University of Southern California Dissertations and Theses

Adoptive cell-based immunotherapy of cancer

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Adoptive cell-based immunotherapy of cancer

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Content ADOPTIVE CELL-BASED IMMUNOTHERAPY OF CANCER

by
Hae Jung Won

A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(PHYSIOLOGY AND BIOPHYSICS)

August 2008

Copyright 2008 Hae Jung Won

ii

Table of Contents
List of Tables iii
List of Figures iv
Abbreviation v
Abstract vi
Introduction 1
Methods: Mice and Cell lines 6
Chemotaxis assay 6
Peptides and cytokines 8
Spleen cell preparations 8
Activation and injection of pmel-1 T cells 9
hgp100
25-33
and PT-treated SPC 10
Flow cytometry 10
B16 challenge 11
DC101 administration 11

Result: Anti-PT antibodies 1B7 and 11E 7 neutralize PT 12
Peptide antigen hgp100
25-33
activates pmel-1 CD8+ T cells 14
IL-2 treated pmel-1 T cells failed to reject B16 melanoma in the 16
presence of PT-treated APC
IL-15 treated pmel-1 T cell is not effective to destroy the B16 18
melanoma
IL-12 treated pmel-1 T cells combined with anti-VEGF-R2 20
antibody DC101 markedly slow growth of established B16
melanoma tumors.
Anti-VEGF-R2 antibody DC101 may markedly potentiate the 23
actions of pertussis toxin on immune responses

Conclusion 25
Bibliography 30

iii
List of Tables
Table 1: Data from the therapeutic arm of Experiment 2008-08 22
Table 2: Data from the prophylactic arm of Experiment 2008-08 24

iv
List of Figures
Figure 1: Antibodies 1B6 plus 11E7 neutralize PT 13
Figure 2: Hgp100
25-33
activates pmel-1 CD8+ T cells 15
Figure 3: IL-2 treated pmel-1 T cells failed to reject B16 melanoma 17
Figure 4: IL-15 treated pmel-1 T cells failed to reject established B16 melanoma 19
Figure 5: Experimental design for Experiment 2008-08 20
Figure 6: IL-12 treated pmel-1 T cells combined with an
anti-VEGF receptor 2 antibody DC101 markedly slow growth of 21
established B16 melanoma tumors

v

Abbreviations
PT pertussis toxin
PT-SPC pertussis toxin-treated spleen cells
hgp100 human form of gp100
CTL cytotoxic T lymphocyte

vi
Abstract
Background: Pertussis toxin (PT) is used to promote autoimmune response. Evidence
suggests this effect arises by improving T cell function. We hypothesized PT or PT-
treated spleen cells (PT-SPC) can improve the efficacy of adoptively transferred T
cells in tumor immunotherapy.
Purpose: Establish experimental systems to test the hypothesis.
Approach: Pmel-1 spleen cells were cultured with various cytokines for different
periods of time to find optimal condition to activate the T cells. The cells were
administrated to mice bearing B16 melanoma. We examined combining
immunotherapy with DC101, anti-VEGF receptor 2 antibody.
Result: Tumor associated T cell primed with antigen in the presence of IL-12 was
effective in rejecting the tumor prophylactically and therapeutically whereas IL-2 and
IL-15 failed. DC101 synergized efficacy. PT and PT-SPC resulted in death of mice.
Conclusion: Culturing T cells with IL-12 for 3 days generated effective effector T
cells and their anti-tumor function was enhanced by co-administration of DC101. PT
holds potential to improve efficacy.

1
Introduction
In the past two decades, cellular based cancer therapy has been actively
studied. However, adoptive transfer of tumor associated CD8
+
T has not been very
successful. In a clinical trial, patients received genetically engineered CD8
+
T cells
which express T cell receptors (TCRs) which recognize melanoma cells, but most of
them failed to reject the tumor (Morgan, Dudley et al.). Tumor-associated CD8
+
T
cells also did not reject tumor in a mouse model. Overwijk et al. inoculated B16
melanoma into pmel-1 transgenic mice that produce CD8
+
T cells reactive to B16
melanoma, but they failed to show the regression of tumor. In their study, naïve or
early tumor recognizing CTLs alone were not sufficient to destroy the tumor, but
when the cells were combined with vaccination, cytokine (IL-2), and irradiation of
the mice, tumor destruction was induced (Overwijk, Theoret et al.). One of the
reasons for this success seems that total body irradiation (TBI) increases the efficacy
of adoptively transferred tumor-specific CD8
+
T cells not only by depleting inhibitory
lymphocytes, but also by inducing infections by disruption of the homeostatic balance
between the host and microbes, which produces systemic inflammation (Paulos,
Wrzesinski et al.). Thus, there is a need to modulate the immune system to enable the
tumor recognizing CD8
+
T cells to destroy the tumor.
One of the possible reasons why tumor–recognizing CD8
+
T cells failed to
eradicate the tumor is that since most of tumor antigen is also self-antigen, the T cells
could be in functionally tolerant state. One way of breaking the tolerance is
immunizing the animal with virus encoding the peptide antigen, thereby both signal 1
2
(epitope) and signal 2 (co-stimulatory signal) from viral compound can be provided.
Overwijk et al. showed that immunization with a virus expressing the peptide
recognized by pmel-1 T cells results in higher numbers of self/tumor antigen-specific
T cells with better anti-tumor efficacy. (Overwijk, Theoret et al.)
When tumor-recognizing T cells are activated in vitro, the length of
incubation and the addition of cytokines influence developing of T cell phenotypes.
Depending on the length of incubation, the T cells differentiate into different cell
types, which have different characteristics, therefore different efficacy of destroying
the tumor cells. Paradoxically, adoptive transfer of naive and early effector T cells
was more effective than transfer of more-differentiated effector T cells to promote
tumor rejection (Gattinoni, Klebanoff et al.). The cytokines incubated with the T cells
influence the profile of T cells as well. Klebanoff et al. demonstrated that culturing
pmel-1 spleen cells with IL-15 can improve the in vivo anti-tumor activity of
adoptively transferred CD8
+
T cells compared to IL-2 (Klebanoff, Finkelstein et al.),
and this is because adoptively transferred central memory CD8
+
T cells (TCM) are
more effective than effector memory CD8
+
T cells (TEM), which can be generated by
IL-15 and IL-2, respectively, in vitro (Klebanoff, Gattinoni et al.). A report was
published that IL-12 enhances anti-tumor activity of CD8
+
T cells (Curtsinger, Lins et
al.; Diaz-Montero, El Naggar et al.), perhaps by mediating cytotoxic activity of CD8
+

