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Self-secretion of checkpoint blockade enhances antitumor immunity by murine chimeric antigen receptor-engineered T cells
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Self-secretion of checkpoint blockade enhances antitumor immunity by murine chimeric antigen receptor-engineered T cells
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Content
Self-secretion of Checkpoint Blockade Enhances Antitumor
Immunity by Murine Chimeric Antigen Receptor-
engineered T Cells
Jiangyue Liu
Department of Molecular Microbiology and Immunology
Master of Science
University of Southern California
December 2018
Table of Contents
ABSTRACT ................................................................................................................................................. 1
INTRODUCTION ....................................................................................................................................... 2
MATERIALS AND METHODS ............................................................................................................... 3
Construction of plasmids .......................................................................................................................... 3
Cell lines and culture media ...................................................................................................................... 4
Virus vector production ............................................................................................................................ 4
Mouse T cell activation, transduction and expansion ............................................................................... 5
Western Blot and ELISA analysis ........................................................................................................... 5
Surface immunostaining ........................................................................................................................... 7
Target cell preparation .............................................................................................................................. 7
Intracellular cytokine staining ................................................................................................................... 7
Cytotoxicity assay ..................................................................................................................................... 8
Competitive blocking assay ...................................................................................................................... 8
Statistical analysis ..................................................................................................................................... 9
RESULTS .................................................................................................................................................... 9
Design and generation of anti-CD19 CAR T cells secreting anti-PD-1 scFv ........................................... 9
Characterization of anti PD-1 scFv secretion of 1D3 PD1 CAR-T cells ................................................ 10
Preparation of target cells ....................................................................................................................... 10
1D3 PD1 CAR-T cells enhances the antigen-specific immune response ............................................... 10
1D3 PD1 CAR T cells downregulated surface PD-1 expression after antigen stimulation .................... 11
DISCUSSION ............................................................................................................................................ 12
ACKNOWLEDGEMENTS ..................................................................................................................... 14
REFERENCES .......................................................................................................................................... 16
FIGURES ................................................................................................................................................... 18
1
ABSTRACT
In melanoma treatment, the immune checkpoint blockade and the adoptive cell transfer
(ACT) have been shown to be associated with favorable prognosis especially in patients with
advanced tumor. Besides, ACT with chimeric antigen receptor T cells (CAR-T cells) is an
uprising therapeutic approach for melanomas and other solid tumors. Among the immune
checkpoint blockade, the blockade of PD-1 pathway, such as anti-PD-1 antibodies, can inhibit
the immune suppressive status in tumor and enhance the T cell function. To overcome T cell
dysfunction in tumor microenvironment (TME), an anti-mouse-CD19 CAR self-secreting anti-
mouse-PD-1 scFv (1D3 PD1 CAR) was designed and expressed on mouse T cells. The secretion
of anti-mouse-PD-1 scFv was measured and the T cell function of 1D3 PD1 CAR-T cells was
analyzed. Besides, the expression of several inhibitory receptors on CAR-T cells was assessed to
demonstrate the potential function of anti-mouse-PD-1 scFv on CAR-T cells in TME. In this
study, 1D3 PD1 CAR-T cells was successfully engineered and the anti-PD-1 scFv can be
efficiently and consistently secreted. The function of 1D3 PD1 CAR-T cells were enhanced after
the specific antigen stimulation and the immune suppress surface marker on T cells were
downregulated. The study indicates the possibility of applying CAR self-secreting immune
checkpoint blockade scFv in solid tumor treatment.
2
INTRODUCTION
Melanoma has been reported as the most aggressive and deadly form of skin cancer. The
treatment of melanoma currently includes surgical removal, chemotherapy, radiation,
immunotherapy and bio-chemotherapy according to the diagnosis for tumor stage and mutation
test.
1
Among these treatments, immune checkpoint blockade and adoptive cell transfer have
shown to be associated with prolonged disease stabilization and longer median survival time.
1
The increasing expressions of several inhibitory receptors, such as cytotoxic T-lymphocyte-
associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), T cell immunoglobulin
and mucin domain (TIM-3), Lymphocyte-activation gene 3 (LAG-3), B- and T-lymphocyte
attenuator (BTLA) and V-domain Ig suppressor of T cell activation (VISTA), are reported to
induce T cell exhaustion in solid tumor microenvironment.
2,3
In ongoing preclinical development
and cancer immunotherapy clinical trials, the immune checkpoint blockade, such as monoclonal
antibody targeting inhibitory receptors, have been shown to ameliorate T cell exhaustion in solid
tumor microenvironment and thus enhance tumor regression.
4
Programmed cell death protein 1 (PD-1), a cell surface protein, plays a role in regulating
immune response through the interaction with its ligand PD-L1/PD-L2. PD-L1 is expressed on T
cells, B cells, macrophages, dendritic cells as well as a wide variety of solid tumor cells
including melanoma cells, while the expression of PD-L1 on normal tissues is barely detectable.
5
Specifically, in the tumor microenvironment, the PD-1 expression on T cell surface is
upregulated after transcriptional activation, and the interaction between PD-1 and PD-L1
expressed on tumor cells causes T cells dysfunction and exhaustion.
6,7
Thus, the blockade of PD-
1 pathway, such as anti-PD-1 antibodies, can inhibit the immune suppressive status in tumor and
enhance the T cell function, which has been tested in colon, renal, lung cancers and
3
melanoma.
8,9,10
In metastatic melanomas, the adoptive cell transfer (ACT) shows durable response and
prolonged disease stabilization. ACT with chimeric antigen receptor T cells (CAR-T cells) is an
uprising therapeutic approach for melanomas and other solid tumors.
11
Single chain variable
fragment (scFv) containing the light and heavy chain variable regions of a monoclonal antibody,
together with the transmembrane domain, the T cell signaling and activation domain were
engineered to be expressed on T cells so that they can become activated by the target antigen and
perform the cytotoxic function.
1
It has been reported that the downregulation of the proliferation and cytokine production in
T cells can be rescued by PD-1 blockade in the subcutaneously implanted melanoma mouse
model.
12
Meanwhile, a study focusing on an anti-human-CD19 CAR-T cells self-secreting anti-
human PD-1 antibody (aPD1-T cells) in the immunodeficient xenograft mouse model shows a
better function and proliferation than parental CAR-T cells.
13
Since the immunodeficient
xenograft mouse model cannot entirely mimic the tumor microenvironment and related immune
status, an allograft mouse model with complete immune function and characterization is
necessary and critical to examine the function and efficacy of the newly engineered CAR-T cells.
Therefore, in this study, an anti-mouse-CD19 CAR with self-secreting mouse PD-1 blockade
was designed and engineered. The target cell was B16 melanoma cells derived from C57BL/6
and the anticipated mouse model would be C57BL/6 for in vivo study.
MATERIALS AND METHODS
Construction of plasmids
The anti-mCD19 CAR (1D3 CAR) was constructed based on the retroviral vector MP71
4
kindly provided by Professor Wolfgang Uckert.
14
The vector consisted of anti-mouse CD19 scFv
derived from the amino acid sequence of mCD19 antibody (clone name 1D3), mCD28, mCD3 ,
P2A and GFP from 5’ to 3’. The anti-mCD19 CAR self-secreting anti-mouse PD-1 scFv (1D3
PD1 CAR) was designed based on 1D3 CAR. The self-cleaving peptide T2A, human IL-2
leading sequence, mouse anti-PD-1 scFv and HA tag was inserted into the 1D3 CAR vector after
the GFP sequence. The lentiviral vector pLVX-Puro was used for the preparation of target cell.
Mouse CD19 sequence was inserted between IRES and CMV promoter in the pLVX-IRES-Puro
vector.
Cell lines and culture media
Virus producer 293T cells (ATCC CRL-3216), non-transduced B16-F10 (ATCC CRL-
6475) and transduced B16-CD19 target cells were cultured in D10 medium consisting 10% fetal
bovine serum (Sigma), 1% GlutaMax (GIBCO) and 1% 100x Penicillin-Streptomycin Solution
in DMEM (Hyclone). Mouse splenocytes were cultured in R10 medium consisting 10% fetal
bovine serum (FBS, Sigma), 1% GlutaMax (Gibco), 10mM HEPES (Gibco) and 1% 100x
Penicillin-Streptomycin Solution in RPMI-1640 (Hyclone) supplemented with 100IU/L
recombinant mouse IL-2 (mIL2, Peprotech).
Virus vector production
Standard calcium phosphate precipitation protocol was used to produce the retroviral
vector 1D3, 1D3 PD1 and the lentiviral vector mCD19. 30mL 0.6 million/mL 293T cells were
seeded in 150*25mm dish one day before transfection. 293T cells were transfected by 37.5 μg
retroviral vector plasmid (40 μg lentiviral vector plasmid), 30 μg packaging plasmid Gag-Pol
5
(20 μg Rev and 20 μg RRE for lentivirus) and 18.75 μg envelope plasmid VSV-G (20 μg VSV-G
for lentivirus). Medium was replaced four hours after transfection. 48 hours after transfection,
virus soup was collected and filtered through 0.45 μm filter (Corning).
Mouse T cell activation, transduction and expansion
Mouse splenocytes were obtained from the spleen of C57BL/6 mice. 8 μg/mL anti-mouse
CD3 (BioLegend) and 2 μg/mL anti-mouse CD28 (Biolegend) were coated at 4℃ overnight in
96-well tissue-culture plate one day before the activation. Tris-buffered ammonium chloride
(TAC) were used to lyse the erythrocytes in the harvested splenocytes. After washing two times
with phosphate-buffered saline (PBS), 3 million/mL cells were suspended in warm R10
supplemented with 0.1IU/mL mIL2. The plate coated with anti-mouse CD3 and anti-mouse
CD28 were washed once with PBS before adding 200 μL cell suspension each well. FITC anti-
mouse CD3 (BD Pharmingen) was used to assess the activated percentage.
