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A mutant recombinant immunotoxin αFAP-PE38 for melanoma treatment in mice

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A mutant recombinant immunotoxin αFAP-PE38 for melanoma treatment in mice

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A mutant recombinant immunotoxin
αFAP-PE38 for melanoma treatment in mice

By
Baiyang Liu
1

Mentor: Pin Wang
2, 3, 4

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfilment of the Requirements for the Degree
MASTER OF SCIENCE
(Biochemistry and Molecular Biology)
December 2017

1
Department of Biochemistry & Molecular Biology, Keck School of Medicine, University of Southern California,
Los Angeles, CA
2
Mork Family Department of Chemical Engineering and Materials Science, University of Southern California,
Los Angeles, CA
3
Department of Biomedical Engineering, University of Southern California, Los Angeles, CA
4
Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA
ii

ACKNOWLEDGEMENTS
I would like to express my appreciation to my advisor Dr. Pin Wang for giving
me the opportunity to join his lab. His guidance and support mean lot to me in the
past year. I would like to thanks my committee members Dr. Peter Danenberg and
Dr. Jian Xu for their kind support.
I would like to thank all members of the Wang’s lab for their kindness and
helpfulness: Si Li, Natnaree Siriwon, John Mac, Xianhui Chen, Paul Bryson, Shuai
Yang, Elizabeth Siegler, Jennifer Rohrs, Yu Jeong Kim, Yun Qu, Jeff. I would like
to especially thank Xiaoyang Zhang and Guanmeng Wang for your support and
friendship.
I would like to express my special thanks to Dr. Richard Roberts for sharing his
lab’s facilities with us.

iii

ABSTRACT
Fibroblast activation protein (FAP) is highly expressed in Tumor associate
fibroblasts (TAFs) in most of human epithelial cancers and the TAFs play an
important role in tumorigenesis, which makes FAP a promising target for cancer
treatment. In previous study, a FAP-targeting immunotoxin aFAP-PE38 has been
designed to deplete TAFs in mouse cancer model. However, this treatment is largely
limited by the early production of neutralizing antibodies to immunotoxin. Thus, we
designed a new immunotoxin aFAP-PE38 with mutant exotoxin PE38 which has less
neutralizing epitopes and less immunogenicity. We test the new immunotoxin on
tumor cell lines and mice B16 melanoma model. The results indicate that the mutant
immunotoxin has a stronger antitumor efficacy and less cytotoxicity than the original
one. We also examine the combined treatment of mutant immunotoxin and PD-1
blockade, which shows a synergy effect on the activation of immune system, leading
to the suppression of tumor development.

Key words: immunotoxin, fibroblast activation protein, tumor-associated
fibroblast, melanoma, tumor microenvironment, PD-1 blockade

iv

TABLE OF CONTENTS
ACKNOWLEDGEMENTS ................................................................................ ii
ABSTRACT ...................................................................................................... iii
TABLE OF CONTENTS .................................................................................... iv
INTRODUCTION ............................................................................................... 1
Tumorigenesis ............................................................................................... 1
Tumor Associate Fibroblasts ......................................................................... 1
Fibroblast Activation Protein ........................................................................ 2
Recombinant Immunotoxin ........................................................................... 3
PD-1 Blockade .............................................................................................. 4
OBJECTIVE OF THE PROJECT ....................................................................... 5
MATERIAL AND METHODS ........................................................................... 6
Mice and Cell Line ........................................................................................ 6
Plasmid Construction and Protein Production .............................................. 6
Protein Purification ....................................................................................... 7
Cytotoxicity Study ........................................................................................ 7
Pharmacokinetic Test .................................................................................... 8
Mouse Xenograft Antitumor Activity Study ................................................. 8
Flow Cytometry Analysis .............................................................................. 8
RNA Isolation and Transcripts Analysis by qRT-PCR .................................. 9
v