T cells. CD8
+
T cells that are activated in the absence of IL-12 can proliferate, but do
not become lytic, and are deficient in the ability to produce IFN-r in response to Ag
(Curtsinger, Schmidt et al.; Curtsinger, Johnson et al.). Díaz-Montero et al. showed
3
IL-12 treated CD8
+
T cells almost completely eradicated 7 day-established B16
tumors (Diaz-Montero, El Naggar et al.). Therefore, in vitro conditioning with IL-12
appears to generate more effective CD8
+
T cells for adoptive cancer immunotherapy.
In addition to generating proper effector CD8
+
T cells, enabling the cells to
infiltrate into the tumor site is critical step in adoptive transfer immunotherapy. It is
known that cytokines such as TNF-α secreted by activated antigen presenting cells
(APCs) or can provide the danger signal at an infection site, which is recognized by
circulating CTLs. However, a tumor microenvironment may be poorly immunogenic
because of immunosuppressive molecule, which could prevent cytokine from
developing the danger signal.
Angiogenic compounds have been studied in tumor therapy not only for their
critical role in tumor growth and metastasis, but also for the immunosuppressive roles
that enable the tumor to escape from host immune surveillance. The vascular
endothelial cells in a tumor express little amount of cell adhesion molecules in
response to TNF-α compared to normal vessel. Dirkx et al. tested the hypothesis that
angiogenic factors such as vascular EC growth factors (VEGFs) and fibroblast growth
factors (FGFs) caused this reduced response. They found that anti-angiogenic
compounds restored leukocyte rolling and adhesion to the vasculature in the tumor by
the up-regulation of endothelial adhesion molecules in tumor vessels (Dirkx, Oude
Egbrink et al.; Dirkx, oude Egbrink et al.).
Pertussis toxin (PT) holds promise to improve the efficacy of cancer-
immunotherapy by providing danger signals at the tumor site. PT is an exotoxin
4
produced by the bacterium Bordetella pertussis. It is composed of six subunits and the
subunits are arranged in A-B structure: the A component is enzymatically active and
is formed from the S1 subunit, while the B oligomer is the receptor-binding portion.
When the B oligomer binds to a cell membrane receptor, the S1 may subunit become
activated and ADP-ribosylate the α-subunits of heterotrimeric G proteins. This
prevents the G proteins from interacting with G protein-coupled receptors on the cell
membrane, thus interfering with intracellular communication, for example,
chemokine-induced migration.
It is known that in several mouse models of autoimmune disease, such as
experimental autoimmune encephalomyelitis (EAE), microbial products are required
along with immunization of the animal with target antigen to trigger pathogenic
autoimmunity. Fujimoto et al. compared various TLR ligands- lipoteichoic
acid/peptidoglycan, zymosan, poly (I:C), LPS, flagellin, and CpG
oligodeoxynucleotide, and PT in the capacity of triggering pathogenic autoimmunity
and PT surpasses other microbial products in this activity and by stimulating
excessive proliferation and polarization toward Th1 of naive T cells (Fujimoto, Yu et
al.). How PT and other microbial compounds induce the autoimmune disease is not
clear, but there are some studies showing possible mechanisms. Kerfoot et al.
indicated that PT alone induces the interaction between leukocytes and endothelium
in CNS and it is mediated by elevated level of P-selectin expression on the blood
vessel. (Kerfoot, Long et al.) Another report has shown that PT might exert its
5
adjuvant effect by modifying DC to promote their maturation and the production of
pro-inflammatory cytokines, thereby eliciting a Th1 response (Hou, Wu et al.).
The above information led us to hypothesize that; 1) IL-12 treated tumor
recognizing CD8
+
T cells will become cytotoxic, therefore, more effective to destroy
the tumor 2) ADP-ribosylation activity of PT will block the migration of APCs to
lymph node, which provides prolonged danger signal systemically including the
tumor site. 3) DC101, an antibody against VEGF receptor 2, enhances the efficacy of
immunotherapy by blocking the immuno-suppressive molecules in the tumor
microenvironment.
To investigate the hypothesis, we inoculated B16 melanoma which prevents a
gp100 peptide on their membrane into C57BL/6 mice. We used pmel-1 transgenic
mice as the source of CD8
+
T cells with engineered TCR. Nearly all (>95%) of their
CD8
+
T cells express a TCR that recognizes and binds to MHC-presented gp100. We
primed pmel-1 T cells in the presence of IL-2, IL-15, or IL-12 as well as human
gp100
25-33
(hgp100
25-33
). Overwijk et al. determined that the 25-33 sequence from
hgp100 was more effective than mgp to activate pmel-1 T cells (Overwijk, Tsung et
al.). Activated pmel-1 T cells were then adoptively transferred to tumor-bearing or
non-tumor bearing mice along with PT or PT-treated antigen spleen cells (PT-treated
SPC) to find out how they work in therapeutic and prophylactic conditions. In
addition, the mice were treated with DC101, a monoclonal neutralizing antibody
targeting the flk-1/KDR (type 2) receptor for VEGF, to test the role of anti-
6
angiogenesis molecules alone or in the combination with immunotherapy in rejecting
tumor.

Materials and Methods
Mice and Cell lines
C57BL/6 mice were purchased from National Cancer Institute. A breeding
pair of pmel-1 mouse was obtained from Jackson Laboratory and a colony established
at USC animal facility. B16 melanoma B16 (H-2b) was provided by Dr. Epstein
(USC) and cultured in complete media (RPMI 1640, 0.1% penicillin/streptomycin,
0.2% L-glutamine, and 2.5% heat-inactivated fetal bovine serum).
Chemotaxis assay
96-well ChemoTx disposable chemotaxis plates with 5μm pore size were from
Neuro Probe, Gaithersburg, MD (cat # 106-5). Pertussis toxin (PT) was from List
Laboratories (cat # 180). To a 50 μg vial of PT was added 0.5 ml 500mM NaCl,
100mM Na
x
PO
4
pH 7.0 and the solution stored in 50 μl aliquots of 5 μg at -80ºC.
Once thawed an aliquout was stored on ice or in the refrigerator and used that day or
discarded. C5a was from Biovision (cat # 4955A). THP-1 cells originally from Dr.
Epstein(USC).
Anti-PT monoclonal antibodies 1B7 and 11E6 were produced from
hybridomas generously provided by Dr. A. Weiss (U. Cincinnati). The hybridomas
were generated by Sato et al. (Sato, Sato et al. 1991). 1B7 recognizes the S1 subunit
of PT; 11E6 recognizes subunits S2 and S3. Both antibodies were reported to
7
neutralize the ability of PT to promote CHO cell clustering, lymphocytosis, and
insulin release (Sato, Sato et al. 1991).
THP-1 cells (10
6
/ml) were maintained by culturing in RPMI-1640 medium
supplemented with 2.5% FBS, 0.1% penicillin/streptomycin, and 0.2% L-glutamine.
To begin an experiment the cells were transferred into supplemented RPMI-1640 plus
15mM HEPES pH 7.4 with or without addition of PT or PT first incubated with
antibodies 1B7 and 11E7. Incubation of PT with antibodies was in supplemented
RPMI-1640 plus HEPES for 1hr in a 37ºC, 5% CO
2
incubator; PT was at 200ng/ml
and the antibodies were each at 20μg/ml. The overnight culture of THP-1 was either
with or without PT at 100ng/ml or the same concentration of PT with each antibody
at 10μg/ml.
After the overnight incubation, the THP-1 cells were collected by
centrifugation (3200rpm, 3 min, 4 ºC), and resuspended in serum-free RPMI-1640
plus HEPES at 10
6
cells/ml. 50μl were then placed on a filter in a chemotaxis plate
above a well containing 30μl serum-free RPMI-1640 with different concentrations of
C5a. After 3 hr incubation in a 37ºC, 5% CO
2
incubator, the media containing cells
on the top of the membrane were removed by absorbing with a tissue (Kim-Wipe).
50μl of PBS containing 2μM EDTA was added on top of each membrane followed by
incubation in a refrigerator for 15min. The PBS-EDTA was removed by absorbing
with a tissue. The media in the bottom chambers containing migrated cells was then
transferred to 500μl tubes by piercing the membrane with a pipette tip and
withdrawing the solution. During this procedure the plate was placed on ice to
8
prevent evaporation. 10μl of the media in the 500μl tube was then mixed with 10μl
of Trypan Blue and the mixture applied to a hemocytometer. Unstained cells in the 4
corner squares of hemocytometer were counted.
Peptides and cytokines
Hgp100
25-33,
KVPRNQDWL, and np
366-374
, ASNENMETM, were synthesized
at USC (order # 50085 and 50086, respectively) to a purity >99%. To make 1mM

solutions, 2mg of hgp100
25-33
was dissolved in 1.73ml of sterile H
2
O and 1.9mg of
np
366-374
was dissolved in 2.72ml sterile H
2
O and the solutions were stored in 100μl
aliquots in a -80ºc freezer.
rIL-2 was from Dr. Epstein (USC). 10ug/ml of rIL-2 was stored in 20ul
aliquots in -20ºc freezer. Recombinant murine IL-15 was purchased from PeproTech
INC (Cat# 210-15). 10μg of IL-15 was dissolved in 1ml PBS with 1% BSA to
generate 10μg/ml solution and stored in 50μl aliquots in a -80ºc freezer. Recombinant
mouse IL-12 combined with BSA as carrier protein was purchased from R&D
Systems (Cat# 419-ML-010). 10μg of rIL-12 was dissolved in 1ml sterile PBS to
generate 10μg/ml solution and stored in 50μl aliquots in a -80ºc freezer. Once thawed
an aliquot was stored on ice or in the refrigerator and used that day or discarded.
Spleen cell preparations
Spleens from pmel-1 mice were obtained to generate anti-tumor T cells and
from C57BL/6 mice to generate APCs loaded with hgp100
25-33.
Spleens were taken
from mice and placed in complete media in different plates and fat and other tissues
around the spleen were removed. A 10ml syringe filled with 10ml complete media
9
was prepared. The media was injected thorough 25gauge into the spleen to make the
splenocytes burst out from the spleen. This is repeated until the spleen color turns to
light pink. The rest of the spleen was ground on the cell collector with a piston until it
almost disappeared.
The cell collector was washed with new media and the flow-through was
collected in a 50ml tube with the cells collected from the plates. The cells were
centrifuged at 1200rpm, and 5min, 4°C (Eppendorf, Centrifuge 5417R). The
supernatant was discarded and the cells were suspended in 30ml complete media. A
10ml syringe filled with 10ml of ‘Histopaque’ (Sigma Aldrich, Lot# 095K6240) was
used to slowly load the Histopaque from the bottom of the tube so that the media and
cells were on the top layer. The cells were centrifuged at 1700 rpm, 20min, 4 °C
(Eppendorf, Centrifuge 5417R). Erythrocytes pelleted down at the bottom of the tube.
Both Histopaque solution and supernatant was transferred into a new 50ml tube and
centrifuged at 1200rpm, 5min, 4°C (Eppendorf, Centrifuge 5417R). The supernatant
was discarded and the cells were resuspended in 5ml of complete media.
Activation and injection of pmel-1 T cells
The culture media was prepared by adding 50ul of 1mM hgp100
25-33
and 50μl
of 10μg/ml rIL-2, 10μg/ml IL-15 or 10μg/ml IL-12 solution to 50ml complete media
to give final concentrations of 1μM of hgp100
25-33
and 10ng/ml of cytokines. About
50 million cells from pmel-1 spleen cell preparation were added to a culture flask and
incubated in a 37ºc, 5% CO
2
incubator for the indicated period of time.
10
After incubation, the cells were centrifuged 1200rpm for 5min at 4 ºC
(Eppendorf, Centrifuge 5417R) and suspended in complete media. Each mouse
received 2-4 million pmel-1 cells in 200μl by i.p injection. Tumor bearing mice were
randomized before the injection.
hgp100
25-33
and PT-treated SPC
50ml complete media with 1μM hgp100
25-33
with or without 100ng/ml PT
were prepared. 50-100 million cells from C57BL/6 spleen preparation were added to
each culture flask and incubated in a 37ºc, 5% CO
2
incubator overnight. The cells
were collected by the same procedure for collecting pmel-1 cells. 340μl of a-PT
antibodies, 1B7 (3.0mg/ml) and 11E7 (3.0mg/ml), were added to the PT-treated
APCs and incubated for 1 hour in a 37ºc, 5% CO
2
incubator prior to collection. The
cells were centrifuged at 1200rpm, and 5min, 4°C (Eppendorf, Centrifuge 5417R)
and washed with 10ml of complete media once at the same speed. Each mouse
received about 1 million cells in 200μl by i.p injection.
Flow cytometry
Spleen cells were prepared from a 6-8 week old male pmel-1 mouse as
described above. The cells were cultured in the indicated conditions. After incubation
the cells were collected in 15ml conical tubes and centrifuged at 1200rpm for 10 min
at 4 ºC followed by washing with 5ml PBS+2%BSA twice at 1200rpm for 5min at 4
ºC. The cells were suspended in 1ml of 5% mouse serum and incubated in ice for 5
min. Each group of cells was divided into five 1.5ml tubes to analyze expression of
four surface proteins; CD8, CD25, CD44, CD62L, and one tube for unstained control.
11
2μl of anti-mouse antibodies against CD8 conjugated with PE (BD Pharmagen, cat#
553033), CD25 conjugate with PE(BD Pharmagen, cat# 554866), CD44 conjugated
with PE(CALTAG, cat# RM5704), and CD62L conjugated with PE(CALTAG, cat#
RM4304) were added to each tube and incubated for 45 min at 4 ºC. The samples
were kept in aluminum foil to block light during the staining reaction and shaken
every 15 min. After incubation, each sample was washed with 1ml PBS + 2% BSA
twice at 3200rpm for 3min at 4 ºC (Eppendorf, Centrifuge 5417R) and resuspended in
500μl PBS + 2% BSA for analysis. The analysis was done using a FACSCalibur flow
cytometer and CELLquest software (both from BD Biosciences).
B16 challenge
To collect B16 melanoma, the culture media was discarded and 10ml of
trypsin-EDTA were added and incubated at 37ºc for 2 minutes. 10ml of complete
media was added to stop the reaction, and the cell suspension was collected in a 50ml
tube to centrifuge at 1200rpm for 5min at 4 ºC (Eppendorf, Centrifuge 5417R). The
cells were resuspended in complete media to generate 2.5 million cells/ml (=0.5
million/200μl). Each mouse was challenged with indicated number of B16 melanoma
cells in 200μl by subcutaneous injection at a shaved site of the back. The tumor
volume was calculated by width x length x height. The mice were euthanized when
one of the diameters exceeded 20mm.
DC101 administration
DC101 was provided by Dr. Epstein (USC). 0.8mg of DC101 was
administrated into each mouse by i.p injection approximately twice a week.
12

Results
Anti-PT antibodies 1B7 and 11E 7 neutralize PT.
Monoclonal α-PT antibody 1B7 is known to bind to the S1 subunit of PT. 11E6
recognizes subunits S2 and S3 in the B-oligomer. Both antibodies were reported to
neutralize the ability of PT to promote CHO cell clustering, lymphocytosis, and
insulin release (Sato, Sato et al.). Before we injected the PT-treated SPC into mice,
the residual PT should be removed or neutralized to prevent it from acting on other
cells in vivo. Therefore, the PT-treated cells were incubated with these two antibodies
for 1hr followed by washing. To demonstrate the antibodies neutralize PT, we used a
chemotaxis assay. As shown in Fig.1, C5a causes migration of THP-1 monocytes in
dose dependent manner. THP-1 monocytes cultured with 20μg/ml of both antibodies
overnight did not show significant difference in migration compared to non-treated
control group. However, THP-1 monocytes that were incubated with 200ng/ml PT
overnight did not migrate towards C5a. In contrast, 200ng/ml PT incubated with
10μg/ml of both antibodies for 1hr had no effect. These results indicate that the
combinations of 1B6 and 11E7 neutralize PT.

13

Figure1. Antibodies 1B6 plus 11E7 neutralize PT.
PT-treated THP-1 monocytes exhibit decreased chemotactic activity toward a
chmoattractant, C5a, and it is increased when the PT is neutralized by a-PT antibodies,
1B6 and 11E7. THP-1 cells were incubated overnight with no-addition, 10ug/ml of
antibodies, 10ng/ml PT, or PT pre-treated with the antibodies for 1hr. the cells were
collected and washed in serum-free RPMI 1640 medium. 50μl of cells suspension
(5.0 X 10
4
cells) were added on the top compartment of ChemoTx disposable
chemotaxis plates whereas the bottom chamber contained indicated concentration of
C5a. After 3hr incubation in a 37
°
c, 5% CO2 incubator, 10ul was taken from the C5a
solution containing migrated cells at the bottom chambers to stain with 10μl of
TrypanBlue and the number of un-stained cells from 4 squares on hemocytometer
was counted.