One day after activation, 500 μL per well 4% RetroNectin (recombinant human fibronectin
fragment, TaKaRa, 1 μL RetroNectin in 25 μL PBS) were added to a 24-well non-treated tissue
culture plated and coated overnight. The next day, the plate was washed softly with PBS and
then blocked with 2% Bovine Serum Albumin Fraction V (BSA, OmniPur, 0.1g BSA to 5mL
PBS) 30 minutes at room temperature. The plate was washed once with PBS before transduction.
0.8 million activated splenocytes and 2mL virus soup per well were added and spun at 2000rpm,
32℃ for 2 hours. Medium was changed after the transduction. Mouse CAR-T cells were cultured
in fresh R10 medium supplemented with mIL2 and expended for up to 7 days.
Western Blot and ELISA analysis
6
The secretion of anti PD-1 scFv by PD-1-CAR was detected by Elisa and Western blot.
Two days after transduction, 1D3 PD1 CAR-T cells were cultured in tissue-culture 96-well plate
at the density of 2 million/mL. The cells were passaged for the next 4 days maintaining the same
density. 100 μL supernatant was taken each day for PD-1 scFv quantification by ELISA. mPD-1
scFv was purified from PD-1 CAR T cell culture supernatant using anti-HA magnetic beads
(Pierce). 25 μL (0.25mg) beads was washed twice with Tris-buffered saline (TBS) supplemented
with 0.05% Tween-20 (TBS-T) and collected with magnetic stand. 4mL supernatant was added
to the pre-washed beads and incubated for one hour at room temperature with mixing. The beads
were washed twice with 300 μL TBS-T and kept in 300 μL ultrapure water for chemical elution.
The sample was incubated with 100 μL of 0.1M glycine (OmniPur, pH 2.0) for five minutes at
room temperature and the supernatant containing anti-mPD-1 scFv was neutralized by adding
15μL neutralization buffer (1M Tris, pH 8.5). Bicinchoninic acid assay (BCA) assay was
performed to determine the protein concentration.
For Western blot, the purified anti PD-1 scFv was subjected to sodium dodecyl sulfate–
polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride
(PVDF, Thermo Scientific) membrane for Western blot analysis. The membrane was incubated
with 5 μg/mL rabbit polyclonal anti-HA tag antibodies (Abcam) and 2ng/mL stabilized goat anti-
rabbit HRP-conjugated antibodies (Pierce) for detection.
For ELISA, 96-well flat bottom Elisa plates (Nunc Maxisorp, Thermo Scientific) was coated
with 5 μg/mL mouse PD-1-Fc chimera protein (R&D System) overnight at 4℃. On the next day,
the plate was first washed with Elisa washing buffer (PBS with 0.05% Tween-20) and then
blocked with Elisa blocking buffer (PBS with 10% FBS) for one hour at room temperature.
Purified mPD-1 was used as the standard curve. Equal volume of cell culture supernatant, serum
7
and blood sample were added to the plate and incubated for 2 hours at room temperature. The
detection antibody 5 μg/mL rabbit polyclonal anti-HA tag antibodies (R&D system) and 2ng/mL
stabilized goat anti-rabbit HRP-conjugated antibodies (Pierce) were added and incubated for 1
hour and 0.5 hour respectively after washing the plate with Elisa washing buffer. 3,3’,5,5’-
Tetramethylbenzidine (TMB) substrate solution (Thermo Scientific) was added for developing
and the plate was read at 450nm after adding stop solution (2N H2SO4).
Surface immunostaining
The expression of 1D3 and 1D3 PD1 CAR-T cells were detected by biotinylated mouse-
anti-rat Fab (Invirtogen) and streptavidin-FITC (BD Pharmingen). 0.1 million cells were
harvested and washed three times with PBS before staining with mouse-anti-rat Fab 30 minutes
at 4℃. Then the cells were washed three times, stained with streptavidin-FITC 15 minutes at 4℃
and then washed two times. The cells were finally resuspended in PBS and assessed using a
Miltenyi Biotec flow cytometer. Non-transduced mouse T cells served as the negative control.
The result was analyzed using the FlowJo software.
Target cell preparation
B16-CD19 was generated by the transduction using lentiviral vector pLVX-mCD19-Puro
and sorting mCD19
positive cells with fluorescence-activated cell sorting (FACS). FITC anti-
mouse CD19 (BioLegend) was used to test mCD19 expression level.
Intracellular cytokine staining
0.1 million none transduced, 1D3 and 1D3 PD1 CAR-T cells were co-cultured with the
8
target cell at the ratio of 1:1 for 24, 48 and 72 hours. PE-anti-mouse PD-1, PE-anti-mouse PD-L1,
APC-anti-mouse TIM3 and FITC-anti-mouse CD8 (all from Biolegend) were used for
immunostaining. IFN- in supernatant was quantified using IFN- mouse ELISA kit (Invitrogen)
according to the kit protocol.