RESULTS .......................................................................................................... 10
Construction and Purification of Mutant αFAP-PE38 ................................ 10
Binding Affinity between Immunotoxins and FAP-expressing Cells ......... 10
Cytotoxicity of αFAP-PE38 ........................................................................ 13
Mutant αFAP-PE38 Treatment Slows B16 Melanoma Growth in vivo ...... 14
Pharmacokinetics Test ................................................................................. 16
Microenvironment Altered by Combine Treatment of Mutant αFAP-PE38 and PD-1
blockade ...................................................................................................... 17
Cytokine Alteration on Transcriptional Level ............................................. 19
DISCUSSION .................................................................................................... 20
REFERENCE .................................................................................................... 24

INTRODUCTION
Tumorigenesis
Tumorigenesis is a complicate multistep process caused by the accumulation of
mutations in tumor cells. Like other normal cells, tumor cells live in a complex
microenvironment consists of growth factors, cytokines, chemokines, immune cells,
inflammatory cells, and stromal cells including endothelial cells and tumor-
associated fibroblasts (TAFs). Previous study reveals that stromal cells have a
profound influence on the development of carcinomas
[1]
. The stromal cells
communicate with tumor cells not only directly via cell interaction, but also
indirectly via protease activity and alteration of extracellular matrix (ECM)
properties
[2]
. The interaction between stromal cells and tumor cells plays a vital role
in the regulation of tumorigenesis.
Tumor Associate Fibroblasts
Tumor associate fibroblasts (TAFs) are important promoters for tumorigenesis,
angiogenesis and metastasis
[3]
. They are responsible for the production of cytokines,
chemokines and growth factors, which induce tumor growth
[4]
. TAFs are also the
principle source of matrix metalloproteinases (MMPs), a ECM-degrading protease
family which alters proteolysis and leads to tumorigenesis
[5]
. In addition, Vascular
endothelial growth factor (VEGF) is primarily secreted by TAFs, though it can be
2

released by tumor cells themselves in a lesser amount
[3]
. VEGF can promote
microvascular permeability, leading to the attraction of inflammatory cells and
endothelial cells
[6,7]
. These cells produce ECM that is rich in type I collagen and
fibronectin, which are crucial for the initiation of tumor angiogenesis
[8,9]
. Due to the
close relation between TAFs and tumor development procession, TAFs become a
promising target for cancer treatment
[10,11]
.
Fibroblast Activation Protein
Fibroblast activation protein (FAP) is an inducible cell surface glycoprotein
firstly identified by monoclonal antibody in 1986
[12]
. FAP is selectively expressed
on the surface of TAFs in most of the human epithelial cancers, though with low
expression in normal fibroblasts and tissues
[13]
. FAP is considered as a crucial
regulator in tumorigenesis and cancer progression
[14,15]
. It has been reported that
FAP-expressing stromal cells promote tumor growth and invasive velocity through
altering ECM properties, e.g., enhancing the production of MMP-2 and MMP-9
[16-
18]
. In addition, FAP-expressing stromal cells show the capability of suppressing
antitumor immunity
[19]
.
FAP is also thought to promote tumor growth by driving angiogenesis
[20]
. A
previous study shows that depletion of FAP decreased blood vessel density in the
tumor tissue of mice
[21]
. Additionally, depletion of FAP-expressing stromal cells
leads to rapid hypoxic necrosis in both stromal cells and tumor cells with the
3

involving of tumor necrosis factor α (TNF-α) and interferon-γ (IFN-γ)
[19]
. Overall,
FAP-expressing stromal cells play a vital role in tumorigenesis, angiogenesis and
metastasis.
Previously, several therapeutic approaches have been reported using FAP as
target. Most of them focus on the inhibition of FAP enzymatic activity by small
molecules
[22,23]
. However, little success has been reported in the clinical trials of
these therapies
[24]
. This could be caused by the non-enzymatic functions of FAP in
tumor development
[25]
.
Recombinant Immunotoxin
Given that the inhibition of FAP enzymatic activity did not effectively suppress
tumor progression as expected, a more rationally designed FAP-targeting
recombinant immunotoxin αFAP-PE38 emerged recently for cancer treatment
[26]
.
Recombinant immunotoxin is a large molecule composed of an antibody Fv
fragment connected with a strong bacterial toxin. It aims to combine the selectivity
of antibody and the cell-killing ability of bacterial toxin to realize tumor specific
treatment
[27]
. Normally, two types of exotoxin are used in making immunotoxins,
including diphtheria toxin (DT) and Pseudomonas aeruginosa exotoxin A (PE), both
effectively inhibit protein synthesis and trigger cell death when bounded to cells
[28,29]
. The recombinant immunotoxin αFAP-PE38 revealed positive results in mice
breast cancer model. However, the production of neutralizing antibodies to
4

