL-2
expression level.
Following a 24hr coculture with antigen presenting tumor cells, NT and CAR T cells
were stained for inhibitory receptors expression. CAR19.aPD1 T cells show less PD-1, LAG-3,
and TIM-3 expression levels compared to CAR19 T cells, and CAR19.aPD1-41BBL T cells
show even less PD-1, LAG-3, and TIM-3 expression levels than CAR19.aPD1 T cells. Secreted
proteins significantly reduced the expression levels of inhibitory receptors and protected the
CAR T cells from exhaustion.
4.3.3 Secreted fusion protein protects parental CAR T cells from exhaustion
To validate that the secreted proteins are the source of protecting CAR T cells from
exhaustion, we collected supernatants from CAR19, CAR19.aPD1, CAR19.aPD1-41BBL cell
cultures and put parental CAR19 cells in the supernatants with or without antigen-presenting
tumor cells SKOV3.CD19. 24 hours later, T cells were again assessed for the expression of
coinhibitory receptors. With CAR19.aPD1-41BBL supernatant, CD8+ T cells showed
significantly lower expressions of PD-1, TIM-3, and LAG-3 after antigen stimulation (Figure 4-
4A). For Figure 4-4B, CAR19 T cells incubated in three different supernatants with or without
antigen-expressing tumor cells were stained for co-expression of inhibitory receptors PD-1, TIM-
3, LAG-3. Percentages of cells expressing all three inhibitory receptors, only expressing 2 out of
3 inhibitory receptors, and only expressing 1 of them were plotted in the stacked bar graph for all
T cells, CD8+ subset T cells, and CD4+ subset T cells in all three groups. CAR19.aPD1-41BBL
supernatant significantly reduced total number of inhibitory receptors expressed on parental
CAR19 T cell both with and without antigen stimulation when compared to CAR19.aPD1 T cells
71
supernatant and CAR19 T cells supernatant. Culturing parental CAR19 T cells in CAR19.aPD1
T cells supernatant and CAR19.aPD1-41BBL supernatant did not affect cytotoxic functions of
parental CAR19 T cells (Figure 4-4C).
Figure 4-4. Secreted fusion protein protects parental CAR T cells from exhaustion. CAR19 T cells were cultured in
CAR19, CAR19.aPD-1, or CAR19.aPD-1-41BBL T cell culture supernatant with or without antigen-presenting
tumor cells. 24 hrs later, T cells were assessed for the expression of coinhibitory receptors. (A) Expression of
inhibitory receptors PD-1, TIM-3, LAG-3 in CD8+ T cells. (B) Number of inhibitory receptors (PD-1, TIM-3, LAG-
3) expressed on T cells with or without incubation with SKOV3.CD19 cells. (C) CAR19 T cells cultured in CAR19,
CAR19.aPD-1, or CAR19.aPD-1-41BBL T cell culture supernatant exhibited comparable cytotoxicity against
SKOV3.CD19 cancer cells.
4.3.4 Phenotype analysis of CAR T cells
CAR T cells were stained for percentages of CD62L+ cells before and after coculturing
with SKOV3.CD19 cells for 24 hours (Figure 4-5A). CAR19.aPD1-41BBL T cells did not
express higher levels of CD62L+ compared to CAR19 T cells and CAR19.aPD1 T cells before
antigen exposure, as shown by the baseline staining. With antigen exposure, CAR19.aPD1-
72
41BBL T cells showed a less differentiated state compared to both CAR19 and CAR19.aPD1 T
cells since CAR19.aPD1-41BBL T cells expressed significantly higher percentages of CD62L+
cells. CAR19.aPD1-41BBL T cells also showed significantly less regulatory T cells compared to
both CAR19 and CAR19.aPD1 T cells after co-culture with SKOV3.CD19 cancer cells for 24
hours (Figure 4-5B).
Figure 4-5. Phenotype analysis of CAR T cells. (A) CD62L positive cells in CAR T cells before and after antigen
exposure. (B) Regulatory T cell percentages in CAR T cells after coculture with SKOV3.CD19 cells for 24 hrs.
4.3.5 In vivo antitumor efficacy of CAR T cells
Figure 4-6. Antitumor efficacy of CAR19.aPD1-41BBL T cells in xenograft mouse model. (A) Tumor growth curve.
(B) Survival analysis. (C) Individual tumor growth curves.
The in vivo antitumor activity of CAR19.aPD1-41BBL T cells were evaluated in a
xenograft tumor model. Once SKOV3.CD19 tumors in NSG mice reached a volume of 50-80
m
3
, we adoptively transferred 1.5x10
6
CAR T cells into mice by intravenous injection. Tumor
growth in all mice were monitored every other day. CAR19.aPD1-41BBL group started showing
73
significantly better tumor growth control at day 7 compared to other groups (Figure 4-6A) and
mice in this group had significantly improved overall survival (Figure 4-6B). From the individual
tumor growth curves (Figure 4-6C), it is obvious that CAR19.aPD1-41BBL T cells reduced
tumor burden for all 6 mice in the group from day 9, while other groups only showed control of
tumor growth rather than tumor burden reduction after the treatment.
4.3.6 Ex vivo analysis of CAR T cells in xenograft mouse model
Figure 4-7. Ex vivo analysis of CAR T cells in xenograft mouse model. T cell population in tumor (A), 50ul of
peripheral blood (B), and spleen (C). (D) Expressions of exhaustion markers on tumor infiltrating lymphocytes. (E)
Percentages of CD62L+ cells in tumor, peripheral blood, and spleen.
Following the same study schematic as the in vivo studies, mice were inoculated with
2.5M SKOV3.CD19 tumor cells. When the tumor sizes reach ~50-80mm
3
, mice were treated
with 1.5M CAR T cells. 10 days after treatment, the mice were sacrificed and their organs
homogenized and analyzed for human T cell populations, phenotypes, and exhaustion markers.
CAR19.aPD1-41BBL T cells expanded significantly more than CAR19.aPD1 T cells and
74
CAR19 T cells, as manifested from the cell count of CD45+ cells in per mg of tumor, per 50ul of
blood, and per spleen (Figure 4-7A-C). In the CAR19.aPD1-41BBL T cells treatment group, the
tumor infiltrating lymphocytes expressed less PD-1 and LAG-3 compared to the TILs in the
parental CAR19 T cell (Figure 4-7D). There were also significantly higher percentages of
CD62L+ cells in tumor, blood, and spleen of the CAR19.aPD1-41BBL T cells treatment group,
characterizing an earlier memory state of human T cells circulating in the mice of the
CAR19.aPD1-41BBL T cells treatment group.
75
4.4 Discussion
4.4.1 Potential mechanisms behind the improved efficacy
The secretion of our novel aPD-1-41BBL fusion protein potentiated CAR T cell therapy.
Our in vitro assays revealed that CAR19.aPD-1-41BBL T cells exhibit comparable cytotoxicity,
reduced T cell exhaustion, and less differentiated state compared to parental CAR19 T cells and
CAR19.aPD-1 T cells. In an engineered solid tumor model using SKOV3 cells transduced to
express CD19 (SKOV3.CD19), CAR19.aPD-1-41BBL T cell treated mice experienced
significantly enhanced tumor control and overall survival, and demonstrated much better
expansion of CAR T cells in vivo compared to other groups. Interestingly, whereas aPD-1
secretion alone enhanced the eradication of modified H292 (H292.CD19) tumors,
147
CAR19.aPD-1 T cells marginally enhanced tumor control and overall survival in our
SKOV3.CD19 model. This is potentially due to the fact that H292 expresses a higher level of
PD-L1 than SKOV3 (data not shown), thus the improvement of therapeutic efficacy by the
introduction of PD-1 blockade into CAR T modality over unmodified CAR T cells is more
prominent in H292 tumor model.
CAR19.aPD-1-41BBL T cells maintained less differentiated status in vitro upon antigen
stimulation compared to CAR19.aPD-1 T cells. A growing body of work supports the notion that
the memory state of CAR T cells influence their clinical efficacy and less differentiated products
cause better clinical results.
167
Central memory T cells or stem cell-like memory T cells for
adoptive cell transfer are known to have sustained in vivo responses because they simultaneously
display great effector functions as well as maintain the expansion potential.
168
There are several
methods for making this possible, such as selecting for certain T cell populations prior to CAR T
cell production, producing CAR T cells in the presence of different cytokines or inhibitors, or
76
altering CAR signaling domains.
169
CAR19.aPD1-41BBL T cells maintained a less differentiated
state upon coculture with target cells and in the animal model, which potentially allow greater
expansion potential compared to control CAR T groups and mediate better antitumor efficacy.
CAR19.aPD1-41BBL T cells exhibit significantly lower regulatory T cell population
upon antigen exposure. Previous studies have illustrated how the balance between effector T
cells and regulatory T cells can influence the success of adoptive immunotherapy, and the
infiltration of CD4+ regulatory T cells into solid tumor decreases the anti-tumor activity of
second-generation CAR T cells co-stimulated by CD28 endodomain.
170-171
It is notable that the
secreted fusion protein downregulated the percentage of regulatory T cells in the CAR T cells
upon antigen stimulation, which may also have contributed to the better performance of
CAR19.aPD1-41BBL T cells in treating the solid tumor.
Tumor microenvironment is notorious for its abundance of inhibitory signaling pathways
through binding receptors such as PD-1, TIM-3, LAG-3, CTLA-4 on T cells, which impair T cell
functions and result in insufficient immune response.
172
Analysis of clinical samples has
identified a population of less differentiated CD8+ CART cells without PD-1 expression that
plays an important role in mediating tumor control.
173
It was also observed that lower expressions
of PD-L1, PD-1, LAG-3, and TIM-3 in lymphoma patients correlate with better responses to
anti-CD19 CAR T cell treatment.
174
In particular, higher expressions of fatigue-related inhibitory
receptors PD-1 and TIM-3 on CD8+ T cells is associated with defects in T cell proliferation,
degranulation, and cytokines production.
175
Protecting infused CAR T cells from inhibitory
receptors signaling is thus crucial to unleash the potentials of CAR-mediated killing effects. The
previous anti-PD-1 scFv secreting CAR T cells in some degree protected cells from PD-1/PD-L1
interactions and other inhibitory receptors signaling, and fusion protein anti-PD-1-4-1BBL
77
mediates even stronger protection than anti-PD-1 scFv alone. Such protection from inhibitory
receptors signaling prevents impairment in T cell function and also contributes to the robust
antitumor efficacy of CAR19.aPD1-41BBL CAR T cells.
4.4.2 Limitations, future outlook and translational value
The lower cytokine expression levels of CAR T cells secreting fusion protein after short
antigen exposure (~24 hours) is contradictory to literature findings that 4-1BB signaling
markedly enhanced IFN-γ production from CD8+ T cells.
159
This is partly due to the
experimental protocol currently used that assessed the percentage of cells positive for the stained
cytokines in the CAR T groups rather than the total amount of secreted cytokines. Additionally,
since effector function is increased upon CD8+ T cell differentiation,
170
it is likely that
CAR19.aPD-1-4-1BBL group exhibits less cytokine expression due to its less differentiated state
compared to parental CAR T groups upon the one-day antigen exposure.
The strategy of combining CAR T cell therapy with immune checkpoint inhibitors has
received great attention in the hope that such combination would overcome some limitations of
CAR T cells in treating hematological malignancies and solid tumors.
176-177
Besides direct
combination of using PD-1/PD-L1 monoclonal antibodies and CAR T cells, there are also a
number of candidates in clinical trials evaluating the antitumor efficacy of PD-1 knock-out or
PD-1 negative receptor CAR T cells. Given the significantly better therapeutic efficacy of
CAR19.aPD1-41BBL T cells over CAR19.aPD1 T cells, we figured that it is of highly
translational value to adopt secretion of aPD1-41BBL fusion protein in other more clinically
relevant solid tumor model. We expect that the novel CAR T product will achieve improved
therapeutic efficacy, especially for solid tumors that are currently resistant to CAR T treatment
with PD-1 blockade.
78
Solid tumor cancer cells transduced to express CD19 as an artificial antigen is commonly
used for proof-of-concept studies that do not involve new designs for extracellular antigen
targeting domain of CARs. Cancer cells are often transduced with 100% antigen expression for
efficient CAR-mediated killing. To evaluate the efficacy of fusion protein secreting CAR T cells
in a more realistic model, we have planned to move anti-PD-1-4-1BBL fusion protein modality
to an anti-mesothelin CAR co-stimulated by CD28 endodomain and will be testing the antitumor
efficacy of the new CAR.meso.aPD1-41BBL against SKOV3 cells that naturally express
mesothelin at a moderate level (~40-50%, data not shown). For animal studies testing
CAR.meso.aPD1-41BBL against SKOV3 cells, we do not expect to see as strong antitumor
response as observed in the artificial SKOV3.CD19 tumor model due to less antigen expression
levels on SKOV3 cells, especially at a low dose of 1.5M CAR T cells infused for treatment. We
plan to inject a higher dose of ~3-5M CAR T cells, and the exact dose will be based upon in vitro
specific killing assay results that evaluate the efficiency of CAR-mediated killing of cancer cells
with low mesothelin expression and whether CAR.meso.aPD1-41BBL T cells may exhibit
bystander killing to mesothelin-negative cancer cells.
79
4.5 Materials and methods
Protocols used for retroviral vector production, T-cell transduction and expansion,
surface immunostaining analysis, and intracellular cytokine staining analysis are based on
protocols in a previous publication.
146
4.5.1 Antibodies
Primary antibodies used in this study include biotinylated goat anti-mouse Fab antibody
(Invitrogen, Carlsbad, CA); PE-anti-CD45, PE-Cy5.5-anti-CD3, FITC-anti-CD4, Pacific Blue
TM
-
anti-CD8, FITC-anti-CD8, PE-anti-IFN-γ, Brilliant Violet 421
TM
-anti-PD-1, PE-anti-PD-L1,
PerCP/Cy5.5-anti-LAG-3, PE-anti-TIM-3 (BioLegend, San Diego, CA), and recombinant Anti-
HA tag antibody (Abcam, Cambridge, UK). The secondary antibodies used were APC-
conjugated streptavidin (BioLegend, San Diego, CA), IRDye
®
680RD Goat anti-Rabbit
IgG Secondary Antibody (LI-COR, Lincoln, NE), and Goat anti-Rabbit IgG antibody with HRP
conjugate (Sigma-Aldrich, St. Louis, MO).
4.5.2 Cell lines and cell culture
SKOV3 and 293T(ATCC) cells were maintained in DMEM with 10% FBS (Gibco), 2
mM L-glutamine, 1% Pen-Strep (Corning, Corning, NY). SKOV3.CD19 cells were generated by
transduction with lentiviral vector encoding the cDNA of human CD19 (hCD19). PBMCs were
cultured in TCM composed of AIM-V medium (Thermo Fisher, Waltham, MA) supplemented
with 5% human AB serum (GemCell, West Sacramento, CA), 10mM HEPES (Gibco, Grand
Island, NY), 1% Pen-Strep (Corning), 1% GlutaMax (Gibco), and 12.25mM N-Acetyl Cysteine
(Sigma-Aldrich, St. Louis, MO).
80
4.5.3 Mice
Female 6-8 weeks old NOD.Cg-Prkdc
scid
IL2Rγ
tm1Wj1
/SZ (NSG) were purchased from The
Jackson Laboratory. All animal studies were performed in accordance with the Animal Care and
Use Committee guidelines of the NIH (Bethesda, MD) and were conducted under protocols
approved by the Animal Care and Use Committee of the USC.
4.5.4 Plasmid design
The retroviral vector encoding anti-CD19 CAR (CAR) was constructed based on the
MP71 retroviral vector kindly provided by Prof. Wolfgang Uckert. The vector encoding anti-
CD19 CAR with anti-PD-1 scFv secretion and aPD-1-4-1BBL fusion protein secretion were then
generated from the anti-CD19 CAR. The CD19 CAR with anti-PD-1 scFv secretion consisted of
the following components in frame 5’ to 3’ end: anti-CD19 CAR, P2A linker, human IL-2 leader
sequence, aPD1 scFv with HA tag. The CD19 CAR with fusion protein secretion consisted of the
following components in frame 5’ to 3’ end: anti-CD19 CAR, P2A linker, human IL-2 leader
sequence, aPD1 scFv, GS linker, 4-1BBL extracellular domain 1, 2
nd
GS linker, 4-1BBL
extracellular domain 2, 3
rd
GS linker, 4-1BBL extracellular domain 3 with HA tag.
4.5.5 Protein isolation and characterization
HEK-293T cells were transduced with CAR19.aPD-1.HA or CAR19.aPD-1-41BBL
retroviral vectors for the stable expression of HA-tagged anti-PD-1 scFv or fusion protein aPD1-
41BBL. Following successful transduction and expansion, the engineered cells were seeded in 10
mL plates in D10. Sixteen hours later the cells were rinsed twice with PBS and then cultured for
48 hrs in 10 mL serum free media (SFM). Supernatants were subsequently collected, clarified,
and then centrifuged in 10 kDa isolation columns (Sigma) for 1 hr at 5000xg 4°C. The remaining
81
supernatant was purified for HA-tagged protein using Dynabeads (Thermo) according to the
manufacturer’s instructions, and standard BCA assay (Sigma) was used for quantification of the
purified proteins.
Binding assays were conducted by testing the affinity of purified proteins to recombinant
human PD-1 or recombinant human 4-1BB. 96 well plates (Maxisorp) were coated with 10
ng/mL human PD-1 Fc chimera or human 4-1BB Fc chimera (Genscript, Piscataway, NJ) in PBS
for 4 hours at 4°C, after which the coated wells were blocked with 1% BSA in PBS with .05%
Tween 20 (PBS-T) for 1 hour at room temperature (RT). Then, wells were washed with PBS-T
and incubated with increasing concentrations (0, 0.098, 0.39, 1.56, 6.25, 25,100 nM in
triplicates) of aPD1 scFv or aPD-1-41BBL fusion protein for 1.5 hours at RT. After three washes
with PBS-T, wells were incubated for 1 hour at RT with rabbit anti-HA (Abcam) antibody,
followed by washes and 1-hour RT incubation with secondary HRP-conjugated anti-rabbit
antibody. After washes, enzyme concentrations were detected with tetramethylbenzidine (TMB)
substrate by measurement of the absorbance at 450 nm with subtraction of the absorbance at 570
nm. The apparent Kd values were obtained by nonlinear regression analysis in Prism software
(version 7, GraphPad) assuming one-site–specific binding.
For Western blotting, 1ug of each purified protein was used per group for SDSPAGE
using 7.5% Mini-PROTEAN precast gel (Bio-rad, Hercules, CA) together with
Chameleon
®
700 Pre-stained Protein Ladder (LI-COR, Lincoln, NE) and transferred to a
nitrocellulose membrane (Thermo Scientific, Waltham, MA) for Western blot analysis. The
membrane was stained with Rabbit anti-HA antibody (Abcam) at 1:1000 in 5% milk in TBS-T
and IRDye
®
680RD Goat anti-Rabbit IgG Secondary Antibody (LI-COR) at 1:10000, and its
chemiluminescence detected with Odyssey
®
Fc imaging unit (LI-COR).
82
4.5.6 Tumor model and animal studies schematics
6-8 weeks old NSG mice were injected subcutaneously with 2.5 M SKOV3.CD19 cancer
cells in 50ul. Tumor volume was determined by caliper measurement (LxW
2
/2). Once tumors
reached an average size of 50-80 mm
3
, CAR T cells were injected intravenously at the dose of
1.5M CAR+ cells per group. Tumor sizes were measured three times a week and mice were
euthanized when they displayed obvious weight loss, ulceration of tumors, or tumor size larger
than 1000 mm
3
.
4.5.7 Statistical analysis
Statistical analysis was performed in GraphPad Prism version 5.01. The differences
among three or more groups were determined with one-way analysis of variance (ANOVA) with
Tukey’s posttest for multiple comparisons. Tumor growth curves were analyzed using two-way
ANOVA with Tukey’s posttest for multiple comparisons. Mice survival curves were evaluated
by the Kaplan-Meier analysis (log-rank test with Bonferroni correction). A P value <0.05 was
considered statistically significant. Significance of findings were defined as: ns = not significant,
P>0.05; * = P<0.05; ** = P<0.01; *** = P<0.001, **** = P<0.0001.
83
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Abstract (if available)
Abstract
This dissertation is a compilation of three projects aiming at engineering chimeric antigen receptor-directed immune cells for enhanced antitumor efficacy in solid tumors. Project one Engineering CAR-expressing Natural Killer Cells with Cytokine Signaling and Safety Switch demonstrated the capacity at which the off-the-shelf candidate NK92 cells can be engineered with synthetic biology tools for enhanced tumor targeting capability, better proliferative potential, and treatment regulation. Project two and Project three focus on armoring CAR-directed T cells with self-secreted immune-modulating compounds to protect CAR-T cells from select immunosuppressive mechanisms in the solid tumor microenvironment. In Project two Engineering CAR-T Cells to Overexpress Adenosine Deaminase 1 (ADA) for Enhanced Anti- tumor Efficacy in Solid Tumor, CAR-T cells were engineered to overexpress ADA1 or express protein modified-ADA1 to target adenosine accumulation in the tumor microenvironment and help ameliorate its suppressive effect on T cells. In Project three Engineering CAR-T Cells to Secrete 4-1BB Ligand Crosslinked to PD-1 Checkpoint Inhibitor for Enhanced Anti-tumor Efficacy in Solid Tumor, CAR-T cells were engineered to overexpress a fusion protein consisting of single-chain 4-1BB ligand crosslinked to anti-PD-1 scFv to simultaneously engage the protective effect of anti-PD-1 scFv against PD-1 checkpoint and the costimulatory effect of 4-1BB ligand interacting with 4-1BB upregulated on activated T cells.
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Qu, Yun
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Core Title
Engineering chimeric antigen receptor-directed immune cells for enhanced antitumor efficacy in solid tumors
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Viterbi School of Engineering
Degree
Doctor of Philosophy
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Chemical Engineering
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03/18/2021
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03/10/2021
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Wang, Pin (
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), Finley, Stacey (
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quy@usc.edu,yunqu54@gmail.com
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immunotherapy
solid tumor