Cytotoxicity assay
Target cells were harvested, washed once and resuspend in PBS at the density of 1
million/mL. CFSE (Invitrogen) supplemented with FBS was added at the ratio of 1 to 1000 and
incubated 30 minutes at 37℃. The same volume of FBS was added to stop staining and the cells
were resuspended in D10. Effector cells including 1D3 CAR, 1D3 PD1 CAR and negative
control none transduced T cells were cocultured with the post-processed target cells at the ratio
of 1:1, 3:1 and 5:1 with 0.05 million target cells each well. After 24 hours incubation at cell-
culture condition, the cells were harvested and stained with 7-AAD. FACS analysis was
performed to quantify the percentage of CFSE
+
7-AAD
+
cells / CFSE
+
cells.
Competitive blocking assay
The 96-well non-treated plate was coated with 8 μg/mL anti-mouse CD3 (BioLegend)
and 2 μg/mL anti-mouse CD28 (Biolegend) overnight at 4℃. The next day, the plate was washed
once with 100 μL PBS and then incubated with 4 μL 100 μg/mL recombinant mouse PD-L1
(rmPD-L1, R&D system). 100uL 3-day post-transduction 1D3-PD-1 CAR conditioned medium
or conditioned medium plus rmPD-L1 was added to corresponding wells. After 4 hours’ cell-
culture conditioned incubation, T cells were harvested and stained with surface marker mCD8,
PD-L1 and intracellular molecule IFN- .
9
Statistical analysis
Graphpad Prism (version 5.01) was used to perform statistical analysis. To assess the
difference among different groups, one-way ANOVA with Tukey multiple comparison was
performed. If p value is less than 0.05, the difference would be considered significant.
Significance of the result is defined as: ns, not significant, p > 0.05; *, p < 0.05; **, p < 0.01; ***,
p < 0.001.
RESULTS
Design and generation of anti-CD19 CAR T cells secreting anti-PD-1 scFv
The scheme of the retroviral vector in the study are shown in Fig. 1A. The retroviral
vector used in the transduction of anti-CD19 CAR, which is designated as 1D3 CAR, is
composed of anti-mouse CD19 scFv (clone name 1D3), transmembrane domain, costimulation
signal CD28, T cell activation domain CD3 as well as the P2A self-cleaving peptide and
fluorescence protein GFP. The retroviral vector for anti-CD19 CAR self-secreting anti-PD-1
scFv, designated as 1D3 PD1 CAR, was constructed based on 1D3 CAR adding T2A self-
cleaving peptide, hIL-2 leading sequence for efficient secretion of downstream protein and anti-
mouse PD-1 scFv before HA tag. Splenocytes from C57BL/6 were activated by anti-mouse
CD3 and anti-mouse CD28 antibodies for 48 hours. T cell marker CD3 was stained after the
activation. As seen in Fig. 1B, T cell proportion was 73% compared to the isotype. After
activation, T cells were transduced with either 1D3 CAR or 1D3 PD1 CAR and the expression
was detected by mouse-anti-rat Fab plus streptavidin. According to Fig. 1C, the expression level
10
of both 1D3 and 1D3 PD1 CAR-T cells were 23.9%. None transduced activated T cells were
used as negative control.
Characterization of anti PD-1 scFv secretion of 1D3 PD1 CAR-T cells
Western Blot and ELISA were performed to assess the expression and secretion of anti
PD-1 scFv. The supernatant of 1D3 PD1 CAR-T cells three days post transduction was collected
and purified for Western Blot and the standard curve of ELISA. The theoretical molecular
weight of anti-mouse PD-1 scFv is supposed to be 30.32 kDa, which is consistent with the size
detected in the blot in Fig. 2A. Starting from day four, the cell density of 1D3 PD1 CAR-T cells
was remained the same and the supernatant was collected for ELISA analysis. According to Fig.
2B, the anti PD-1 scFv secretion ranged from 200ng/mL to 400ng/mL and the secretion was
increasing gradually.
Preparation of target cells
The target cell used in this study was B16-CD19 mouse melanoma cells. The lentiviral
vector used for the transduction of wild type B16 was designed based on pLVX-IRES-Puro
vector. The mouse CD19 sequence was inserted between the CMV promoter and IRES. After
transduction, the cells were stained with FITC-anti-mouse CD19 and the most positive 10% were
sorted by FACS. As seen in Fig. 3A and Fig. 3B, the proportion of mCD19 positive cells were
90.6% after sorting compared with 84.9% before sorting, and the expression remained consistent
after freezing thawing procedure. None transduced B16 cells were used as the negative control.
1D3 PD1 CAR-T cells enhances the antigen-specific immune response
11
To analyze the effect of 1D3 PD1 CAR-T cells through antigen-specific stimulation, 1D3,
1D3 PD1 CAR-T cells, and non-transduced T cells were co-cultured respectively with target
cells for different durations. IFN- in supernatant were quantified by ELISA. In all three
stimulation periods, 1D3 and 1D3 PD1 CAR-T cells have significantly more IFN- than non-
transduced T cells (Fig. 4A). Specifically, the IFN- produced by 1D3 and 1D3 PD1 showed no
significantly difference for longer cocultures, which were 48 hours and 72 hours.
To assess the blocking function of anti-PD-1 scFv secreted by 1D3 PD1 CAR-T cells, the
competitive blocking assay was performed by analyzing the expression of intracellular IFN- and
surface marker PD-L1 in none activated and activated T cells incubated with different
conditioned medium. In Fig. 4B and Fig 4C, both IFN- and PD-L1 on CD8
+
T cells were
significantly higher after activation. After incubating activated T cells with 1D3 PD1 CAR-T
conditioned medium, IFN- secreted by T cells significantly increased and the block effect
remained with rmPD-L1 in medium, indicating that anti-PD-1 scFv secreted by 1D3 PD1 CAR T
cells effectively reverse the immune suppress effect by rmPD-L1 on T cells.
The cytolytic function of 1D3 and 1D3 PD1 CAR-T cells were examined by cytotoxicity
essay. T cells were cocultured with marked target cells at different effector-to-target ratios for 24
hours and cell killing were measured by flow cytometry. Non-transduced T cells were used as
negative control. The ability of 1D3 and 1D3 PD1 CAR T cells to trigger cytotoxic effects was
stronger than control cells at all effector-to-target ratios (Fig. 4D). However, the difference
between 1D3 and 1D3 PD1 CAR T cells were not significant.
1D3 PD1 CAR T cells downregulated surface PD-1 expression after antigen stimulation
To assess the effect of PD-1 blockade in 1D3 PD1 CAR T cells on helping T cells from
12
dysfunction and exhaustion after antigen stimulation, the expression of PD-1 was analyzed after
coculturing 1D3 and 1D3 PD1 CAR-T cells with target cells for 24, 48 and 72 hours. At all time
periods, the expression of PD-1 on both 1D3 and 1D3 PD1 CAR-T cells was significantly higher
than non-transduced T cells (Fig. 5A). However, the PD-1 expression on 1D3 PD1 CAR-T cells
were significantly lower than 1D3 CAR T cells at 48-hour and 72-hour incubation time, which
indicated the PD-1 blockade effect.
In addition to PD-1, other inhibitory receptors characterized in T cells such as TIM-3 and
LAG-3 were assessed using similar methods as PD-1. In Fig. 5B and 5C, the expression of both
TIM-3 and LAG-3 were significantly higher in 1D3 and 1D3 PD1 CAR-T cells. Within 1D3 and
1D3 PD1 groups, the difference is not significant for 24-hour and 72-hour coculture on TIM-3
and for 24-hour coculture on LAG-3.
Moreover, it has been reported that the survival of GD2 CAR T-cells were promoted by PD-
1 blockade after stimulated by PD-L1 negative target cells, indicating that PD-1 scFv secreted by
CAR T cells would inhibit the interaction between T cells expressing PD-1 and T cells
expressing PD-1 ligands (PD-L1), which may help to inhibit the suppression of T cell function.
15
Thus, the expression of PD-L1 was analyzed similarly. The expression of PD-L1 in both CAR-T
cells were significantly higher than non-transduced cells after antigen stimulation (Fig. 5D). The
difference was significant within 1D3 and 1D3 PD1 CAR-T cells at 72-hour coculturing.
DISCUSSION
In this study, the anti-mouse-CD19 CAR-T cells secreting anti-mouse-PD-1 scFv was
successfully engineered. It was demonstrated that the anti-PD-1 scFv can be efficiently and
consistently secreted. Besides, they can block the PD-1/PD-L1 interaction either between T cells
13
and tumor cells or between T cells themselves even supplied with additional PD-L1. The
function of 1D3 PD1 CAR-T cells was enhanced after the specific antigen stimulation and the
immune suppress surface markers on T cells were downregulated.
In previous study, the PD-1 blockade benefits the CAR-T cells in expansion, function and
resistance of regulatory immune cells within B16 melanoma tumors.
12
The repeated injection of
antibodies would be one of the disadvantages considering the patient convenience in future
clinical practice. Human CAR-T cells with the intrinsic PD-1 blockade has been reported to be
resistant to tumor-mediated inhibition.
13,16,17
However, none of these studies have ever examined
the practical function of CAR-T cells in the endogenous immunosuppressive tumor
microenvironment. Thus, this study and upcoming allograft in vivo study would provide the
possibility of applying CAR self-secreting immune checkpoint blockade scFv in solid tumor
treatment.
In the T cell function analysis, the secreted IFN- and the cytotoxicity of two CAR-T
cells group were significantly higher than non-transduced T cells, while the difference between
two CAR-T cells were not significant at most data point. These results indicate the restriction of
the in vitro study and the importance of applying the treatment to immunocompetence murine
model with complete tumor microenvironment.
In the inhibitory surface marker analysis including PD-1, TIM-3 and LAG-3, the inhibitory
surface marker level expressed on CD8
+
T cells significantly increased after the specific antigen-
stimulation at all time points. While a trend of a lower expression was observed on 1D3 PD1
CAR-T group than 1D3 CAR-T group for longer incubation time. The lower expression of PD-1
contributes from the anti-PD-1 blockade function and the downregulation of PD-1 expression.
Since the upregulation of PD-1 is related to the dysfunction of tumor-infiltrating T cells, the anti-
14
PD-1 scFv may enhance the function of effect T cells at tumor site.
18
The simultaneously
expression of multiple inhibitory receptors has been reported to be associated with T cell
dysfunction in the progression of lung cancer.
19
Thus, the decrease expression of other inhibitory
markers such as TIM-3 and LAG-3 in 1D3 PD1 CAR-T group indicated the benefit of anti-PD-1
scFv to the function of T cells. The lower expression of PD-L1, on the other hand, indicated the
decrease of PD-1/PD-L1 interaction induced by tumor cells and/or T cells and increase of T cell
function.
Further in vivo studies are needed to evaluate the function of 1D3 PD1 CAR-T cells in tumor
microenvironment. For the pilot study plan, 0.5 million B16-CD19 target cells would be
inoculated per mouse.
20,21
The treatment group would be 1D3 CAR-T cells only, the combination
of 1D3 and anti-mouse-PD-1 antibody and 1D3 PD1 CAR-T cells. The dose of CAR-T cells
would be 10 million per mouse.
13
The expected data would be the tumor growth curve, the body
weight, the distribution of CAR-T cells in organs, the level of anti-PD-1 scFv in blood and the
surface marker analysis. The enhanced function and infiltration of 1D3 PD1 CAR-T cells
consistent with the in vitro results is expected.
ACKNOWLEDGEMENTS
Foremost, I would like to express my special appreciation to Professor Wang for offering
me the opportunity to study in his laboratory. His advice on research as well as my Ph. D
application have been priceless. I would also like to express my gratitude to my thesis committee
members Professor Jing-Hsiung James Ou, Professor Pinghui Feng and Professor Weiming Yuan
for their encouragement and insightful comments and questions.
15
My sincere thanks also go to senior Ph. D student Xianhui Chen for everything not
limited to technique training, experiment designing and troubleshooting. I would also appreciate
Ph. D student Yun Qu and master student Chumeng Chen for their collaboration on experiment
and review of the thesis.
Last but not the least, I would like to thank my parents: Xiaohang Liu and Yumei Guo,
and my boyfriend Jianhang Zhou, for supporting my master’s study economically and spiritually.
16
REFERENCES
1. Domingues B, Lopes J, Soares P, Populo H. Melanoma treatment in review.
Immunotargets Ther. 2018;Volume 7:35-49. doi:10.2147/itt.s134842
2. Fuertes Marraco S, Neubert N, Verdeil G, Speiser D. Inhibitory Receptors Beyond T Cell
Exhaustion. Front Immunol. 2015;6. doi:10.3389/fimmu.2015.00310
3. Pardoll D. The blockade of immune checkpoints in cancer immunotherapy. Nature
Reviews Cancer. 2012;12(4):252-264. doi:10.1038/nrc3239
4. Dine J, Gordon R, Shames Y, Kasler M, Barton-Burke M. Immune checkpoint inhibitors:
An innovation in immunotherapy for the treatment and management of patients with
cancer. Asia Pac J Oncol Nurs. 2017;4(2):127. doi:10.4103/apjon.apjon_4_17
5. Yamazaki T, Akiba H, Iwai H et al. Expression of Programmed Death 1 Ligands by
Murine T Cells and APC. The Journal of Immunology. 2002;169(10):5538-5545.
doi:10.4049/jimmunol.169.10.5538
6. Postow M, Callahan M, Wolchok J. Immune Checkpoint Blockade in Cancer Therapy.
Journal of Clinical Oncology. 2015;33(17):1974-1982. doi:10.1200/jco.2014.59.4358
7. Topalian S, Drake C, Pardoll D. Immune Checkpoint Blockade: A Common Denominator
Approach to Cancer Therapy. Cancer Cell. 2015;27(4):450-461.
doi:10.1016/j.ccell.2015.03.001
8. Tumeh P, Harview C, Yearley J et al. PD-1 blockade induces responses by inhibiting
adaptive immune resistance. Nature. 2014;515(7528):568-571.
doi:10.1038/nature13954
9. Cho J, Ahn S, Yoo K et al. Treatment outcome of PD-1 immune checkpoint inhibitor in
Asian metastatic melanoma patients: correlative analysis with PD-L1
immunohistochemistry. Invest New Drugs. 2016;34(6):677-684. doi:10.1007/s10637-
016-0373-4
10. Goff S, Dudley M, Citrin D et al. Randomized, Prospective Evaluation Comparing
Intensity of Lymphodepletion Before Adoptive Transfer of Tumor-Infiltrating
Lymphocytes for Patients With Metastatic Melanoma. Journal of Clinical Oncology.
2016;34(20):2389-2397. doi:10.1200/jco.2016.66.7220
11. Klemen N, Feingold P, Goff S et al. Metastasectomy Following Immunotherapy with
Adoptive Cell Transfer for Patients with Advanced Melanoma. Ann Surg Oncol.
2016;24(1):135-141. doi:10.1245/s10434-016-5537-0
12. Curran M, Montalvo W, Yagita H, Allison J. PD-1 and CTLA-4 combination blockade
expands infiltrating T cells and reduces regulatory T and myeloid cells within B16
melanoma tumors. Proceedings of the National Academy of Sciences.
2010;107(9):4275-4280. doi:10.1073/pnas.0915174107
17
13. Li S, Siriwon N, Zhang X et al. Enhanced Cancer Immunotherapy by Chimeric Antigen
Receptor–Modified T Cells Engineered to Secrete Checkpoint Inhibitors. Clinical
Cancer Research. 2017;23(22):6982-6992. doi:10.1158/1078-0432.ccr-17-0867
14. Engels B, Cam H, Schü ler T et al. Retroviral Vectors for High-Level Transgene
Expression in T Lymphocytes. Hum Gene Ther. 2003;14(12):1155-1168.
doi:10.1089/104303403322167993
15. Gargett T, Yu W, Dotti G et al. GD2-specific CAR T Cells Undergo Potent Activation
and Deletion Following Antigen Encounter but can be Protected From Activation-
induced Cell Death by PD-1 Blockade. Molecular Therapy. 2016;24(6):1135-1149.
doi:10.1038/mt.2016.63
16. Cherkassky L, Morello A, Villena-Vargas J et al. Human CAR T cells with cell-intrinsic
PD-1 checkpoint blockade resist tumor-mediated inhibition. Journal of Clinical
Investigation. 2016;126(8):3130-3144. doi:10.1172/jci83092
17. Suarez E, Chang D, Sun J et al. Chimeric antigen receptor T cells secreting anti-PD-L1
antibodies more effectively regress renal cell carcinoma in a humanized mouse model.
Oncotarget. 2016;7(23). doi:10.18632/oncotarget.9114
18. John L, Devaud C, Duong C et al. Anti-PD-1 Antibody Therapy Potently Enhances the
Eradication of Established Tumors By Gene-Modified T Cells. Clinical Cancer
Research. 2013;19(20):5636-5646. doi:10.1158/1078-0432.ccr-13-0458
19. Thommen D, Schreiner J, Muller P et al. Progression of Lung Cancer Is Associated with
Increased Dysfunction of T Cells Defined by Coexpression of Multiple Inhibitory
Receptors. Cancer Immunol Res. 2015;3(12):1344-1355. doi:10.1158/2326-6066.cir-
15-0097
20. Hu B, Ren J, Luo Y et al. Augmentation of Antitumor Immunity by Human and Mouse
CAR T Cells Secreting IL-18. Cell Rep. 2017;20(13):3025-3033.
doi:10.1016/j.celrep.2017.09.002
21. Wang Q, Yu Z, Hanada K et al. Preclinical Evaluation of Chimeric Antigen Receptors
Targeting CD70-Expressing Cancers. Clinical Cancer Research. 2016;23(9):2267-2276.
doi:10.1158/1078-0432.ccr-16-1421
18
FIGURES
Figure 1. Design and generation of anti-CD19 CAR T cells secreting anti-PD-1 scFv. A. Scheme
of the construction of the retroviral vector MP71 encoding anti-mCD19 CAR (1D3 CAR) and
anti-mCD19 CAR secreting anti-PD-1 scFv (1D3 PD1 CAR). B. Activation of T cells.
Splenocytes from C57BL/6 were activated by anti-mouse CD3 and anti-mouse CD28
antibodies for 48 hours. Cells were stained with FITC-anti-mouse CD3 . FITC-isotype was used
as negative control. C. Expression of 1D3 and 1D3 PD1. The expression was detected by
biotinylated mouse-anti-rat Fab plus FITC-conjugated streptavidin. Non-transduced but activated
T cells were used as negative control.
A
B
C
19
Figure 2. Characterization of anti-PD-1 scFv secretion of 1D3 PD1 CAR-T cells. A. Western
Blot for anti-PD-1 scFv secreted by 1D3 PD1 CAR-T cells. The sample was purified using anti-
HA magnetic beads. B. Expression of anti-PD-1 scFv in 1D3 PD1 CAR-T cells supernatant. The
samples were analyzed by ELISA. The protein concentration of purified anti-PD-1 scFv was
assessed by BCA assay and used as the standard curve.
A
B
1D3 PD1
20
Figure 3. Preparation of target cells. A. Expression of surface marker mCD19 on target cells
before cell sorting. B. Expression of surface marker mCD19 on target cells after cell sorting.
Both cells in A. and B. were stained with FITC-anti-mouse CD19. Isotype was used as negative
control.
A
B
21
Figure 4. 1D3 PD1 CAR-T cells enhances the antigen-specific immune response. A. Expression
of IFN- by 1D3 and 1D3 PD1 CAR-T cells after antigen-specific stimulation for different
durations. 0.1 million CAR-T cells were cocultured with the same number of target cells for 24,
48 and 72 hours at 37℃ and IFN- in supernatant were quantified by ELISA. Non-transduced T
cells were used as negative control (n=3, mean± SEM; ns, not significant, p > 0.05; *, p < 0.05;
A
B
C
D
22
**, p < 0.01; ***, p < 0.001). B. Competitive blocking assay for anti-PD-1 scFv secreted by 1D3
PD1 CAR-T cells. The percentage of CD8
+
T cells expressing IFN- or PD-L1 over the total T
cells were illustrated. None activated splenocytes were used as the negative control (n=3,
mean± SEM; ns, not significant, p > 0.05; *, p < 0.05; **, p < 0.01). D. Cytotoxicity of 1D3 and
1D3 PD1 CAR-T cells against target cells. Two groups of effector cells were cocultured with
different ratio of target cells at 37℃ for 24 hours. The percentage of CFSE
+
7-AAD
+
cells /
CFSE
+
cells was defined as the cytolytic ability. None-transduced T cells were used as the
negative control (n=3, mean± SEM; **, p < 0.01; ***, p < 0.001).
23
Figure 5. 1D3 PD1 CAR T cells downregulated surface PD-1 expression after antigen
stimulation. A, B, C, and D. PD-1, TIM-3, LAG-3 and PD-L1 expression on CD8
+
CAR-T cells.
The expression was analyzed after coculturing 1D3 and 1D3 PD1 CAR-T cells with target cells
for 24, 48 and 72 hours. The cells were then harvested and stained with PE-anti-mouse PD-1,
APC-anti-mouse TIM-3, PE-anti-mouse LAG-3, PE-anti-mouse PD-L1 and FITC-anti-mouse
CD8. Non-transduced T cells were used as negative control (n=3, mean ± SEM; ns, not
significant, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001).
A
B C
D
Abstract (if available)
Abstract
In melanoma treatment, the immune checkpoint blockade and the adoptive cell transfer (ACT) have been shown to be associated with favorable prognosis especially in patients with advanced tumor. Besides, ACT with chimeric antigen receptor T cells (CAR-T cells) is an uprising therapeutic approach for melanomas and other solid tumors. Among the immune checkpoint blockade, the blockade of PD-1 pathway, such as anti-PD-1 antibodies, can inhibit the immune suppressive status in tumor and enhance the T cell function. To overcome T cell dysfunction in tumor microenvironment (TME), an anti-mouse-CD19 CAR self-secreting anti-mouse-PD-1 scFv (1D3 PD1 CAR) was designed and expressed on mouse T cells. The secretion of anti-mouse-PD-1 scFv was measured and the T cell function of 1D3 PD1 CAR-T cells was analyzed. Besides, the expression of several inhibitory receptors on CAR-T cells was assessed to demonstrate the potential function of anti-mouse-PD-1 scFv on CAR-T cells in TME. In this study, 1D3 PD1 CAR-T cells was successfully engineered and the anti-PD-1 scFv can be efficiently and consistently secreted. The function of 1D3 PD1 CAR-T cells were enhanced after the specific antigen stimulation and the immune suppress surface marker on T cells were downregulated. The study indicates the possibility of applying CAR self-secreting immune checkpoint blockade scFv in solid tumor treatment.
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Asset Metadata
Creator
Liu, Jiangyue
(author)
Core Title
Self-secretion of checkpoint blockade enhances antitumor immunity by murine chimeric antigen receptor-engineered T cells
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Molecular Microbiology and Immunology
Publication Date
11/12/2018
Defense Date
10/16/2018
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
CAR-T,chimeric antigen receptor-engineered T cells,immune checkpoint blockade,mouse tumor microenvironment,OAI-PMH Harvest,PD-1,programmed cell death protein 1,solid tumor
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Language
English
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Electronically uploaded by the author
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Advisor
Ou, James (
committee chair
), Feng, Pinghui (
committee member
), Yuan, Weiming (
committee member
)
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jiangyul@usc.edu,vennaliu0503@gmail.com
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https://doi.org/10.25549/usctheses-c89-102358
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Tags
CAR-T
chimeric antigen receptor-engineered T cells
immune checkpoint blockade
mouse tumor microenvironment
PD-1
programmed cell death protein 1
solid tumor