immunotoxin has largely limited its efficacy
[26]
. To lower the immunogenicity of
immunotoxin αFAP-PE38, we introduced a mutant PE38 with seven T-cell epitopes
modified, which is required for the generation of neutralizing antibodies
[30]
.
PD-1 Blockade
Program death 1 (PD-1) is a 288-amino acid transmembrane protein discovered
in 1992 in a T cell hybridoma undergoing cell death
[31]
. PD-1 receptor is required in
the induction and maintenance of T cell tolerance, and its ligand PD-L1 can block
PD-1 pathway and inhibit T cell response, protecting tissues from immune-mediated
cell death. The tumor cells have taken advantage of PD-1: PD-L1 pathway to evade
tumor immunity
[32]
. PD-1 significantly affects the production of cytokines that
associated with immune stimulation and cell death, such as IFN-γ, TNF-α, and IL-
2. Additionally, PD-1 inhibits the expression of transcription factors related to
effector cells, e.g., Tbet, Eomes and GATA-3
[33]
. Due to the crucial role that PD-1:
PD-L1 pathway plays in cancer immunity tolerance, several antibodies targeting PD-
1: PD-L1 pathway has been adopted as new cancer therapy
[34-36]
. Clinical trials show
that Anti-PD-1 Antibody is a promising drug for non-small cell lung cancer,
melanoma, and renal-cell cancer
[37]
.
5

OBJECTIVE OF THE PROJECT
In this study, we improved the recombinant immunotoxin αFAP-PE38 by using
a mutant exotoxin PE38 with less immunogenicity
[30]
. The cytotoxicity of the new
immunotoxin is examined by cytotoxicity assay in FAP-expressing 293T cell line.
Antitumor efficacy of the immunotoxin is tested in mouse B16 melanoma xenograft
model. We also investigated the molecular mechanism of immunotoxin on both
mRNA and protein level. Finally, considering the functional similarity of TAFs and
PD-1 in cancer immunity, we explored the anti-tumor efficacy of the combination
therapy of mutant immunotoxin αFAP-PE38 and Anti-PD-1 antibody in mouse
melanoma xenograft model (Fig. 1).

Fig. 1. Combination treatment of αFAP-PE38 and Anti-PD-1 antibody
6

MATERIAL AND METHODS
Mice and Cell Line
C57BL/6 mice were purchased from Charles River Laboratories (Wilmington,
MA). All animal experiments were completed following the guidelines set by NIH
and the University of Southern California on the Care and Use of Animals.
HEK293T and B16-F10 cells were purchased from ATCC (Manassas, V A). The
culture medium is prepared with high-glucose Dulvecco’s Eagle medium (Hyclone,
Logan, UT) adding 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO) and L-
glutamine (Hyclone Laboratories, Omaha, NE).
Plasmid Construction and Protein Production
The sequence of mutant Pseudomonas exotoxin A (PE38) fused with species-
cross reactive FAP-specific scFv were cloned into the pET-28a(+) vector. The
plasmid is transformed into Escherichia coli BL21. Then the E. coli were cultured
in Luria-Bertani (LB) liquid medium with 100 μg/ml kanamycin at 37℃. Isopropyl-
b-D-1-thiogalactopyranoside (IPTG, 1 mM) is added to induce the protein
production when the OD
600
reached 0.6. Cell were harvested 4 hrs after IPTG
induction, then the targeted protein αFAP-PE38 were isolated as inclusion bodies.
We renatured the protein by dissolving the inclusion bodies in 8M urea and gradually
changing buffer to 4M urea, 2M urea, and finally PBS.
7

Protein Purification
The αFAP-PE38 protein was purified by his-tag affinity chromatography using
HisTrap HP column (GE Healthcare, Uppsala, Sweden) on ÄKTA start purification
system (GE Healthcare, Uppsala, Sweden). The column and system were firstly
washed with distilled water, then equilibrated with binding buffer. The sample was
applied with 5 mL syringe. After washing with binding buffer in 10 column volumes,
the protein was eluted with a linear gradient elution buffer in 20 column volumes.
Cytotoxicity Study
We used a commercial kit from Roche Scientific, standard XTT
assays to examine the cytotoxicity of mutant αFAP-PE38 immunotoxin in cultured
cells. The HEK293T cells were transfected with lentivirus plasmid and the lentivirus
vectors were assembled and secreted into the medium. Then the HEK293T cells
were treated with lentivirus vectors taking FAP genes and we got FAP-expressing
HEK293T cell lines. The cells were plated on 96-well dishes 1 day before XTT assay
treatment. The mutant αFAP-PE38 immunotoxin treatment was performed on day 2
and XTT assay detection on day 4. PBS was used as 0 % cell death as control. The
OD values were normalized between 100% cell death and PBS controls (0% cell
death).

8

Pharmacokinetic Test
C57BL/6 mice (n = 3 per group) were injected intravenously with 10µg of either
original or mutant αFAP-PE38. The blood was obtained by retro-orbital bleeding at
several intervals between 2 and 60 minutes from the time of injection. The
concentration of immunotoxin in blood is tested by His-Tag ELISA Detection Kit
(GenScript, Piscataway, NJ).
Mouse Xenograft Antitumor Activity Study
Female C57BL/6 mice (n = 5 per group) were injected subcutaneously with
2×10
5
B16-F10 cells on the right flank. Tumor treatment was started 10 days after
tumor inoculation. Mutant αFAP-PE38 protein and anti-PD-1 antibody were diluted
with 0.9% NaCl to 0.5 mg/kg then administrated to mice through i.v. injection every
2 days. Tumor size was measured every time before injection and calculated by
following equation: volume = (L×S
2
)/2, where L is the long dimension of tumor and
S is the short dimension. Survival end is set when the tumor size reaches 1,000 mm
3
.
Flow Cytometry Analysis
Tumor tissue were harvested from treated mice. The tissue is minced to get
single suspended cells and filtered by 0.7 μm nylon strainers (BD Falcon, Franklin
Lakes, NJ). The cells were first washed with cold PBS for twice times and incubated
9

by anti-mouse CD16/CD32 mAbs (BD Biosciences) in 4℃ for 10 min. Then various
monoclonal antibodies conjugated with fluorescent dyes (purchased from
eBioscience or BioLegend) were used in cell incubation, including anti-CD45, anti-
CD3, anti-CD4, anti-CD8, anti-F4/80, anti-CD206, anti-PD-1, anti-CD25, anti-
FoxP3, anti-CD11b, and anti-Gr-1. CD4 cells were identified by CD45
+
CD4
+

markers; CD8 cells were identified by CD45
+
CD8
+
markers; Tregs were identified
by CD45
+
CD4
+
CD25
+
Foxp3
+
markers; MDSCs were identified by
CD45
+
CD11b
+
Gr-1
+
markers. Data were acquired using MACSquant cytometer
(Miltenyi Biotec, San Diego, CA).
RNA Isolation and Transcripts Analysis by qRT-PCR
Total tissue RNA was extracted from mice flank tumor using RNesay Mini Kit
(QIAGEN, Hilden, Germany). The cDNA was synthesized from total RNA using
High Capacity cDNA Transcription Kit (Applied Biosystems, Foster City, CA).
Each PCR sample has a volume of 20 μl including 4 μl distilled water, 4ul Primers
(2M), 2μl cDNA template (75ng/μl) and 10ul 2X Power SYBR Green PCR Master
Mix (Applied Biosystems, Foster City, CA). The qPCR is conducted on 7300 Real
Time PCR System (Applied Biosystems, Foster City, CA) follow the guideline.
Gene expression level is calculated by ΔΔCt method, and the results were
normalized with the reference gene of GAPDH.

10

RESULTS
Construction and Purification of Mutant αFAP-PE38
To reduce the immunogenicity of immunotoxin αFAP-PE38, we introduced a
mutant Pseudomonas Exotoxin A (PE38) into our new immunotoxin, which has a
region deleted and seven epitopes modified (Fig. 2B). The variable regions of the
heave chain and light chain of anti-FAP antibody (αFAP) were linked with mutant
Pseudomonas Exotoxin A (Mutant PE38) with CD8α hinge region (Fig. 2A). A his-
tag was fused to αFAP for later protein purification. The sequence of mutant αFAP-
PE38 was cloned into the pET-28a(+) vector and the plasmid was transformed into
Escherichia coli BL21 for protein expression. After raw proteins obtained from the
renaturation of inclusion body, we purified mutant αFAP-PE38 using His-tag affinity
chromatography. Western blot was conducted to verified the molecular weight of
target proteins (Fig. 2C)
Binding Affinity between Immunotoxins and FAP-expressing Cells
To examine the binding affinity and specificity of mutant αFAP-PE38 to the
FAP-expressing cells, we generated a FAP-expressing 293T cell line through
transduction with lentiviral vectors encoding murine FAP (mFAP) and human FAP
(hFAP) genes. Flow cytometry with anti-FAP antibody was used to verify the
expression of FAP in these two cell lines. Then we analyzed the binding affinity of
11

both original and mutant αFAP-PE38 to mFAP- or hFAP-expressing 293T cells using
flow cytometry-based assay. The K
D
of the interaction between immunotoxins and
FAP was calculated by Lineweaver–Burk kinetic analysis [38]. The K
D
of original
αFAP-PE38 against mFAP and hFAP were 1.77±0.2×10
-9
and 7.27±0.3×10
-9
, while
the K
D
of original αFAP-

Fig. 2. Construction and Purification of mutant α-FAP-PE38. (A) Schematic representation of
mutant α-FAP-PE38 coding region in pET-28a(+). (B) Ribbon drawings of the original α-FAP-
PE38 and the mutant α-FAP-PE38. (C) SDS-PAGE of purified immunotoxins. Lane 1, purified
original aFAP-PE38 (75kDa) after His-tag affinity chromatography; Lane 2, purified mutant aFAP-
PE38 (53kDa) after His-tag affinity chromatography.
12

PE38 against mFAP and hFAP were 2.41±0.6×10
-9
and 7.77±0.4×10
-9
, indicating
that mutant αFAP-PE38 has the same high binding affinity to FAP as original αFAP-
PE38 (Fig. 2).

Fig. 3. The KD value of the interaction between original/mutant αFAP-PE38 and cell-surface
mFAP/hFAP, as determined by Lineweaver-Burk analysis. All the assays were conducted in
triplicate for each cell line. Data are representative of mean±SEM.

13

Cytotoxicity of Original and Mutant αFAP-PE38
The cytotoxic effect of αFAP-PE38 is evaluated by performing XTT assay on
FAP-expressing 293T cells in vitro. Cells were cultured in 96 wells plate and
incubated with original and mutant αFAP-PE38 for 48 h. Then XTT reagent was
added in to detect the cell death rate. A 293T cell line without FAP expression was
used as control. The half maximal inhibitory concentration (IC
50
) of original αFAP-
PE38 to mFAP- and hFAP-expressing 293T cells were 5.5ng/ml and 67ng/ml, while
the IC
50
of mutant αFAP-PE38 to mFAP- and hFAP-expressing 293T cells were
255ng/ml and 3.18ug/ml, indicating that mutant αFAP-PE38 has a lower cytotoxicity
toward FAP-expressing cells (Fig. 3).

Fig. 4. The cell cytotoxicity of original/mutant αFAP-PE38 against 293T, 293T-mFAP and 293T-
hFAP cells was performed by a standard XTT assay with a 48-hr treatment procedure. Data are
given as an IC50 value, the concentration of immunotoxin that causes a 50% inhibition of cell death
after a 48-hr incubation with immunotoxin. All the assays were conducted in triplicate for each
cell line. Data are representative of mean±SEM.

14

Mutant αFAP-PE38 Treatment Slows B16 Melanoma Growth in vivo
The antitumor efficacy of mutant αFAP-PE38 is tested in a B16 melanoma
mouse model. Firstly, B16 cells were inoculated to the right flank of BALB/c mice
to induce tumor growth. Approximately 8 days later, the average tumor size reached
50 mm3 and the treatment was started. Mice were separated as 4 groups (n = 5) and
each group was given five sequential intravenous injection every two days with PBS,
original αFAP-PE38 (0.5mg/kg), mutant αFAP-PE38 (0.5mg/kg) and mutant αFAP-
PE38(1.5mg/kg), respectively. The tumor size and body weight were measured over
time. Mice in all immunotoxin treated groups showed significant tumor suppression
efficacy. Additionally, low-dose mutant αFAP-PE38 (0.5mg/kg) and original αFAP-
PE38 (0.5mg/kg) revealed similar capability in slowing down the tumor growth.
High-dose mutant αFAP-PE38 (1.5mg/kg) group showed a little improvement in
antitumor efficacy than low-dose group. No significant body weight loss was
detected over time.
In addition, combined treatment of mutant αFAP-PE38 and anti-PD-1 antibody
showed significant antitumor effect on tumor development, while the single
treatment of mutant αFAP-PE38 or anti-PD-1 antibody showed less antitumor
efficacy.

15

Fig. 5. Antitumor efficacy of original/mutant αFAP-PE38 in B16-F10 tumor-bearing mice. Effect
of original/mutant αFAP-PE38 on the growth of established B16-F10 melanoma cancer model.
Female C57BL6 mice were inoculated s.c. with 2×105 B16-F10 cells in the right flank and then
treated with original αFAP-PE38 (0.5 mg/kg) or mutant αFAP-PE38 (0.5 mg/kg)/(1.5 mg/kg) 10
days after tumor implantation through i.v. injection for total of five times at the indicated days.
Tumor volume (A) and body weight was monitored every 2/3 days posttreatment. Error bars,
average tumor volume±SEM, n=3 for each treatment group.

16

Pharmacokinetics Test
The pharmacokinetic test was conducted on female C57BL/6 mice (n = 3 per
group). After the injection of samples, blood was obtained from mice at the timepoint
of 2, 5, 10, 20, 30, 60 min and the concentration of immunotoxin was determined by
His-tag ELISA. We have observed that mutant αFAP-PE38 has a shorter half-life
time than original αFAP-PE38.

Fig. 6. Pharmacokinetics of original/mutant αFAP-PE38. CL57BL6 mice were injected
intravenously with 10µg of either original or mutant αFAP-PE38 and bled at several intervals
between 2 and 60 minutes from the time of injection. The concentration of immunotoxin in the
plasma at the various intervals was determined by His-Tag ELISA and fit to a single exponential
decay function. The corresponding half-life (t1/2) is indicated.

17

Microenvironment Altered by Combine Treatment of Mutant αFAP-PE38
and PD-1 blockade
Tumor stromal cells plays a vital role in tumor development by producing
different types of cytokines, chemokines and growth factors which supports
tumorigenesis and metastasis. It is reported that depletion of FAP-expressing stromal
cells leads to rapid hypoxic necrosis of tumor cells with the involving of TNF-α and
IFN-γ [19]. Additionally, PD-1 affects the production of cytokines that associated
with immune stimulation and cell death, such as IFN-γ, TNF-α, and IL-2. This
prompted
us to generate a hypothesis that the combination of PD-1 blockade and mutant αFAP-
PE38 treatment will increase antitumor activity and immune response. To test this
hypothesis, we investigated the immune cells ratios in mouse tumor tissue using flow
cytometry. The combine group showed a significant increase in all ratios including
CD4/Treg, CD8/Treg, CD4/MDSC, CD8/MDSC.

18

Fig. 7. Combination treatment of mutant αFAP-PE38 and anti-PD-1 antibody enhanced tumor-
infiltrating T cells and increased the ratio of T effector cells versus Treg cells or myeloid-derived
suppressor cells within tumor. Cells were obtained from mice tumor tissue 3 weeks after B16
melanoma cells injected. Flow cytometry was conducted on MACSquant cytometer.
19

Cytokine Alteration on Transcriptional Level
The mRNA expression of cytokines, including ICOS, IL-12p35, IL-12p40,
TNF-α, TGF-β and Perforin, was examined by RT-qPCR. For ICOS, TNF-α, IL-
12p40 and Perforin, similar results have been observed that both PD-1 blockade and
mutant αFAP-PE38 groups improved the expression of these cytokines and the
combine group showed the highest expression. For IL-12p35, combine group
showed a significant increase in its expression while no significant difference was
observed between other groups and control group. For TGF-β, no significant change
was observed among four groups.

Fig. 8. Transcription expression of ICOS, IL-12p35, IL-12p40, TNF-α, TGF-β and Perforin
within tumor tissue. Except for TGF-β, combination treatment group has a significant increase in
the mRNA expression of other cytokines when compared with control and single treatment
groups.
20

DISCUSSION
In this study, we developed an FAP-targeting immunotoxin mutant αFAP-PE38.
The immunotoxin is composed of the FAP antibody and a mutant PE38 with lower
immunogenicity. We constructed and expressed the immunotoxin mutant αFAP-
PE38 in E. coli and then purified it using His-tag chromatography. The affinity
between mutant αFAP-PE38 and FAP-expressing cells is confirmed by flow
cytometry-based assay. Then XTT assay is used to demonstrated the cytotoxicity of
the mutant immunotoxin. We compared the pharmacokinetic attribute of the original
and mutant immunotoxin by His-tag ELISA. Additionally, we examined the
antitumor efficacy of the mutant αFAP-PE38 by tumor challenge in mice melanoma
model. Finally, the results from flow cytometry and RT-QPCR showed that the
combine treatment of mutant αFAP-PE38 and anti-PD-1 antibody enhanced the
antitumor activity through the increase of immune response.
Recently, tumor stromal cell is emerged as a potential target for cancer therapy
[11]
. It is indicated that tumor stromal cells play a vital role in the formation of tumor
microenvironment. Furthermore, the tumor stromal cells are much more genetically
stable than tumor cells, which makes it a good target for cancer treatment.
FAP is highly expressed on the surface of tumor associated stromal cells in most
of epithelial cancers. It is also considered as an important regulator in tumorigenesis
and cancer progression
[14]
. A former FAP-targeting immunotoxin αFAP-PE38 has
21

been designed to suppress tumor development
[26]
. However, its antitumor efficacy
is greatly affected by its high immunogenicity. To solve this problem, we introduced
a mutant PE38 to create a new immunotoxin. The mutant PE38 has the domain II
deleted and seven epitopes mutated, which were related to T-cell responses (Fig. 1).
Our data showed that this new immunotoxin can bind to FAP-expressing cells with
high affinity as the original αFAP-PE38 (Fig. 2). The cytotoxicity of mutant αFAP-
PE38 is tested by XXT assay in vitro. The mutant αFAP-PE38 showed a reduced
cytotoxicity comparing with the original immunotoxin (Fig. 3). The decrease in
cytotoxicity is probably due to the deletion of domain II of PE38
[39]
.
Mouse xenograft antitumor activity is examined in C57BL/6 melanoma mice
model. Both original and mutant αFAP-PE38 showed strong capability to suppress
the development of tumor (Fig. 4a). There is no significant difference observed
between the antitumor efficacy of original and mutant αFAP-PE38, which indicated
that the antitumor activity is not affected by the modification of exotoxin PE38 in
mutant αFAP-PE38. Additionally, the results showed that the combine treatment of
mutant αFAP-PE38 and anti-PD-1 antibody enhanced antitumor efficacy
significantly, which verified our hypothesis that there is a synergy effect between
mutant αFAP-PE38 and PD-1 blockade.
It is well accepted that tumor microenvironment, including growth factors,
cytokines, chemokines and matrix metalloproteinases, plays an important role in
tumorigenesis, angiogenesis and metastasis
[40]
. To understand the influence of the
22

combine treatment on tumor microenvironment, we investigated immune cells ratios
in mouse tumor tissue using flow cytometry. The percentage of CD4 and CD8 T cells
in tumor infiltrating lymphocytes (TILs) were significantly increased in combine
treatment group, suggesting that the combination treatment has probably enhanced
the immune cell infiltration and antitumor immune response. The results also
showed that the ratios of CD4/Treg, CD8/Treg, CD4/MDSC, CD8/MDSC were
increased in combination treatment group. The ratio of CD8/Treg were reported to
be linked with tumor progression and therapeutic outcomes in human and mice
[41,42]
.
The MDSCs are considered as an immune suppressor in tumor microenvironment
that can reduce T-cell response toward tumors
[43]
.
Furthermore, RT-qPCR was conducted to investigate the mRNA expression of
cytokines in tumor tissue. IL-12 is the stimulator of T-cell growth and function. It
also stimulates the production of TNF-α and IFN-γ. TNF-α participates in the
induction of inflammation and apoptotic cell death, which inhibits tumorigenesis.
Perforin is secreted by killer cells that form transmembrane pore on targeting cells.
ICOS, like IL-12, is a T-cell stimulator. Our data showed that the mRNA expression
of IL-12p35, IL-12p40, TNF-α, Perforin and ICOS were significantly increased in
the tumor tissue obtained from combine treatment group. Meanwhile, the mRNA
expression of TGF-β kept same in all groups. TGF-β is a cytokine belonging to
transforming growth factor superfamily. It is reported that the increased level of
23

TGF-β often correlates with the malignancy of various cancer types and indicate a
defect in the cellular growth inhibition response to TGF-β.
During the experiment, we noticed an intriguing phenomenon that the mice
treated with mutant αFAP-PE38 showed less side effects than the mice treated with
the original αFAP-PE38. It may indicate that the mutant αFAP-PE38 has less
systematic toxicity than the original one, while it sustains the same antitumor activity.
Thus, we plan to examine the systematic toxicity of both immunotoxins by testing
the LD
50
on mice.
In conclusion, our study has designed a new FAP-targeting immunotoxin
mutant αFAP-PE38. Comparing with the original immunotoxin, the mutant αFAP-
PE38 has less immunogenicity and better antitumor efficacy. We tested the combine
treatment of Anti-PD-1 antibody and mutant αFAP-PE38, which showed a
significant increase in antitumor activity. We also investigated the mechanism of
combine treatment on cell level and transcription level.

24

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Abstract (if available)

Abstract Fibroblast activation protein (FAP) is highly expressed in Tumor associate fibroblasts (TAFs) in most of human epithelial cancers and the TAFs play an important role in tumorigenesis, which makes FAP a promising target for cancer treatment. In previous study, a FAP-targeting immunotoxin aFAP-PE38 has been designed to deplete TAFs in mouse cancer model. However, this treatment is largely limited by the early production of neutralizing antibodies to immunotoxin. Thus, we designed a new immunotoxin aFAP-PE38 with mutant exotoxin PE38 which has less neutralizing epitopes and less immunogenicity. We test the new immunotoxin on tumor cell lines and mice B16 melanoma model. The results indicate that the mutant immunotoxin has a stronger antitumor efficacy and less cytotoxicity than the original one. We also examine the combined treatment of mutant immunotoxin and PD-1 blockade, which shows a synergy effect on the activation of immune system, leading to the suppression of tumor development.

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Asset Metadata

Creator Liu, Baiyang (author)

Core Title A mutant recombinant immunotoxin αFAP-PE38 for melanoma treatment in mice

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publication Date 09/15/2017

Defense Date 07/20/2017

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

Tag immunotoxin, fibroblast activation protein, tumor-associated, fibroblast, melanoma, tumor microenvironment, PD-1 blockade,OAI-PMH Harvest

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Danenberg, Peter (committee chair), Wang, Pin (committee chair), Xu, Jian (committee member)

Creator Email Baiyang.Liu@rice.edu,lby921207@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-428997

Unique identifier UC11264492

Identifier etd-LiuBaiyang-5725.pdf (filename),usctheses-c40-428997 (legacy record id)

Legacy Identifier etd-LiuBaiyang-5725.pdf

Dmrecord 428997

Document Type Dissertation

Rights Liu, Baiyang

Type texts

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

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA