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Novel design and combinatory therapy to enhance chimeric antigen receptor engineered T cells (CAR-T) efficacy against solid tumor
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Novel design and combinatory therapy to enhance chimeric antigen receptor engineered T cells (CAR-T) efficacy against solid tumor
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Content
Novel Design and Combinatory Therapy to Enhance
Chimeric Antigen Receptor Engineered T cells (CAR-
T) Efficacy against Solid Tumor
By
Shuai Yang
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirement for the Degree
MASTER OF SCIENCE
(Biochemistry and Molecular Biology)
August 2018
Copyright 2018 Shuai Yang
Acknowledgment
I would like to present my sincere gratitude to my advisor Dr. Pin Wang
for allowing me to join his laboratory and offering me encouragement,
support and guidance to my research project. I appreciate his creativity, focus
and positive attitudes towards scientific research, which will benefit in my
future life.
I would also like to thank all other lab members of Dr. Wang’ laboratory
for their generous help during my project. In particular, I would like to thank
Dr. Si Li who was an excellent mentor and offered practically advise for my
project. Also, I would like to thank Dr. Natnaree Siriwon who, together with
Dr. Li, taught me techniques on mouse experiments. It was a pleasure to work
with them.
Finally, I would like to thank my committee members, Dr. Peter
Danenberg and Dr. Yves Albert Declerck for their time and support. I learned
the background about the development of chemotherapy from Dr.
Danenberg and knowledge about tumor microenvironment from Dr. Declerck.
I was honored to have them as my committee members.
Table of Contents
Abstract------------------------------------------------------------------------------------------------------1
Introduction------------------------------------------------------------------------------------------------2
Design and development of chimeric antigen receptors (CAR)------------------------------2
Success and limitations of CAR therapy-----------------------------------------------------------3
Project 1. Engineer CAR-T cells to secrete anti-PD-1 scFv------------------------------------4
Project 2. Design chimeric antigen receptor utilizing CD3 epsilon signal-----------------5
Results--------------------------------------------------------------------------------------------------------5
Characterization of anti-CD19 CAR-T cells with anti-PD-1 antibody secretion----------5
CAR-T cells showed elevated antigen-specific responses when PD-1 pathway was
blocked---------------------------------------------------------------------------------------------------------6
CAR-T cells with anti-PD-1 secretion exhibited enhanced antitumor efficacy in
xenograft model----------------------------------------------------------------------------------------------6
CAR-T cells with anti-PD-1 antibody secretion expanded more efficiently in tumor-
bearing mice--------------------------------------------------------------------------------------------------7
Characterization and antigen-specific immune responses of chimeric antigen receptor
utilizing CD3 epsilon signal (ChCD3e) ------------------------------------------------------------------8
Discussion---------------------------------------------------------------------------------------------------8
Methods----------------------------------------------------------------------------------------------------13
Figures------------------------------------------------------------------------------------------------------20
References------------------------------------------------------------------------------------------------30
1
Abstract
Chimeric antigen receptor engineered T cells (CAR-T) therapy, has achieved
remarkable success in treatment against hematopoietic malignancies, while it exhibited
limited efficacy against solid tumors. To overcome the limitation, we developed
combination therapy by blocking program death 1 (PD-1) pathway and a novel design of
antigen-specific receptor utlilizing CD3-epsilon signal.
Immunosuppressive tumor microenvironment played an essential role in inhibiting T
cell functions at the tumor site. High expression of PD-L1 on tumors, like breast tumor or
ovarian tumor, can suppress and exhaust T cells through the PD-1 pathway. To evaluate
CAR-T cell function against tumor cells, we engineered anti-CD19 CAR T cells (CAR) to
secrete anti-PD-1 scFv which can block the binding of PD-1 and program death ligand 1
(PD-L1). According to both in vitro and in vivo results, anti-CD19 CAR T cells with anti-PD-
1 scFv secretion (CAR.aPD1) showed enhanced proliferation, promoted cytolytic activity
and elevated tumor inhibition in comparison with parental CAR T cells. Also, CAR.aPD1
cells dramatically prolonged overall survival of tumor-bearing mice.
To explore the possibility of chimeric receptor utilizing signals other than CD3 zeta,
we designed and constructed chimeric CD3 epsilon (ChCD3e) receptor by linking anti-
CD19 scFv to extracellular end of CD3e. Anti-CD19 ChCD3e T cells can be specifically
activated by CD19 antigen and demonstrated significant cytotoxicity against H292-CD19
cells.
2
Introduction
Traditional treatment methods like surgery, chemotherapy, and radiotherapy have
been developed for decades as main therapeutic strategies against most kinds of cancer.
However, limited efficacy and substantial side effects are still unavoidable due to non-
specificity of these methods [1-3]. To recognize cancer cells specifically and to utilize
human’s own immune system against tumors, immunotherapy has gained great success
in cancer treatment these years. Immunotherapy mainly includes cytokine therapy,
monoclonal antibodies and adoptive cell transfer (ACT). The basic workflow of ACT is to
collect and isolate immune cells from patients, then activate, engineer and expand them
in vitro. And infuse the treated cells back into patient circulation to kill tumor cells. The
immune cells used for immunotherapy can be dendritic cells (DCs), T cells and natural
killer (NK) cells [4-6].
Design and development of chimeric antigen receptors (CAR)
Among all the ACT methods, chimeric antigen receptor engineered T cells (CAR-T)
cells showed significant potential in cancer treatment. Chimeric antigen receptor is a kind
of artificially designed receptor consists of antigen recognition domain, a hinge domain, a
transmembrane domain and intracellular signaling domain. Antigen recognition domain
is usually derived from single chain variable fragment (scFv) of monoclonal antibodies.
Hinge domain influences length, flexibility, and function of the receptor. Transmembrane
domain at first was considered to stabilize the structure of the receptor, but several
studies have shown it also is a factor that could potentially increase the function of the
receptor. Intracellular signaling domain triggers downstream T cell activation pathways.
Unlike natural T cell receptors (TCRs), CAR can recognize antigens in MHC independently.
Thus antigen presenting progress is not mandatory for CAR-T cells recognition and
3
activation [6-9].
First generation CAR only has CD3zeta subunit of TCR complex as intracellular
signaling domain of CAR-T cells, but hyporesponsiveness has been shown in vivo due to
lack of co-stimulation factors on and around tumor cells. To improve CAR-T cell function,
co-stimulation domain such as CD28, 4-1BB, OX40, and CD27 are also introduced into CAR
structure. Second generation CAR has one of those co-stimulation domains, and third
generation CAR could have two co-stimulation molecule sequences. Although third
generation CAR seems to have higher function due to one extra stimulation molecule,
there is still no conclusion about which of the generation is better [6-9]. What is more,
different co-stimulation may have different effects on the function of CAR-T cells. Take the
most commonly used two stimulation domain CD28 and 4-1BB for example. When having
CD28 in CAR molecule, CAR-T cells respond faster and gain higher cytotoxicity against
tumor cells. However, exhaustion begins sooner; the proliferation status sustains much
shorter than CAR-T cells with 4-1BB [10-11].
Success and limitations of CAR therapy
The most successful CAR-T therapy now is anti-CD19 CAR-T against B-cell
malignancies such as acute lymphoblastic leukemia (ALL) and Non-Hodgkin lymphoma
(NHL) [6, 12]. CD19 is a molecule expressed on most mature B cells and is not presented
on other types of cells. This characteristic makes it a promising target for CAR-T therapy.
In previous clinical trials treating ALL among adolescences, the overall remission rate
reached 83%, and CR of B cell lymphoma patients is about 53%. Considering the patients
who received CAR-T treatment did not have any responses for or relapsed from the
previous chemo- and radiotherapy, the remission rates are remarkable. Last year, FDA
approved anti-CD19 CAR-T as formal treatment methods against adolescences ALL and B-
4
cell lymphoma [12-14].
Although CAR-T has gained remarkable results in treating blood cancer, the efficacy
is limited when treating solid tumors. The possible reason for the limited efficiency is the
complex tumor microenvironment. In tumor microenvironment, many factors suppress T
cell proliferation, activation, and cytotoxicity. For example, the high concentration of
adenosine can lead to less IL-2 and IFN-gamma secretion by T cell and suppress T cell
functions. Other factors like higher level IL10, tumor-associated fibroblasts, hypoxia
condition and Treg cells all inhibit T cell function in the tumor microenvironment. Immune
checkpoints CTL-4 pathway and PD-1/PD-L1 pathway are two of major immune inhibition
pathways that limit T cell functions. In normal conditions, Program Death 1 (PD-1) is
upregulated on T cells after T cells activation to avoid hyper-responsive immune system
from damaging healthy tissue. Most solid tumor cells have upregulated PD-L1, PD Ligand
1, which binds to PD-1 molecule on T cells leading to T cell exhaustion and apoptosis.
Therefore, if the binding of PD-1 and PD-L1 can be blocked, T cells function would be
sustained. Recent studies have shown that anti-PD-1 monoclonal antibodies can prevent
PD-1 pathway and inhibit tumor growth [7-9]. FDA approved several anti-PD-1 or anti-PD-
L1 antibody product last few years for treatment against different types of solid tumors
[15-17].
Project 1. Engineer CAR-T cells to secrete anti-PD-1 scFv
Our first project is to realize the possibility of combination therapy of CAR-T and PD-
1 blockade. Here we engineered T cells to express CAR molecules and to secrete anti-PD-
1 scFv at the same time. Theoretically, CAR-T cells would suppress tumor growth more
significantly with anti-PD-1 secretion. In our study, we found anti-PD-1 scFv can interrupt
PD-1/PD-L1 binding preventing T cells exhaustion and hypofunction. And anti-PD-1 scFv
5
secretion enhanced CAR-T cell proliferation, cytotoxicity and tumor cell growth inhibition
both in vitro and in vivo.
Project 2. Design chimeric antigen receptor utilizing CD3 epsilon signal
As we looked back into the structure of CAR, CD3zeta is the most commonly used
stimulation domain [6-9]. Although the mechanism is not clear right now, CAR can activate
T cell via triggering multiple downstream signals in TCR signaling pathways, like Lck, Fyn,
LAT, etc. [18]. While, there is another critical subunit in TCR complex, CD3epsilon.
CD3epsilon is a transmembrane protein. In the CD3 complex, there are two CD3epsilon
subunits, each of which pairs with CD3delta or CD3gamma. Not like CD3zeta with 3 ITAMs,
CD3epsilon has only one ITAM domain in its structure [19, 20]. However, when treated
with anti-CD3epsilon antibodies, T cells can be significantly activated [4-6]. Therefore, I
linked anti-CD19 scFv part to the extracellular domain of CD3epsilon, to generate chimeric
CD3epsilon (ChCD3e). ChCD3e should be able to activate T cells after interacting with
CD19 molecules. And results showed specificity and high responsiveness of novel
designed ChCD3e against tumor cells in vitro.
Results
Characterization of anti-CD19 CAR-T cells with anti-PD-1 antibody secretion
Main retrovirus vector sequence encoding CARs is shown in Fig 1A. We designed
second generation anti-CD19 CAR with CD28 as co-stimulation domain as CAR19.
CD19.aPD1 encodes both anti-CD19 CAR and secretive anti-PD-1 scFv. After transduction
with retroviruses carrying target genes, CD19 CAR molecules of both constructs can be
expressed on T cells. And anti-PD-1 scFv expression by CD19.aPD1 is confirmed by
Western Blot and ELISA.
6
CAR-T cells showed elevated antigen-specific responses when PD-1 pathway was
blocked
To test the function of CAR with the PD-1 blockade, we performed antigen-specific
stimulation assays. CAR19 cells and CAR19.aPD1 cells were both co-cultured with H292-
CD19 cells which have a relatively high PD-L1 expression on the surface. At different time
points, the supernatant of co-culture was harvested and analyzed by ELISA to quantify the
amount of IFN gamma secreted by T cells. The result showed similar IFN gamma secretion
by CD19 and CD19.aPD1 at 24h, however, at 72h and 96h, CD19.aPD1 secreted
significantly more IFN gamma.
Next, the cytotoxicity assay was performed to compare the cytolytic ability of CAR19
and CAR19.aPD1. Both effector cells were co-cultured with H292-CD19 cells for 6 hours at
E/T (effector/target) ratios as 1, 5, 10 and 20. There is nearly no difference in cytolytic
ability between CAR19 T cells and CAR19.aPD1 cells at each E/T ratio.
To further compare functions of CAR19 and CAR19.aPD1, proliferation assay was
performed. T cells were stained with carboxyfluorescein diacetate succinimidyl
ester (CFSE) before co-culture with H292-CD19 cells at E/T ratio as 1. After 96 hours, both
CAR-T cells proliferated remarkably faster than non-transduced T cells, and proliferation
rate of CD19.aPD1 T cells (75.9±5.5%) was significantly higher than that of CD19 T cells
(57.9±10.2 %).
CAR-T cells with anti-PD-1 secretion exhibited enhanced antitumor efficacy in
xenograft model
To assess the ability to inhibit tumor growth, we established xenograft tumor model
on NSG mice using H292-CD19 cells. When average tumor size reaches around 100mm
3
,
7
CAR-T cells were infused into tumor-bearing mice via i.v. injection. The timeline for in vivo
study is shown. The result showed that anti-PD-1 scFv along with non-treated cells did not
inhibit tumor growth effectively. All T cells expressing anti-CD19 CAR suppressed tumor
growth significantly. CAR19 and CAR19 with extra anti-PD-1 scFv soluble protein
demonstrated the similar efficacy of antitumor effect. Notably, CAR.aPD-1 T cells showed
most dramatic tumor shrink after treatment, and nearly all the tumors disappeared after
day 17. Tumor growth was monitored during the procedure, and waterfall plot showed
that all tumors in NT and NT with combined anti-PD-1 scFv treatment continued to grow.
Tumors from CAR19 and CAR19 with combined anti-PD-1 scFv treatment were inhibited,
and half of the tumor shrank by various degree. Surprisingly, the size of all the tumors
from CAR19.aPD1 group decreased by nearly 100%. We keep the mice in the following
over 70 days and found that none of the mice from NT and NT with anti-PD-1 scFv survived
30 days. The mice from CAR19 and CAR19 with anti-PD-1 scFv survived longer than NT
groups, with 20% mice survived over 70 days. Excitingly, all the mice from CAR.aPD1 group
survived over 70 days.
CAR-T cells with anti-PD-1 antibody secretion expanded more efficiently in tumor-
bearing mice
We further evaluated the engraftment and expansion of CAR-T cells with or without
anti-PD-1 scFv secretion. After two days CAR-T cells were infused into tumor-bearing mice,
mice were sacrificed, and cells were collected from blood, spleen, bone marrow and
tumor site. After analysis, the results showed that T cells nearly did not expand and there
was no significant difference between non-transduced T cells and CAR-T cells. Most T cells
homed at spleen at a ratio around 2%, and less than 0.5% T cells remained in circulation.
However, ten days after T cells infusion, CAR-T cells expanded significantly, while non-
transduced T cells only existed in the spleen. In all examined tissues, CAR19.aPD1 T cells
8
showed higher proliferation rate than CAR19 T cells, which is consistent with previous in
vitro results.
Characterization and antigen-specific immune responses of chimeric antigen
receptor utilizing CD3 epsilon signal (ChCD3e)
The schematic figure of ChCD3e is shown in Figure. CD3epsilon leading sequence is
substituted by anti-CD19 scFv with CD8 leading sequence, to provide proper
transmembrane and antigen recognition ability. After transduction, T cells were able to
express ChCD3e protein, although the expression is relatively low (around 24%).
Antigen-specific activation of ChCD3e T cells was evaluated by intracellular cytokine
staining (ICCS). Cytokine secretion was blocked by GolgiPlug before T cells were co-
cultured with target cells. Here we used two types of CD19 positive cells, H292-CD19 and
SKOV-CD19, and 293T cells were introduced as the negative control. After 24-hour co-
culture, cells were collected and analyzed by Flow Cytometry. The results showed non-
transduced T cells barely expressed IFN-gamma, while 5%-15% ChCD3e T cells expressed
IFN-gamma after activated by CD19 antigen. Then, the cytolytic efficacy of ChCD3e T cells
was evaluated by co-culture with antigen presenting cells H292-CD19 cells at E/T ratio of
1, 2 and 5. After overnight co-culture, ChCD3e T cells showed significantly high cytotoxicity
against tumor cells.
Discussion
CAR-T therapy, as a promising method of cancer treatment, has achieved remarkable
success in hematopoietic malignancies. However, the efficacy against solid tumors is
limited. One of the primary reason for the limitation is immunosuppressive
9
microenvironment of solid tumors. PD-1/PD-L1 pathway plays a vital role in T cell
exhaustion in the tumor microenvironment [7-9]. The PD-1 level is upregulated on TILs
with low prognosis solid tumors, leading to tumor escape. The interrupted binding of PD-
1 and PD-L1 can release T cells from the inhibition of this immune checkpoint [21-23].
Various methods of PD-1 pathway inhibition have been developed, such as intrinsic
PD-1 shRNA to downregulate PD-1 expression of T cells and treatment with anti-PD-1 or
anti-PD-L1 antibodies [23-25]. Antibody treatment has been a topic of interest for a long
time and has gained notable achievements in animal studies and clinical trials. However,
the efficacy of treatment with antibodies is still limited due to multiple problems. For
example, antibodies, as relatively large protein product, can hardly infiltrate into tumor
mass and unlikely to encounter PD-1 positive TILs. To address this problem, a higher dose
of anti-PD-1 antibody is required, but high concentration foreign protein can lead to side
effects like diarrhea, autoimmune hepatitis, and colitis. Moreover, a high dosage anti-PD-
1 antibody is likely to cause overresponse of immune cells when interaction with other
pathogens resulting in damage to healthy tissues [26, 27]. Therefore, we managed to
engineer CAR-T cells to secrete anti-PD-1 scFv instead of injecting soluble anti-PD-1
antibodies.
The results showed CAR19.aPD1 T cells gained higher proliferation rate and stronger
tumor inhibition ability both in vitro and in vivo when compared with CAR19 T cells. And
the mixed treatment of CAR19 and injecting anti-PD-1 antibodies hardly improved
immune response against tumor cells compared to CAR19 itself, however, CAR19.aPD1 T
cells eradiated tumor size and prolonged tumor-bearing mice survival dramatically.
Multiple factors contributed to this high anti-tumor efficacy. For example, the localized
concentration of anti-PD-1 antibodies secreted by CAR19.aPD1 T cells at the tumor site is
probably higher than anti-PD-1 antibody injection. The amount of anti-PD-1 antibody in
10
circulation was measured (not shown in the thesis), and it is confirmed that antibody
concentration of the combination group (about 0.7 μg/ml) was 15-fold higher than that
of CAR.aPD1 group [21]. This high dose of circulative antibody is not enough to affect T
cells at tumor site effectively. As mentioned above, poor infiltration ability of antibody
proteins limits the amount entering tumor mass, causing difficult interaction between TILs
and antibodies. However, CAR19.aPD1 T cells can infiltrate into tumor mass and provide
anti-PD-1 scFv for TILs. The result above suggests CAR-T cells with anti-PD-1 antibody
secretion may be a safer and more effective treatment method to block PD-1/PD-L1
signaling and enhance antitumor response against solid tumors.
In vivo study showed tumor regression in the treatment group of CAR-T cells with
anti-PD-1 antibody secretion. However, multiple other factors might contribute to the
enhanced efficacy against the tumor. Firstly, the xenograft model was established in NSG
mice which are immune-insufficient, but the situation would be more complicated in
actual solid tumors due to T cell suppressive factors like Tregs. Thus, immunocompetent
mice should be introduced to generate tumor models. Meanwhile, mouse T cells should
be engineered to express CAR that targets at mouse tumor antigens as well. For example,
we can establish tumors with B16-mCD19 cells which are murine melanoma cell lines with
mouse CD19 expression, and create anti-mouse CD19 CAR-T cells to perform in vivo
studies. Also, the toxicity of secreted anti-PD-1 antibodies cannot be thoroughly assessed
in NSG mice lacking lymphocytes. The side effect of PD-1 blockade CAR-T cells to healthy
tissues should be studied in mice with normal immune systems.
Furthermore, in this thesis, we injected tumor cells subcutaneously to establish the
tumor model. Nonetheless, the tumor microenvironment under skin is different from that
of the tissues where solid tumors locate in patients. Therefore, to evaluate the function
of anti-PD1 secretion CAR-T cells more accurately, we should generate orthotopic tumors
11
at various tissues in immune-competent mice in future studies. Another limiting factor
contributing to the significant results is that we used a well-established anti-CD19 CAR
and engineered target cells with high CD19 expression. To further confirm the enhanced
immune response of CAR-T cells with PD-1 blockade, other tumor associate antigens like
HER2 or GD2 that express on actual solid tumor cells in breast cancer or neuroblastoma
patients should be explored.
To explore and develop possibilities of chimeric receptors that could be involved in
targeted treatment. We focused on CD3-epsilon, an essential subunit in TCR-CD3 complex.
In the complex, there are two CD3-epsilon molecules, one of which pairs with CD3-gamma
and the other one pairs with CD3-delta, forming two heterodimers. One of the reason for
that choosing CD3-zeta as stimulation domain in most CAR structures is that three ITAMs
regions and binding sites for ZAP70 and Lck/Fyn are located in the CD3z structure [18, 20].
Although there is only one ITAM region in CD3-epsilon structure and no such binding sites
for downstream signaling, T cell functions can be activated by anti-CD3e antibodies, and
it is currently the most commonly used method of T cell activation. To utilize signaling of
CD3e in a chimeric receptor, we linked anti-CD19 scFv sequence to the extracellular end
of CD3e. To evaluate the function of the anti-CD19 ChCD3e receptor, ICCS and cytotoxicity
assay were performed, and the results showed that ChCD3e could activate T cells in an
antigen-specific manner. Therefore, ChCD3e is a promising design for antigen-specific
treatment.
However, many problems are remaining unclear with ChCD3e. For example, it is not
clear whether ChCD3e will be able to dimerize with CD3g/CD3d and form CD3 complex or
not. Since the extracellular domain of CD3e is required for dimerization [28, 29], CD19
scFv would probably influence the transmembrane ability of ChCD3e and interaction with
other CD3 subunits. These problems may contribute to the low expression of ChCD3e
12
examined by FACS. Therefore, optimization of the ChCD3e structure is essential for higher
expression. Leading and linker sequence can be substituted by a sequence derived from
other molecules, such as CD3e itself, CD4, CD3d or CD3g.
We are surprised to observe the significant cytolytic activity of ChCD3e T cells when
interacting with H292-CD19 cells. To further compare the function difference between
ChCD3e and CAR, multiple paralleled experiments should be performed. Besides
cytotoxicity assay, ICCS examining IFNg and IL2 and proliferation assay, downstream
pathway signal molecules should also be assessed to differentiate the signaling of ChCD3e
and CAR. Primarily, the phosphorylation levels of ITAMs in parental TCR/CD3 complex
should be examined in a short timeline after interaction between T cells and antigens
happens. These results would help to compare the response speed and strength of
ChCD3e and CAR signals. Moreover, whether ChCD3e will join CD3 complex or not is still
unknown. The location of ChCD3e can be monitored by in situ hybridization, and the
different response speed would be explained.
In conclusion, with anti-PD-1 antibody secretion, CAR-T cells demonstrated enhanced
cytolytic ability, higher proliferation rate, more IFNg secretion and improved tumor
inhibition both in vitro and in vivo. In xenograft mouse models, CAR19.aPD1 T cells
eradiated most tumors and prolonged tumor-bearing mice survival dramatically
compared to CAR19 cells. Tumors with a natively high expression of PD-L1, such as ovarian
cancer or breast cancer, will be the targets in next steps to achieve better immune
responses.
Moreover, chimeric receptor utilizing CD3e signaling exhibited antigen-specific
responses and dramatic cytolytic toxicity against H292-CD19 cells. Although further
exploration is still required to optimize the structure design, new experiments should be
13
performed to evaluate the function and signaling of ChCD3e, and it has excellent potential
to become a promising antigen-specific receptor in clinical treatment.
Methods
Cell culture and antibodies
H292 lung cancer cell line was kindly provided by Dr. Ite laird-Offringa (University of
Southern California, Los Angeles, CA). SKOV3 and 293T cells lines were purchased from
ATCC. SKOV3 and H292 cell lines were transduced by lentivirus vector encoding human
CD19 to generate SKOV3-CD19 and H292-CD19. Transduced cells were stained with anti-
human CD19 antibodies (BioLegend, San Diego, CA) and sorted via flow cytometry to yield
cells with high CD19 expression. H292, SKOV3, H292-CD19 and SKOV3-CD19 cells were
cultured in R10 consisting of RPMI-1640 medium with 10% fetal bovine serum (FBS), 2mM
L-glutamine, 10mM HEPES, 100 U/ml penicillin and 100μg/ml streptomycin. 293 T cells
were cultured in D10 consisting of DMEM medium supplemented with 10% FBS, 2 mM L-
glutamine, 10 mM HEPES, 100 U/ml penicillin and 100μg/ml streptomycin. All the medium
and supplements were obtained from Hyclone (Logan, UT). Human peripheral blood
mononuclear cells (PBMCs) were cultured in T cells medium (TCM), composed of X-Vivo
15 medium (Lonza, Walkersville, MD) with 5% human AB serum (Gemcell, West
Sacramento, CA), 1% HEPES (Gibco, Grand Island, NY), 1% Pen-Strep (Gibco), 1% GlutaMax
(Gibco), and 0.2% N-Acetyl Cysteine (Sigma-Aldrich, St. Louis, MO).
Primary antibodies used in this study include biotinylated Protein L
(GeneScript,Piscataway, NJ) FITC-Goat-anti-Mouse IgG F(ab’) 2 (Invitrogen, Rockford, IL);
PE-anti-CD45, PE-Cy5.5-anti-CD3, FITC-anti-CD4, Pacific BlueTM-anti-CD8, FITC-anti-CD8,
PE-anti-IFN-γ, Brilliant Violet 421TM-anti-PD-1, PE-anti-PD-L1, PerCP/Cy5.5-anti-LAG-3,
14
and PE-anti-TIM-3 (BioLegend, San Diego, CA); and Rabbit anti-HA tag antibody (Abcam,
Cambridge, MA). The secondary antibodies used were FITC-conjugated streptavidin
(BioLegend, San Diego, CA) and goat anti-rabbit IgG-HRP (Santa Cruz, San Jose, CA). The
SuperSignal® West Femto Maximum Sensitivity Substrate used for Western blot analysis
was from Thermo Fisher Scientific (Waltham, MA).
Plasmid construction
MP71 retroviral vector encoding anti-CD19 CAR (CAR) was kindly provided by Prof.
Wolfgang Uckert. The vector encoding anti-CD19 CAR with anti-PD-1 scFv (CAR.aPD1) was
based on MP71 structure. An EcoRI site, a leader sequence derived from human IL-2, the
anti-PD-1 scFv light chain variable region, a GS linker, the anti-PD-1 scFv heavy chain
variable region, the HA-tag sequence, and a NotI site was inserted at 3’ end of anti-CD19
CAR sequence through Gibson assembly method. The anti-PD-1 scFv region in the
CAR.aPD1 sequence was derived from human monoclonal antibody 5C4 specific against
human PD-1. The coding sequence of anti-PD-1 scFv was then optimized by online codon
optimization tool and synthesized by Integrated DNA Technologies (Coralville, IA).
The CD3e sequence was synthesized by Integrated DNA Technologies (Coralville, IA).
The chcd3e sequence was composed of a NotI site CD8 leading sequence, anti-CD19 scFv,
a GS linker region, the CD3e sequence without its leading sequence and an EcoRI site.
ChCD3e and MP71 vector was digested with NotI and EcoRI, then ligated by T4 Ligase.
Retroviral vector production
293T cells were transfected with retroviral vectors based on a standard calcium
phosphate precipitation protocol. 293T cells in 150 mm petri dish were transfected with
15
37.5 μg retroviral backbone plasmid, 18.75 μg envelope plasmid pGALV and 18.75 μg
packaging plasmid gag-pol. The supernatant was harvested 48 hours post-transfection and
filtered through 0.45 μm filter (Corning, Corning, NY) before transduction.
T cell transduction and expansion
Human PBMCs were obtained from AllCells (Alameda, CA). Before transduction,
PBMCs were activated for 2 days by 50ng/ml OKT3, 50 ng/ml anti-CD28 antibody, and
10ng/ml recombinant human IL-2 (PeproTech, Rocky Hill, NJ). 12-well plates were coated
with 15 μg retronectin (Clontech Laboratories, Mountain View, CA) overnight. Freshly
harvested retrovirus was span down onto the coated plates by centrifuging for 2 hours at
2000g at 32°C. 5 × 10
5
cells/ml activated PBMCs were added to the vector-loaded plates
with fresh TCM supplemented with 10 ng/ml human IL-2. The plates were then
centrifuged at 1000g at 32°C for 10 minutes and incubated at 37°C and 5% CO2 overnight.
The CAR expression was detected every 48 hours after transduction. During ex vivo
expansion, the culture medium was replenished, and cell density was adjusted to 5-10 ×
10
5
cells/ml every two days.
Surface immunostaining and flow cytometry
To detect anti-CD19 CAR or ChCD3e expression on CAR-T cell surface, T cells were
stained with biotinylated protein L or FITC-Goat-anti-Mouse IgG F(ab’) 2. 5 × 10
5
cells were
harvested and washed with FACS buffer (PBS containing 5% bovine serum albumin
fraction V) and stained with 1 μg biotinylated protein L or FITC-Goat-anti-Mouse IgG
F(ab’) 2 for 30 minutes at 4°C. Cells stained with protein L were washed and then stained
with FITC-conjugated streptavidin at 4°C for 10 minutes. After cells were washed and fixed
with Transfix cellular antigen stabilizing reagent (Thermo Scientific, Waltham, MA) at 4°C
16
for 10 minutes, cells were washed and stained with anti-CD3, anti-CD4, and anti-CD8
antibodies at 4°C for 10 minutes. Finally, cells were washed and resuspended in PBS and
analyzed by MACSquant cytometer (Miltenyi Biotec, San Diego, CA). All FACS data were
analyzed by FlowJo software (Tree Star, Ashland, OR).
Intracellular cytokine staining (ICCS)
T cells (1 × 10
6
) were co-cultured in TCM with target cells (H292-CD19 or SKOV3-CD19)
at a ratio of 1:1 for 6 hours with GolgiPlug (BD Biosciences, San Jose, CA) in 96-well round
bottom plates. Then cells were harvested and stained with Cy5.5-anti-CD3, FITC-anti-CD4,
Pacific Blue-anti-CD8 and PE-anti-IFNg antibodies. Cytofix/Cytoperm Fixation and
Permeabilization Kit (BD Biosciences was used to permeabilize cell membrane and
perform intracellular staining.
Western Blot
The supernatant of CAR.aPD1 T cells was harvested, and anti-PD-1 scFv was purified
with Pierce
TM
anti-HA magnetic beads (Thermo Scientific, Waltham, MA). SDS-PAGE was
performed with the subject of purified antibody; then protein was transferred to
nitrocellulose membrane (Thermo Scientific, Waltham, MA). Western blot was analyzed
with anti-HA tag antibody.
ELISA
IFNg secreted by T cells was measured by human IFNg ELISA kit (BD Biosciences, San
Jose, CA). 96-well ELISA plates (Thermo Scientific, Waltham, MA) were coated with 200
ng/well of capture antibodies at 4°C overnight. Wells were washed with wash buffer (PBS
17
with 0.05% Tween-20) and blocked with assay buffer (PBS with 10% FBS) for 2 hours at
room temperature. After wells were washed, cell culture supernatant or samples were
added to the coated wells for 2 hours at room temperature. Then wells were washed and
were incubated with detection antibodies for another hour at room temperature. Finally,
HRP reaction substrates were added to the well; the results were quantified by a micro
reader. To measure the amount of anti-PD-1 scFv secreted by T cells, a similar procedure
was involved, recombinant human PD-1 (rhPD-1) was used to coat the plates. Anti-HA tag
antibodies and goat anti-mouse IgG1-HRP were used as detection antibodies.
Cytotoxicity or antigen-specific cell lysis assay
Lysis of target cells (H292-CD19) was measured by comparing the survival of target
cells to the survival of the negative control cells (NCI-H292). This method has been
described previously (22). NCI-H292 cells were labeled by suspending them in R10
medium with 5 μM CellTracker Orange (5-(and-6) -(((4-chloromethyl) benzoyl) amino)
tetramethylrhodamine) (CMTMR), a fluorescent dye for monitoring cell movement
(Invitrogen, Carlsbad, CA), at a concentration of 1.5 × 10
6
cells/mL. The cells were
incubated at 37°C for 30 minutes and then washed twice and suspended in the fresh R10
medium. H292-CD19 cells were labeled by suspending them in PBS+0.1% BSA with 5 μM
carboxyfluorescein succinimidyl ester (CFSE) fluorescent dye at a concentration of 1 × 10
6
cells/mL. The cells were incubated for 30 minutes at 37°C. After incubation, the same
volume of FBS was added into the cell suspension and then incubated for 2 minutes at
room temperature. The cells were then washed twice and suspended in the fresh R10
medium. Equal amounts of NCI-H292 and H292-CD19 cells (5 × 10
4
each) were combined
in the same well for each culture with effector CAR-T cells. Co-cultures were set up in
round bottom 96-well plates in triplicate at the following effector-to-target ratios: 1:1 and
5:1. The cultures were incubated for 4 hours at 37°C, followed by 7-AAD labeling,
18
according to the manufacturer’s instructions (BD Biosciences). Flow cytometric analysis
was performed to quantify remaining viable (7-AAD-negative) target cells. For each co-
culture, the percent survival of H292-CD19 cells was determined by dividing the
percentage of live H292-CD19 cells by the rate of live NCI-H292 cells. In the wells
containing only target and negative control cells without effector cells, the ratio of the
percentage of H292-CD19 cells to the percentage of NCIH292 cells was calculated and
used to correct the variation in the starting cell numbers and spontaneous cell death. The
cytotoxicity was determined in triplicate and presented in mean ± SEM.
Cell proliferation assay
3 × 10
5
H292-CD19 cells were suspended in TCM medium and then seeded in 6-well
plates. Once target cells attached to the plates, T cells were harvested, washed with PBS
and then labeled with 10 μM CFSE at a cell density of 1 × 10
6
cells/ml. After a 60-minute
incubation at 37°C, the cells were washed and resuspended in fresh TCM. T cells were
added to the target cells at 1:1 ratio. After 96-hour incubation, the co-cultures were
analyzed by flow cytometry to quantify the intensity of CFSE.
Xenograft tumor model and adoptive transfer
At 6 to 8 weeks after birth, each NSG mice was inoculated subcutaneously with 3 ×
10
6
H292-CD19 cells. When the average tumor size reached 100-120 mm
3
at day 10 to 13
post-inoculation, the mice were treated with 1 × 10
6
or 3 × 10
6
CAR-transduced T cells in
100 μl PBS via i.v. injection. CAR expression was normalized to 20% in both CAR groups by
adding donor-matching non-transduced cells. Soluble anti-PD-1 antibodies were injected
into mice twice a week after treatment. Tumor size was measured twice a week with
calipers and calculated by the approximate formula: W2 × L / 2. Once symptom like
19
noticeable weight loss, ulceration of tumors or tumor size larger than 1000 mm
3
was
observed, mice were considered dead and euthanized.
Statistical analysis
Statistical analysis was performed in software GraphPad Prism, version 5.01. One-way
ANOVA with Tukey’s multiple comparisons was performed to evaluate the differences
among different groups in the in vitro assays and tumor growth curve. Mouse survival
curve was assessed by Kaplan-Meier analysis (log-rank test with Bonferroni correction). A
P value less than 0.05 was considered statistically significant. Significance of findings was
defined as: ns = not significant, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
20
Figures
21
Figure 1. construction and characterization of CAR19 and CAR.aPD1.
(A) Schematic figure showing the gene structure of anti-CD19 CAR (CAR19) and CAR19
with anti-PD-1 secretion (CAR19.aPD1); (B, C) Expression of CAR molecule of transduced
human T cells. T cells were stained with biotinylated protein L, and FITC-conjugated
streptavidin to monitor CAR expression on T cell surface. NT stands for non-transduced T
cells, here as a negative control. Histogram (B) shows CAR expression of viable CD3+ T
cells, while plot figure (C) shows CAR expression in CD4+ or CD8+ cell group; (D) the
amount of IFNg secreted by CAR19 or CAR19.aPD1 was analyzed by ELISA and Western
blot.
22
23
Figure 2. CAR19.aPD1 exhibited enhanced antigen-specific immune responses in
comparison to CAR19.
(A) CAR19 and CAR19.aPD1 T cells were co-cultured with H292-CD19 for various length
of time. IFNg secreted by T cells was measured by ELISA; (B) T cells were co-cultured
with H292-CD19 cells for 6 hours at E/T ratio of 1:1, 5:1, 10:1 and 20:1. The cytolytic
ability of two groups of CAR T cells is similar; (C) Proliferation rate of CAR19.aPD1 is
higher than that of CAR19 T cells. T cells were stained with CFSE before co-cultured with
H292-CD19 cells at 1:1 ratio. After 96-hour incubation, CFSE intensity of T cells was
measured by flow cytometry. (D) Analyzed data from proliferation result in (C) were
presented in bar graphs.
24
25
Figure 3. CAR T cells with anti-PD-1 scFv secretion enhanced tumor inhibition efficacy
in xenograft mice model.
(A) In vivo procedure is shown, including tumor inoculation, T cell adoptive transfer, and
antibody treatment. 3 × 10
6
H292-CD19 cells were injected s.c. into each NSG mice. The
average tumor size reached 100 mm
3
20 days- post-injection. 1 × 10
6
of CAR19 or
CAR19.aPD1 T cells were adoptively transferred into tumor-bearing mice through i.v.
injection. Supplementary anti-PD-1 antibodies were injected at day 1, 5, 9 and 12 for
group NT + aPD-1 and CAR19 + aPD-1. Tumor growth was monitored every two days; (B)
Tumor growth curve represents the average tumor size change of mice from 5 groups,
namely NT, NT + aPD1, CAR19, CAR19 + aPD1, and CAR19.aPD1. Two mice from each
group were sacrificed and the tumors were separated on day 12 post-infusion, from left
to right, NT, NT + aPD1, CAR19, CAR19 + aPD1, and CAR19.aPD1; (C) Waterfall plot shows
the tumor eradiation of each mice in multiple treatment groups; (D) Survival of tumor-
bearing mice after treatment is shown.
26
27
Figure 4. CAR19.aPD1 T cells expanded more efficiently than CAR19 T cells in tumor-
bearing mice.
The percentage of human CD45+ T cells in tumor, blood, spleen and bone marrow of
tumor-bearing mice was examined by flow cytometry at day 2 (A) and day (10) post
adoptive transfer; (C) FACS scatter plot shows the percentage of human CD45+ T cells in
tumor, blood, spleen and bone marrow of mice from different groups.
28
29
Figure 5. Construction, characterization of chimeric antigen receptor utilizing CD3
epsilon signal (ChCD3e).
(A) Schematic representation of anti-CD19 ChCD3e molecule construct; (B) Expression of
ChCD3e in human T cells after transduction. NT and ChCD3e T cells were stained with FITC
conjugated anti-mouse F(ab’) 2 antibodies. The viable CD3+ T cells were gated; (C)
Cytotoxicity of ChCD3e T cells against H292-CD19 is shown. T cells were co-cultured with
H292-CD19 for 12 hours at E/T ration of 1:1, 1:2 and 1:5. Cell lysis of H292-CD19 was
measured. (D)(E) The antigen-specific activation of T cells was monitored through ICCS. T
cells were co-cultured with H292-CD19, SKOV3-CD19 and 293T cells supplemented with
GolgiPlug for 6 hours. The percentage of IFNg expression of CD4+ or CD8+ T cells was
measured by flow cytometry.
30
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Abstract (if available)
Abstract
Chimeric antigen receptor engineered T cells (CAR-T) therapy, has achieved remarkable success in treatment against hematopoietic malignancies, while it exhibited limited efficacy against solid tumors. To overcome the limitation, we developed combination therapy by blocking program death 1 (PD-1) pathway and a novel design of antigen-specific receptor utlilizing CD3-epsilon signal. ❧ Immunosuppressive tumor microenvironment played an essential role in inhibiting T cell functions at the tumor site. High expression of PD-L1 on tumors, like breast tumor or ovarian tumor, can suppress and exhaust T cells through the PD-1 pathway. To evaluate CAR-T cell function against tumor cells, we engineered anti-CD19 CAR T cells (CAR) to secrete anti-PD-1 scFv which can block the binding of PD-1 and program death ligand 1 (PD-L1). According to both in vitro and in vivo results, anti-CD19 CAR T cells with anti-PD-1 scFv secretion (CAR.aPD1) showed enhanced proliferation, promoted cytolytic activity and elevated tumor inhibition in comparison with parental CAR T cells. Also, CAR.aPD1 cells dramatically prolonged overall survival of tumor-bearing mice. ❧ To explore the possibility of chimeric receptor utilizing signals other than CD3 zeta, we designed and constructed chimeric CD3 epsilon (ChCD3e) receptor by linking anti-CD19 scFv to extracellular end of CD3e. Anti-CD19 ChCD3e T cells can be specifically activated by CD19 antigen and demonstrated significant cytotoxicity against H292-CD19 cells.
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Creator
Yang, Shuai
(author)
Core Title
Novel design and combinatory therapy to enhance chimeric antigen receptor engineered T cells (CAR-T) efficacy against solid tumor
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Biochemistry and Molecular Biology
Publication Date
08/02/2020
Defense Date
06/19/2018
Publisher
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chimeric antigen receptor engineered T cells (CAR-T),chimeric CD3 epsilon (ChCD3e),OAI-PMH Harvest,program death 1 (PD-1),solid tumor,tumor microenvironment
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Tags
chimeric antigen receptor engineered T cells (CAR-T)
chimeric CD3 epsilon (ChCD3e)
program death 1 (PD-1)
solid tumor
tumor microenvironment