0
5
10
15
20
25
30
0 10 30 100
C5a concentration(nM)
# cells migrated
control
+Abys
+PT
+PT+Abys
14
Peptide antigen hgp100
25-33
activates pmel-1 CD8+ T cells
The B16 melanoma expresses the mouse homologue of human gp100
(mgp100), an enzyme involved in pigment synthesis that is expressed by the majority
of malignant melanoma cells, as well as by normal melanocytes.
FACS analysis was performed to demonstrate the epitope specificity of the
pmel-1 splenocyte preparation. Pmel-1 splenocytes were cultured with 1μM of
peptide antigen; hgp100
25-33
, an irrelevant peptide; np
366-374
, or no addition. In
addition, to further characterize the phenotypic polarization by cytokines, the cells
were cultured in the presence of either 10ng/ml rhIL-2 or 10ng/ml rhIL-15. After 7
day incubation, the cell preparations were analyzed by flow cytometry for CD8, and
activation markers CD25 and CD44 and lymph node homing marker CD62L. The
pmel-1 splenocytes cultured for 7 days without peptide or with np
366-374
showed a
naïve T cell profile with about 15% of the cells staining for CD8. The preparation
exhibited an intermediate level of CD44 expression and low levels of CD25 and
CD62L expression (Fig 2). Adding hgp100
25-33
caused a marked increase in CD8
+

cells, due to prolonged survival or proliferation of CD8
+
T cells. The expression of
CD25, CD44, and CD62L was increased, indicating the cells became Ag-experienced.
When IL-2 was added along with hgp100
25-33
, all of the markers are maintained
positive but level of lymph node homing marker, CD62L, was down regulated by
about 20%. When the cells were treated with IL-15, the percentage of CD8 + T cells
15

Figure2. Hgp100
25-33
activates pmel-1 CD8+ T cells.
Flow cytometry analysis of pmel-1 spleen cells cultured for 7 days with indicated
combination of peptides and cytokines. Pmel-1 splenocytes were harvested as
described above and cultured with no-addition, np
366-374;
irrelevant peptide
,
hgp100
25-
33
; self/tumor antigen, with or without IL-2 and IL-15 for 7 days in a 37
°
c, 5% CO
2

incubator. Cells were collected and stained with the antibodies recognizing CD8,
CD25, CD44, and CD62L conjugated with fluorescent dyes. The analysis was done
after gating for lymphocytes.

45.20%
15.42%
15.82%
87.12%
0.38%
0.55%
99.10%
75.91%
74.86%
69.04%
1.20%
1.24%
88.45%
31.16%
74.90%
81.29%
97.11%
98.89%
88.07%
52.15%
CD25 CD44 CD62L
Control
Hgp100
25-33

+ IL-15
CD8
NP
366-374

Hgp100
25-33

+ IL-2
Hgp100
25-33

16
in the IL-15 treated pmel-1 cells was markedly increased up to 88% but no significant
changes were observed in other markers. These results indicated that pmel-1 T cells
are reactive to the tumor specific peptide, hgp100
25-33
, and IL-2 and IL-15 guide the
cells to polarize to slightly different phenotype T cells.

IL-2 treated pmel-1 T cells failed to reject B16 melanoma in the presence of
PT-treated APC
IL-2 is a T cell growth and activation factor and it has been used extensively
for the treatment of patients with metastastic melanoma and other cancers
(Rosenberg). Overwijk et al. observed that combination of pmel-1 T cell, vaccination,
and cytokine eradicated established tumor in irradiated mice (Overwijk, Theoret et
al.). To mimic the effect of vaccination, the mice received administration of antigen-
loaded spleen cells. According to Paulos et al. irradiation enhances the efficacy of
pmel-1 T cells by activating innate immune system caused by gut microflora and this
can be mimicked by administration of LPS (Paulos, Wrzesinski et al.). We
hypothesized that PT could replace the irradiation because PT is a potent adjuvant,
and its ability to trigger pathogenic autoimmunity surpasses other microbial products
including LPS (Fujimoto, Yu et al.). Also, Hou et al. reported that PT might exert an
adjuvant effect on DC to promote their maturation and the production of
proinflammatory cytokines, thereby eliciting a Th1 response (Hou, Wu et al.).
To test IL-2 treated pmel-1 T cells and PT-treated SPC, about 2 million of
both pmel-1 T cells and antigen-loaded SPCs treated with or without PT were
17

B
Mouse #
Tumor volume (mm3) at
Day 16

Average
1 952
2 432
3 1140
Control

4 3312
1459

1 3060
2 1080
3 3570
IL-2 treated pmel T cell

4 ~0
1928

1 810
2 196
3 9375
4 9720
5 5184
IL-2 treated pmel T cell
+ PT-treated APC

6 ~0
4214

Figure 3. IL-2 treated pmel-1 T cells failed to reject B16 melanoma
IL-2 treated pmel-1 T cells failed to reject established B16 tumors. (A) 1.0 x 10
6
B16
melanoma cells were injected subcutaneously at the back of C57BL/6 mice on Day 0.
Pmel-1 splenocytes were harvested and cultured with the peptide antigen, hgp100
25-33
,
and IL-2 for 7 days to activate the T cells. Splenocytes from C57BL/6 mice were also
primed with hgp100
25-33
with or without PT overnight. PT-treated cells were
incubated with α-PT antibodies, 1B7 and 11E6, to neutralize the residual PT. About 2
million Pmel-1 T cells and 1 million of APC were injected to tumor bearing mice i.p.
at Day 5. (B) Tumor sizes were measured on the mice that received IL-2 treated
pmel-1 T cells with or without PT-treated APC 16 days since the tumor challenge.
.

1 million B16
Antigen-loaded Spleen cells
Antigen primed-pmel T cell with IL-2
Day 0 1 3 2 5 4 7 6 9 8 10
A
18
injected into mice 5 day after implants B16 tumor cells (Fig 3A). However, as shown
in Fig 3B, 16 days after tumor challenge, the average tumor volumes did not show
significant difference among the groups that received no treatment, IL-2 treated pmel-
1 T cells, and IL-2 treated pmel-1 T cells with PT treated APCs. Therefore, IL-2
treated pmel-1 T cells were not sufficient to reject 5 day established B16 melanoma
and PT-treated APC failed to improve the efficacy of the pmel-1 T cell preparation.
IL-15 treated pmel-1 T cell is not effective to destroy the B16 melanoma
IL-15 and IL-2 possess similar properties. Both cytokines bind to and signal
through a common, intermediate affinity receptor complex composed of CD122 and
CD132 subunits; thus, IL-2 and IL-15 can share similar in vitro activities (Burton,
Bamford et al.; Grabstein, Eisenman et al.). Despite similarities, it is now clear that
IL-2 and IL-15 can play very different roles in T cell biology by interacting with
distinctive receptors. Although IL-2 can promote T cell activation and proliferation,
signaling through the IL-2 receptor (IL-2R) complex may trigger the elimination or
suppression of activated lymphocytes (Refaeli, Van Parijs et al.; Van Parijs, Refaeli et
al.; Shevach). By contrast, signaling through the IL-15R complex is necessary for the
development of elements of the innate immune system, and it contributes to the
maintenance of memory CD8+ T cells (Waldmann and Tagaya; Judge, Zhang et al.).
Therefore, we tested whether IL-15 treated pmel-1 T cells rejected established B16
melanoma tumors. The experiment procedure was similar to the previous experiment,
except 10ng/ml IL-15 replaced IL-2 and the B16 challenge was reduced from 1.0
19
A

B
Day11 Day13
Treatment
Tumor
volumes(mm3) Average
Tumor
volumes(mm3) Average
32 126
224 500
70 252
147 480
Control

147
124

96
290.8

168 429
168 484
28 308
320 64
IL-15 treated
pmel T cells
112
159.2

216
300.2

240 500
144 500
196 112
IL-15 treated
pmel T cells
+SPC
84
166
84
299

256 336
60 200
240 336
240 168
IL-15 treated
pmel T cells
+ PT-SPC
50
169.2

147
237.4

Figure 4. IL-15 treated pmel-1 T cells failed to reject established B16 melanoma.
(A) 0.5 x 10
6
B16 melanoma cells were injected subcutaneously at the back of
C57BL/6 mice on Day0. Pmel-1 splenocytes were harvested and cultured with the
peptide antigen, hgp100
25-33
, and IL-15 for 7 days to activate the T cells. Splenocytes
from C57BL/6 mice were also primed with hgp100
25-33
with or without PT overnight.
PT-treated cells were incubated with α-PT antibodies, 1B7 and 11E6, to neutralize
the residual PT. About 2 million pmel-1 T cells and 1 million of APC were injected to
tumor bearing mice i.p. at Day 5. (B) Tumor sizes were measured from each mouse
that received IL-15 treated pmel-1 T cells with or without PT-treated APC on day 11
and 13 days since the tumor challenge.
0.5 million B16
Antigen-loaded Spleen cells
Antigen primed-pmel T cell with IL-15
Day 0 1 3 2 5 4 7 6 9 8 10
20

million to 0.5 million cells (Fig 4A). As shown in Fig 4B, IL-15 treated pmel-1 T
cells also failed to eradicate tumor in the presence and absence of PT-treated APC.

IL-12 treated pmel-1 T cells combined with anti-VEGF-R2 antibody DC101
markedly slow growth of established B16 melanoma tumors.
As this work was in progress, Diaz-Montero et al. reported that a spleen cell
preparation from pmel-1 mice cultured for three days with hgp100
25-33
and IL-12
could alone eradicate seven-day established B16 melanoma tumors without injection
of IL-2 or irradiation of the mice (Diaz-Montero, El Naggar et al.). This result was
surprising as VEGF from established B16 tumors(Culp, Neal et al.) should cause
B16: therapeutic arm
0 3 5 7 9 10 14 18 21
DC101:
therapeutic arm only
PT
SPC
PT-SPC pmel-1
B16: prophylactic arm
day:
B16: therapeutic arm
0 3 5 7 9 10 14 18 21
DC101:
therapeutic arm only
PT
SPC
PT-SPC pmel-1
B16: prophylactic arm
day:

Figure 5. Experimental design for Experiment 2008-08.
The experiment has two arms: B16 cells were injected before (therapeutic
arm) or after (prophylactic arm) administration of compositions to be tested
for ability to reject B16 tumors. All compositions were injected ip. DC101 =
0.8 mg aby blocking VEGF-R2. PT = 200ng pertussis toxin. SPC = spleen
cell preparation cultured overnight with 1 µm hgp100
25-33
(2 million). PT-
SPC = SPC cultured overnight with both 1 um hgp100
25-33
and 100 ng/ml PT.
pmel-1 = spleen cell preparation from pmel-1 mice cultured three days with
10 ng/ml IL-12 and 1 µm hgp100
25-33
(2 million). For details see Methods
section.
21
pmel-1 T cells to fail to adhere to the vasculature in the tumor (Dirkx, Oude Egbrink
et al.). A failure to adhere should cause the T cells to fail to infiltrate the tumor and
kill the B16 cells. We thus decided to determine if we could reproduce the result
reported by Diaz-Montero et al. and also test whether or not an
antibody, termed DC101, that blocks VEGF receptor 2 (VEGF-R2) on the vasculature
(Manning, Ullman et al.) improved the cytotoxic function of the pmel-1 T cells.
The experimental design for the experiment (termed Experiment 2008-08) is
shown in Figure 5. The experiment has two arms: B16 cells were injected before
(therapeutic arm) or after (prophylactic arm) administration of compositions to be
tested for ability to reject B16 tumors.

Figure 6. IL-12 treated pmel-1 T cells combined with an anti-VEGF
receptor 2 antibody DC101 markedly slow growth of established B16
melanoma tumors.
Tumor growth eventually caused the death of all mice. The data producing
this chart are from Table 1.
22
Date 5/7 5/8 5/9 5/10 5/11 5/12 5/13 5/15 5/17 5/19 5/22
DC101 DC101
Days since
tumor inj: 16 17 18 19 20 21 22 24 26 28 31
2250 2805 euth
1800 2560 euth
euth
euth
Control

euth
864 720 975 1125 1200 euth
252 450 440 600 360 euth
147 400 400 196 845 720 euth
108 144 324 550 600 1372 1568 euth
pmel-1

48 108 108 1050 1125 1008 1632 2890 euth
384 2040 3230 1638 3610 euth
384 726 2304 1768 1872 euth
280 700 825 2754 2176 euth
196 440 520 dead
DC101

dead
1020 1190 euth
40 300 600 816 300 euth
4 50 40 40 50 360 672 euth
1 20 32 42 60 50 96 297 704 880 euth
pmel-1 +
DC101

1 12 25 98 405 60 126 270 616 910 euth
216 576 810 euth
8 80 210 dead
1 16 50 dead
dead
pmel-1 +
DC101+ PT

dead
30 126 189 120 308 600 825 dead
18 112 147 144 400 360 560 euth
18 70 120 216 400 900 900 euth
18 60 60 210 288 320 360 euth

pmel-1 +
DC101
+ SPC

8 50 50 140 280 308 700 2160 euth
4 18 18 18 12 72 120 210 840 1260 euth
4 9 9 dead
dead
dead

pmel-1
+ DC101
+ [PT-SPC]

dead
Table1. Data from the therapeutic arm of Experiment 2008-08.

23

Table1. Data from the therapeutic arm of Experiment 2008-08 (continued).
Values are volumes (mm3) of tumor for individual mice
“dead” = mouse found dead. “euth” = mouse euthanized due to size of tumor.
All compositions injected ip as per the experimental design shown in Figure 5. PT =
pertussis toxin. Pmel-1 = spleen cell preparation from pmel-1 mice cultured 3 days
with IL-12 and hgp100
25-33
. DC101 = antibody blocking VEGF receptor 2. SPC =
spleen cell preparation cultured overnight with hgp100
25-33
. [PT-SPC] = spleen cell
preparation cultured overnight with hgp100
25-33
and pertussis toxin and then treated
with antibodies neutralizing pertussis toxin. For details see Methods section.

In the therapeutic arm, DC101 and the IL-12-treated pmel-1 cell preparation
alone modestly reduced growth of B16 tumors whereas the combination was more
effective in blocking growth of B16 tumor. However, tumors began to grow in mice
receiving the combination of DC101 and pmel-1 T cells once the administration of
DC101 ended, and death of the mice ensued (Table 1). The results indicate that while
the combination of pmel-1 cells and DC101 had a marked effect, it was not sufficient
to completely kill all B16 cells.
Anti-VEGF-R2 antibody DC101 may markedly potetiate the actions of
pertussis toxin on immune responses
Anticipating that the pmel-1 preparation combined with DC101 would not be
sufficient to completely eradicate B16 tumors, pertussis toxin (PT) or a spleen cell
preparation loaded with hgp100
25-33
cultured without (SPC) or with PT (PT-SPC) was
administered to determine if these compositions would further boost the anti-tumor
cytotoxic effect of pmel-1 T cells. SPC without PT did not promote rejection of the
24
B16 tumor. Surprisingly, however, several mice injected with PT or PT-SPC died
before tumors appeared (Table1). This result was not expected as in our previous
experiments death was not associated with administration of PT or PT-SPC, and a
vast literature lacks reports that this dose of PT is lethal. Furthermore, PT was not
lethal in the prophylactic arm of the experiment (Table 2). The main difference
between the mice receiving PT in the prophylactic and therapeutic arms of the
experiment (in addition to the different time of administration of B16 cells) is that in
the therapeutic arm the mice received DC101 and in the prophylactic arm they did not.
Date 5/10 5/12 5/15 5/17 5/19 5/22 5/27 5/30 6/2 6/5
Days since
tumor inj: 10 12 15 17 19 22 27 30 33 36
40 216 780 dead
24 126 440 520 euth
24 56 360 495 1728 euth
9 40 320 836 825 euth
Control

4 24 198 520 480 euth
0 0 0 0 240 768 euth
0 0 0 0 32 96 840 1445 euth
0 0 0 0 0 24 192 480 1326 euth
0 0 0 0 0 0 0 0 0 0
pmel-1

0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 420 770 euth
0 0 0 0 0 0 98 405 715 euth
0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0
pmel-1
+PT

0 0 0 0 0 0 0 0 0 0

Table 2. Data from the prophylactic arm of Experiment 2008-08
Values are volume of tumor for individual mice.
“dead” = mouse found dead. “euth” = mouse euthanized due to size of tumor.
All compositions injected i.p as per the experimental design shown in Figure 5.
PT = pertussis toxin. Pmel-1 = spleen cell preparation from pmel-1 mice
cultured 3 days with IL-12 and hgp100
25-33
. For details see Methods section.

25
A recent report suggests that factors from tumors act on the VEGF-R2 to produce
immunosuppressive responses (Manning, Ullman et al.). The results reported above
are consistent with PT (and PT-SPC) promoting an autoimmune response towards
melanocytes that is normally dampened by autologous factors acting via VEGF-R2.
When DC101 blocked that response, the autoimmune response may have become so
intense it killed the mice.

Conclusion
The results in this study suggest three findings. While additional work is
needed to firmly establish them, this study provides evidence suggesting continuing
to do so would be worthwhile.
1. Culturing CD8
+
T cells with IL-12 prior to injection improves their
cytotoxic function in the adoptive immunotherapy of cancer.
IL-12 is a well-known modulator of immune response. It polarizes the
differentiation of naïve T cells toward Th1 response. In vitro or in vivo treatment with
IL-12 results in enhanced activity of T and NK cells which is characterized by
increased secretion of IFN-r (Trinchieri) (Watford, Moriguchi et al.).. The enhanced
ability of IL-12 treated CD8
+
T cells can be due to increased primary expansion of
CD8
+
T cells by reducing cell death, resulting in a larger CD8+ T cell memory pool
(Chang, Cho et al.) (Diaz-Montero, El Naggar et al.).
Furthermore, IL-12 enhances the activity of anti-tumor CD8
+
T cells to
reverse the functional tolerance against host. Since the tumor antigen, gp100, is also a
26
self antigen, the host body may be tolerant to the T cells reactive to the self/tumor
antigen. In fact, Overwijk et al. failed to reject the tumor when they challenged B16
melanoma directly into pmel-1 transgenic mice and demonstrated the needs of
vaccination of the host and co-administration of a T cell growth and activation factor,
IL-2, along with the tumor-specific CD8 T cells to overcome functional tolerance of
adoptively transferred T cells [2]. Therefore, pre-treatment of T cells with IL-12 not
only enable the T cell become more cytotoxic but also break the tolerant state.
Due to its superior ability in caner immunotherapy, IL-12 has been tried in
clinics. However, the clinical application of systemic IL-12 has been hindered by
considerable toxicities (Cohen; Car, Eng et al.). Therefore, in vitro conditioning of the
T cells prior to administration is a potent alternative approach because it could
prevent the systemic effect or toxicity of IL-12. Considering the efficacy of IL-12
treated T cells, there are reports showing ex vivo culture with IL-12 also improves
CD8+ T cell immunotherapy (Emtage, Clarke et al.; Macgregor, Li et al.; Diaz-
Montero, El Naggar et al.). Diaz et al. showed almost complete rejection of 10 day
B16 melanoma with CD8 T cells cultured with IL-12. Although we failed to
reproduce their result perfectly, our result also showed significantly delayed tumor
growth. The difference in the result could be due to the difference in B16 melanoma
cell line or in injecting the cells or; i.p and i.v. However, according to Petersen et al.,
the route of injection affected neither the total number of adoptively transferred cells
found in tumor tissue nor the ability of these cells to control tumor growth (Petersen,
Petersen et al.).
27
It has also been proposed that IL-12 provides a “3
rd
signal”. According to
recent reports, the immune system requires 3 rd signal along with 1
st
and 2
nd
signal,
which is provided by antigen and co-stimulation, respectively, for optimal clonal
expansion and acquisition of effector function of T cell (Curtsinger, Lins et al.)
(Curtsinger, Johnson et al.) . IL-12 is secreted from antigen-presenting cells upon the
interaction with TCR on CD4 helper T cells. Therefore, exogenously provided IL-12
can substitute the adjuvant in the adjuvant-free environment of tumor.
We tried priming the T cells in the presence of the peptide antigen with IL-2,
IL-15 or IL-12, and only IL-12 treated T cells succeeded in rejecting tumor and other
cytokines failed, which further supports the idea that IL-12 plays a role as a 3
rd
signal.

2. Blocking VEGF-R2 improves the function of adoptively-transferred
cytotoxic CD8
+
T cells.
Angiogenesis, defined as the development of new vasculature from
preexisting blood vessels, is involved in tumor growth and metastasis (Ellis, Liu et
al.) and it has been a target for treating cancer. Blocking angiogenesis prevents the
formation of new vessels and it has been proposed to improve the efficacy of cancer
immunotherapy. There are several mechanisms explaining how anti-angiogenesis
drugs help the immune response against tumor. First, they may enhance the cross-
presentation of tumor antigen by inducing tumor cell apoptosis (Ackerman and
Cresswell). Second, they up-regulate co-stimulatory molecules or cytokines within
the tumor microenvironment which improves direct priming of T cells (Sojka,
28
Donepudi et al.; Foss). Third, evidence suggests anti-angiogenesis drugs induce the
infiltration of lymphocytes in tumor vessel (Dirkx, oude Egbrink et al.). Consistent
with these reports, we observed regression of tumor when we combined IL-12 treated
T cells and DC101.

3. Blocking VEGF-R2 potentiates the autoimmune responses promoted
by PT and PT-treated APCs.
We hypothesized that PT has potential to provide prolonged 3
rd
signal at the
tumor site by preventing activated APCs from homing to lymph nodes. Wakim et al.
further support this hypothesis by showing that memory CD8
+
T cell responses can be
initiated not only in lymph nodes but also in peripheral tissues through CD4
+
T cells
and recruited dendritic cells (Wakim, Waithman et al.). At this moment, the data do
not clearly prove this hypothesis because most of our mice that received PT treatment
had died very early, perhaps because PT promoted such a strong immune response
that it caused excessive secretion of cytokines leading to a systemic shock. Therefore,
PT may be promising to further improve immunotherapy of adoptive transfer of T
cells.

4. Future studies.
Several things need to be done in the future. First of all, some critical control
experiments are missing in this study. For example, in tumor experiments, the number
of B16 cells and the length of incubation of pmel-1 spleen cells were not identical in
29
all experiments. For studying the behavior of pmel-1 T cells in vivo, imaging
technique will be useful. We transfected lenti-virus with a gene expressing green
fluorescent protein (GFP) into B16 cells. Also, there are many fluorescent dyes that
can label lymphocytes. Thus, by using B16-GFP and labeling pmel-1 T cells with
fluorescent dye, we expect to image how the T cells distribute throughout the animal
and infiltrate into tumor. One way of testing whether the combination of DC101 and
PT triggers a strong immune response is treating mice with PT and DC101 along with
pmel-1 T cells and seeing if it causes death or autoimmune response. If it kills the
mice again, cytokine levels should be measured to confirm the death is due to shock.
Since gp100 is self-peptide, pmel-1 T cell would have responded to more
systemically. Therefore, it would be interesting to see if the treatment lead the same
response when white mouse (Balb/C) is used which has less melanocytes. Also, it can
be tested in different experimental system such as OVA-expressing tumor and OT-1
cells. It is possible that PT is too strong immune-booster. Perhaps, we can reduce the
side effect by using less PT or PT treated SPC. Also, it is worthwhile to test other
cytokines or adjuvant, such as TNF-α, to enhance the immune response.

In conclusion, three factors that can improve the efficacy of cell-based
immunotherapy appear to be IL-12, PT and α-VEGF antibody. This study
demonstrates the potential benefit of combining different compositions to maximize
the efficacy of cancer immunotherapy.

30
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Abstract (if available)

Abstract Background: Pertussis toxin (PT) is used to promote autoimmune response. Evidence suggests this effect arises by improving T cell function. We hypothesized PT or PTtreated spleen cells (PT-SPC) can improve the efficacy of adoptively transferred T cells in tumor immunotherapy.

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Development of TCR-like antibody and novel chimeric antigen receptor for cancer immunotherapy

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Development of immunotherapy for small cell lung cancer using iso-aspartylated antigen

Asset Metadata

Creator Won, Hae Jung (author)

Core Title Adoptive cell-based immunotherapy of cancer

School Keck School of Medicine

Degree Master of Science

Degree Program Physiology

Publication Date 07/31/2008

Defense Date 06/10/2008

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag angiogenesis,immunotherapy,OAI-PMH Harvest,pretussis toxin,T cell

Language English

Advisor Kaslow, Harvey (committee chair), Epstein, Alan L. (committee member), Peti-Peterdi, Janos (committee member)

Creator Email haejungw@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m1473

Unique identifier UC1447479

Identifier etd-Won-20080731 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-90365 (legacy record id),usctheses-m1473 (legacy record id)

Legacy Identifier etd-Won-20080731.pdf

Dmrecord 90365

Document Type Thesis

Rights Won, Hae Jung

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu