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Tri-specific T cell engager immunotherapy targeting tumor initiating cells

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Tri-specific T cell engager immunotherapy targeting tumor initiating cells

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Content Copyright 2022 Nuan Wang
Tri-specific T cell engager immunotherapy targeting tumor initiating cells

By

Nuan Wang

A Thesis Presented to the
FACULTY OF THE USC KECK SCHOOL OF MEDICINE
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(MOLECULAR MICROBIOLOGY AND IMMUNOLOGY)

August 2022

ii
Acknowledgments

I want to first appreciate Dr. Keigo Machida for accepting me as a member into his laboratory
and taught me everything of the lab; he provided continuous support throughout all my master
program. I also want to appreciate Dr. Choi and Carlos for teaching me the operations of the
ent and training me in various experiments. Lastly, I would like to appreciate Dr.
Alan Epstein and Dr. Weiming Yuan For their guidance and supports.

iii
Table of Contents
Acknowledgements ........................................................................................................................ ii
List of Tables ................................................................................................................................ iv
List of Figures ..................................................................................................................................v
Abstract .......................................................................................................................................... vi
Chapter 1: Introduction ....................................................................................................................1
Chapter 2: Materials and Methods ...................................................................................................4
Chapter 3: Specific Aims ...............................................................................................................11
Chapter 4: Results and Discussion .................................................................................................12
Chapter 5: Summary ......................................................................................................................26
Chapter 6: Future Directions ..........................................................................................................27
References ......................................................................................................................................31

iv
List of Tables
Table 1: groups of anti-TIC ScFv FACS assay ...............................................................................8
Table 2: Primers of candidate genes for RT-qPCR .......................................................................10
Table 3: sequence of shRNA targeting the candidates ........................................................... 10-11
Table 4: The input volume and outcome volume of every round of group one ............................15
Table 5: the input volume and outcome volume of every round of group two .............................15
Table 6: summary location, alternation, and number of alternation of candidate gene in TCGA
data
........................................................................................................................................................19

v
List of Figures
Figure 1: structure and mechanism of TriTE ...................................................................................4
Figure 2: The overview of phage display library screening ............................................................7
Figure 3: flowchart of transduction of lentivirus into Huh7 cells ..................................................11
Figure 4: The immunohistochemistry of CD3 and CD14 in tumor inflammatory liver tissue ......13
Figure 5: The immunohistochemistry of CD3 and CD14 in non-tumor liver tissue .....................13
Figure 6: the positive percentage of CD3 and CD14 surrounding the inflammatory tumor liver
tissue and non-tumor liver tissue
........................................................................................................................................................14
Figure 7: The FACS assay results ..................................................................................................16
Figure 8: The PCR result (the band indicated by the red arrow is the PCR product) ................... 17
Figure 9: The Western-blot result of TRITE .................................................................................18
Figure 10: multi-gene summary of target candidate of TriTE on surface of Huh7 CD133+ TIC
.................................................................................................................................................. 20-23
Figure 11: qRT- CD133+ Huh7 cell line comparing to CD133- Huh7 cell line
....................................................................................................................................................... 25
Figure 12: In vivo experiment on comparing TriTe killing ability with BiTE and single anti-CD3
ScFv
........................................................................................................................................................29
Figure 13: Knockout candidate gene on PDX tissue that implanted on mouse model ..................30

vi
Abstract
Hepatocellular carcinoma (HCC) is a malignancy of hepatocytes that develops in the liver.
Patients with a history of liver illness, such as liver fibrosis and cirrhosis, develop HCC. About
80% of all liver cancers are HCC, making it one of the most prevalent malignancies worldwide.
Liver tumor initiating cells (TICs) are responsible for liver tumor formation, metastasis,
medication resistance, and relapse. As a result of their capacity for self-renewal and
differentiation, liver TICs can recur and produce new tumors once medication treatment is
stopped. CD133, wnt/beta-catenin, and nanog are stemness genes expressed by both TICs and
pluripotent stem cells. CD133, a glycoprotein with a 5-transmembrane domain, is expressed in
both normal and malignant tissues. In recent years, cancer immunotherapy has expanded
significantly in terms of agents that give a prognosis benefit by stimulating the immune system
to mount a response against growing tumors, but have been unsuccessfully explored for decades
in HCC. Tri specific T cell engager protein (TriTE) is created based on BITE in this study. When
attached to a tumor cell, TriTE has the ability to attract two distinct types of immune cells to
enhance cytotoxicity. The previous work of our lab screened out a series of DNA sequences
generating single-chain antibodies with strong binding affinity to tumor initiating cells (TIC) and
confirmed the antibody's high affinity for CD133+ Huh7 cells. The TriTE was designed to
incorporate sequences encoding single-chain anti-human CD14, anti-human CD3, and anti-TIC
antibodies. It connects human tumor initiating cell, myeloid cell, and T-lymphocyte by acting as
a "bridge." We hypothesized that the design of Tri-specific T cell engager protein (TRITE) with
specific binding capacity of myeloid cell, T cells, and TICs will increase the efficiency of
immunotherapy against HCC. In our study, we identified four candidates for the binding target of
anti-TIC ScFv of Trite on TICs. These candidates were validated by multiple bioinformatics
vii
analyses and quantitative reverse transcription polymerase chain reaction (qRT-PCR), which
demonstrated that their expression was higher in tumor initiating cells than in non-tumor
initiating tumor cells. In subsequent investigations, we will evaluate the specific binding target of
anti-TIC ScFv of TriTE on TIC using FACS analysis on knockdown cell lines and knockdown
mice models. Using a TUNEL assay, we'll then determine the TriTE killing mechanism. Then,
we will evaluate the cytotoxicity of TriTE using an in vitro FACS analysis and a myeloid cell
depletion test, as well as an in vivo mice model including BiTE and a single anti-CD3 ScFv.
Finally, in vivo and in vitro toxicity tests will be conducted on TriTE.

1
Chapter 1: Introduction
Hepatocellular carcinoma (HCC) is a malignancy of hepatocytes that arises within the liver.
HCC occurs in patients with background of liver disease such as liver fibrosis and liver cirrhosis.
HCC accounts for approximately 80% of all liver cancers and is one of the most prevalent
malignancies worldwide.
[1]
The survival rate of patients with primary liver cancer and
intrahepatic bile ducts is between 2-7%.
[2]
Liver transplantation is the preferred surgical
treatment for HCC, but due to a lack of organ donors, the majority of patients are instead treated
with partial hepatic resection.
[3]
Despite advancements in surgery and patient care, the 5-year
survival rate is approximately 40 to 50 percent
[4]
In addition, the cumulative 5-year recurrence
rate is between 75% and 100% and HCC ranks as the second leading cause of cancer death
worldwide.
[5]
Thus, HCV-associated HCC stays an incurable malignancy and is an urgent unmet
medical need. These tumors are comprised of a heterogeneric mix of different cells but most
importantly , a subset of cell can generate new tumors, called tumor initiating cells (TICs). Liver
TICs account for liver tumorigenesis, metastasis, drug-resistance and relapse. With self-renewal
and differentiation capacities, liver TICs can relapse and generate new tumors when cytolytic
drugs are no longer being needed.
[6]
TICs express stemness genes that are also expressed in
pluripotent stem cells, such as CD133, wnt/beta-catenin, nanog. CD133, a 5-transmembrane
domain glycoprotein, expresses in normal and tumor tissues. The function of CD133 is not
entirely known yet, however, it has been used as a marker of TICs in many solid tumors,
including liver cancer.
[7]
CD133 has been used as a surface marker of Huh7 TICs because of
highly expressesed in Huh7 cells.
[8]
In the clinic, there are a number of anti-CD133+ ScFv
conjugated anti-tumor medicines with FDA approval that demonstrate high anti-tumor therapy
efficacy.

2

In recent years, cancer immunotherapy has expanded quickly in terms of agents that improve
prognosis by stimulating the immune system to develop a response against developing
malignancies, with particular success in metastasis melanoma. Immunotherapeutic techniques
are now based upon two fundamental tenets: the capacity to unmask current immune
responses; and the capacity to trigger new or alternative immune responses. Immune therapies
have been continuously but unsuccessfully studied for decades in HCC. Sorafenib (Nexavar), the
first clinically approved targeted drug therapy for HCC, was only able to increase overall
survival by two to three months. Additionally, many patients permanently withdraw from
treatment because of severe skin toxicity.
[9]
Later, there are many first line drugs and second line
drugs for advance stage of HCC, such as tyrosine kinase inhibitors, Regorafenib and Lenvatinib,
or checkpoint inhibitors, Anti-PD1, Anti-PDL1, and Anti-CTLA4, but even with these advance
drugs, the survival rate of patients with advance stage of HCC is still between 8 and 13
months.
[11][12]
Consequently, there remains an unmet need for tolerable, life-prolonging
strategies for patients in the management of HCC.
Bispecific T cell engagers (BITEs) have shown great promise for the immunological treatment of
cancer. This reagent has been explored as a new means to recruit cytotoxic T cells to kill targeted
tumor cells. In 2014, the FDA approved the first BiTE antibody, anti-CD19-CD3 BiTE
blinatumomab (BlincytoTM), for the treatment of patients with Philadelphia chromosome-
negative relapsed/refractory B cell precursor ALL, indicating that BiTE is indeed a promising
option for treating this type of malignancy.
[10]
However, there is no comparable progress of its
applications in solid tumors has been proven beneficial yet because T cells cannot easily
penetrate and survive in the tumor microenvironment.

3

Tri specific T cell engager protein (TriTE) is designed in this study based on previous studies on
the development of BITEs. In order to achieve higher cytotoxicity, TriTE was designed
to recruit two distinct types of immune cells when linked to a tumor cell. Our previous research
identified a series of DNA sequences encoding TIC specific single-chain antibodies that bind to
CD133+ Huh7 cells. TriTE was designed with sequences encoding anti-human CD14, anti-
human CD3, and anti-TIC single chain antibodies based on this research (Figure 1). It functions
a human tumor initiating cell, a myleoid cell and a T-lymphocyte together.
With presence of Antigen presenting cells (APC), the TriTE could be able to work in two kinds
of mechanism: direct tumor killing, and indirect tumor killing. In direct tumor killing, the CD4+
T cell will directly produce granzyme perforin killing the tumor, and in the indirect killing, the
there are IFN-gemma, TNF and IL-2 produced by CD4+ T cell binding to APC to activate B cell,
inhibite angiogenesis, and tumor senescence, and activate microphages and recruit CD8+
cells.
[15]
In recent decades, tumor associated macrophages (TAMs) have gained a lot more
interest because their remarkable capacity to either inhibit or promote tumor formation. In many
malignancies, TAM invasion is typically related with poor prognosis and short survival.
Numerous studies have shown that a higher density of M2-type macrophages correlates with
enhanced tumor cell proliferation, vascularity, immune suppression, treatment resistance,
induced histological malignancy, and a poor prognosis. Due to their heterogeneous character, it
should be evident that the impact of TAMs on tumor formation may fluctuate and be determined
by the diversity of the TME. TriTE may be able to recruit more M1 type macrophages to produce
more IL-12 in the tumor microenvironment, thereby converting M2 type macrophages into M1
type macrophages to limit tumor growth in the presence of anti-CD14.
[14]
We anticipated that the
development of a Tri-specific T cell engager protein (TRITE) with the ability to attach

4

specifically to myeloid cells, T lymphocytes, and TICs will increase the efficacy of cell therapy
in targeting HCC.

Figure 1: Structure and mechanism of TriTE. (A)genetic structure of the TriTE formed by fusing anti-CD3 scFv (orange box) N
to the anti-TIC scFV (yellow box) and to anti-CD14 scFv (brown box).The anti-CD3 ScFv will recruit T cell and anti-CD14
ScFv will recruit myeloid cell when anti-TIC ScFv bind to tumor initiating cells.
In this study, we found four promising candidates as target ligand of anti-TIC ScFv of Trite to
TICs and validated this concept using bioinformatic analyses and qRT-PCR. We evaluated the
specific binding target of anti-TIC ScFv of TriTE on TIC by FACS analysis in vitro on
knockdown cell lines and in vivo on knockdown mouse models. The killing mechanism of TriTE
were quantitated by the TUNEL assay. TriTE cytotoxicity was verified in vitro by FACS
analysis and a myeloid cell depletion assay, and in vivo using a mouse model with BiTE and
single anti-CD3 ScFv as control. Lastly, we tested the toxicity of TriTE in vivo and in vitro.
1.B
1.A

5

Chapter 2: Materials and Methods
Immunohistochemistry
In prior studies, immunohistochemistry was done on paraffin-embedded tissue sections supplied
by the USC liver disorders research center. Human tissue samples were supplied by a patient
volunteer with HCC in HCR Los Robles Hospital who signed the informed permission form for
our laboratory's research. In this study, both HCC tumor tissue and normal hepatic tissue were
examined as tissue samples.

After removing the paraffin melt, all the slides were baked in an incubator at 37°C, and three
Coplin jars containing 90% xylene and six jars containing alcohol in concentration gradients
(100% alcohol, 95%, 75%, 50%, and 25%) were produced. After baking, the slides were
immersed for 10 minutes in 90 percent xylene solutions in each jar. The slides were then
rehydrated by soaking them for 5 minutes in alcohol solutions of decreasing concentration. The
slides were submerged in water for 15 minutes. The slides were then placed in a jar containing
Citrate buffer (2.94 g sodium citrate, 500 l of Tween 2.0 diluted in 1000 ml DW) and heated in
the microwave for 30 minutes to retrieve antigens. The slides were then removed and cleaned
with care using dry tissuedry. The slides were then placed in a blocking solution containing 5%
goat serum and 10% bovine serum albumin in PBS for 2 hours at room temperature before being
rinsed in DW. According the recommendation, primary antibodies against rat anti-human CD14
antibody and goat anti-human CD3 antibody (both acquired from Santa Cruz Biotechnologies)
were diluted twenty times in 10% PBS and 1% BSA solution. On the slide, the well-prepared
primary antibodies were deposited until the entire tissue was coated by the solution. All slides
were stored overnight in the cold room. On the second day, the slides were washed in DW and

6

treated with secondary antibodies mouse anti-rat antibody (Alexa Fluor 568
®
) and chicken anti-
goat antibody (FITC) by coating the tissue dropped on the slides to cover the entire tissue for a
two-hour incubation at room temperature in a light-tight box. The slides were then washed with
DW and DAPI was added to counterstain the nucleus. Indirect immunofluorescence microcopy
The USC Liver disorders research center's Cell and Tissue Imaging Core conducted the
microscope observation.

Phage Display Library screening
In prior study conducted by our laboratory, an array of filamentous phage M13 carrying multiple
types of single-chain antibody sequences was obtained. E. coli (Rosetta cells) is the host strain
for transfection, and the host strain was injected in LB medium on a shaking bed until mid-log
phase. Before transfection, the phage was pre-warmed to 37°C and tenfold diluted to 1x10
10
;
fast vortex and incubated
for 5 minutes. The mixture was then evenly distributed on LB plates and incubated overnight.

Purified Huh7 CD133+ TICs are cultivated in a sterile 12- transfected E. coli was injected to each well for binding for two hours, and unbound E. Coli cells
were washed in each culture medium. The bound E. Coli was collected and introduced to a new

7

well of grown purified Huh7 CD133(+) TICs culture, then the unbound E. Coli were removed by
washes, and this process was repeated six times.

Figure 2. The overview of phage display library screening. A huge amount of phages expressing antibodies was treated with
target ligands. After three hours, unattached phages were washed away and attached phages were eluted. The eluted phages were
used to infect C. component cells for amplification, after which they entered the subsequent screening cycle until all remaining
phages express antibodies with high specific binding activity.
Approximately 10
2
E. Coli with specialized binding capacity were harvested and delivered to the
Genome company for high-throughput DNA sequencing. The company deciphered the sequences
and returned anti-TIC ScFv sequences, which were subsequently utilized in our study.

FACS Analysis
In earlier laboratory research, anti-TIC binding ability was analyzed by using FACs. Four groups
were designed for the FACS assay. In the experimental groups, the purified anti-TIC ScFv
antibody was used to link to the Huh7 CD133(+) cell line or the Huh7 CD133(-) cell line. As the
second fluorescent antibody, the rabbit anti-6X His tag antibody combination with DyLight®488
(purchased from Abcam) was used. Human normal IgG antibody labeled with Alexa Fluor® 458
(purchased from Abcam) was utilized to bind to the human Huh7 CD133(+) cell line for the
negative control group. Goat anti-human CD133 antibody conjugated with FITC (purchased from
Abcam) was used to bind to Huh7 CD133(+) TICs as a positive control.

8

Huh 7 CD133(+) TICs were cultured in DMEM. In a 12-well plate, 10
7
co-cultured cells were
incubated for 24 hours. Next, the medium was removed and replaced with new medium in each
well for an additional 24 hours in the incubator. After 24 hours, the co-cultured cells were washed
with PBS using a scraper and their concentration was adjusted to 106/ml in cold FACS buffer (1
percent BSA, 0.1 percent NaN3, PBS). The cells were placed in a 10 ml conical vial and
centrifuged (1500rpm for 5 minutes at 4°C) at a speed of 1,500rpm. The supernatant was discarded,
and the remaining cells were PBS-washed and divided into three Eppendorf tubes.

The concentration of anti-TIC ScFv was determined using the Bradford assay. In each group, 30µg
of primary antibody was added, and then the volume was refilled with PBS to 300µl. All samples
were incubated in the dark at 4°C for 30 minutes. Next, the unbound antibodies were removed by
centrifugation (1500rpm, 5min, 3 times) and resuspended in 200µl of cold FACS buffer. Flow
cytometry was performed at the USC Liver Disease Research Center flow cytometry facility.

Group design Antibody Cell line
Positive control Goat anti-human CD133 antibody
conjugated with FITC
Huh7 CD133(+) cell line
Negative control Normal human IgG antibody
conjugated with Fluor
®
458
Huh7 CD133(+) cell line
Experimental groups Anti-TIC ScFv Huh7 CD133(+) cell line
Anti-TIC ScFv Huh7 CD133(-) cell line
Table 1 groups of anti-TIC ScFv FACS assay

9

Bioinformatic Analysis
Co-IP and MS based protein identification Analysis report (Creative proteomics) were analyzed
for possible candidates based on LFQ intensity and score. The score was derived from peptide
posterior error probabilities and LFQ Intensity which showed the relative abundance of protein
in each sample. Gene alternation and mRNA expression level of liver hepatocellular carcinoma
were compared with normal sample based on TCGA database. The Survival rate between
candidate gene alternation and normal sample are generated based on TCGA database. The gene
expressions of the candidate genes were analyzed based on HCCDB.

Cell culture
Huh7 cells are used in the study for the HCC TICs model, while 293T cells and ChoK1 cells
were used as transfected-target cells to help produce the shRNA construct and engager protein
TriTE. For H supplemented
with 10% FBS, 1% streptomycin antibiotics,1% penicillin, 1% glutamine and 1% non-essential
amino acids was used as the culture medium. For ChoK1 cells, DMEM/F12 supplied with 10%
FBS, 1% of streptomycin antibiotic, 1% penicillin, 1% glutamine and 1% non-essential amino
acid was used as culture medium. To sort the CD133+ Huh7 cells, CD133 microbeads from
Miltenyi Biotec were used.

qRT-PCR
Total RNA of CD133+ Huh7 cell, CD133- Huh7 cells, and primary human hepatocytes was
prepared by the Trizol procedure. RNAs were reverse transcript with oligo dT primers. Primers
used for PCR reactions are shown below and samples and controls were analyzed in triplicate.

10

The level of mRNA expression was normalized to the housekeeping gene, Gapdh. qRT-PCR
by the test.
Table 2: Primers of candidate genes for RT-qPCR
TBCE -AGGCCAACAGATGTTCTCCAG-
-CAGGGGGTTTCTTAGGCAGG-
XPO5 5'-CACAACAAGGAGAGGTGATGAG- 3'
5'-AAGGTGAGAAGACGGAACAGAG-3'
PEA15 -GTTCTGTAGTCAACCACCAT-
-ACCAACAACATCACCCTT-
CEP170 5'-ACAGGTGCAGGGCATGCTTCA-3'
5'-TCTACTCCAAACACAACAGCTTGGTC-3'
MRPL15 5'-TTGAACTCGCCAGGAAGTATG-3'
5'-CCTGGAGCAAGACCAAAGAA-3'

Construction shRNA
The shRNA targeting sequence was designed and synthesized as below. BamHI and EcoR1
digestion sites were introduced into the positive-sense and antisense strands, and annealed to
form a double-stranded structure. The annealed oligonucleotide fragment was cloned into the
pGreenPuro plasmid to establish the pGreenPuro shRNA lentiviral vector, which was then
transfected into competent cells. The positive recombinant plasmid DNA was identified by PCR.
Table 3: sequence of shRNA targeting the candidates
Name Sequence
TBCE

XPO5

CEP17
0

11

CTGACAG

MRPL
15

Transfection
Transfection of shRNA pgreenpuro vectors and TriTE. HEK-293T cells and CHOK1 cells were
plated in 6 cm dishes in fresh media and transduced with 0.3ml of supernatant containing
collected. The Ni-NTA beads was used for the His Tag binding purification process. The
lentivirus was transduced into Huh7 cells by adding 4ml of supernatant and polybrene and
incubated for 48-72 hours. Fresh medium is changed and puromycin was added for positive
selection for incubation for 24 hours. Knockdown efficiency is measured by qRT-PCR.

Figure 3: flowchart of transduction of lentivirus into Huh7 cells.

12

Chapter 3: Specific Aims
1. Evaluating the specific binding target of anti-TIC ScFv of TriTE to TICs in vitro and in
vivo
a. The previse work showed that anti-TIC ScFv has the potential to bind specifically to
TICs. The specific binding domain on the surface of TIC is still unknow. I propose to use
TICs lysis combined with anti-TIC ScFv plus FC part protein and identify its binding
domain via mass-spect technology. Through series of bioinformatic analyses determining
the final candidate of specific binding domains will be identified and tested by the
binding efficiency of TriTE on sh-candiadate genes Huh7 cell line to TriTE on normal
Huh7 cell lines.
2. Identifying the killing mechanism of TriTE
a. To understand the killing mechanism of TriTE, I purpose to use the TUNEL assay
to detect whether apoptotic cells that undergo extensive DNA degradation during
the late stages of apoptosis.
3. Evaluating the specific killing efficiency of anti-TIC ScFv of TriTE to TICs
a. FACS assay is used to detect live/dead cell after TriTE bind to Huh7 cell lines .
For these studies, rabbit anti-6x his tag antibody conjugate with Dylight 488 and
goat anti-human CD133 antibody conjugate with FITC and propidium iodide will
be used. Huh7 cells are cocultured with Jurkat cells or PBMCs in the presence of
different concentrations of purified TriTE and BiTE. After 24 hours, the surface
expression of the T cell activation marker CD69 was determined by FACS
analysis.

13

Chapter 4: Results and Discussion
The number and distribution of dendritic cells and T cells surrounding tumor inflamed liver
tissue are significantly greater than those surrounding non-tumor liver tissue

Figure 4. The immunohistochemistry of CD3 and CD14 in tumor-inflamed liver tissue. CD3 (green) and CD14 (red) fluorescence
confocal images of inflamed liver tissue. Scale car 50µm.

Figure 5. CD3 and CD14 immunohistochemistry in non-tumorous liver tissue. CD3 (green) and CD14 (red) fluorescence
confocal images of liver tumor tissue was shown. Scale car 50µm.

Anti-CD14

14

Figure 6. Positive proportions of CD3 and CD14 lymphocytes surrounding inflammatory liver tumor tissue and normal liver
tissue
In the immunohistochemical assay, the CD14 and CD3 positive percentages surrounding
inflammatory liver tumor tissue and nontumor liver tissue are compared. In "hot tumor" HCC
tissues, the percentages of positive staining for CD14 (34.6%) and CD3 (26.2%) are significantly
higher than for CD14 (18.2%) and CD3 (13.7%) in "cool tumor" HCC tissues, as determined by
the measurement of markers. These findings demonstrate that immune cells in "cold tumor"
microenvironments have limited ability and affinity to capture and identify tumor cells
efficiently, resulting in dysfunctional immune activation and immune response. The low number
of immune cells in "cold tumor" samples indicates the restriction of existing BiTE treatment and
supports our concept of stimulating the immune response by simultaneously connecting TICs
and T lymphocytes with antigen-presenting cells via the engager protein TRITE.

15

Phage-display library screening uncovered novel TIC-targeting immunoglobulin variable
chain segments.

Previously, a phage display library of human immunoglobulins was utilized to identify and isolate
TIC-specific antibodies in our lab's study. The top ScFv candidates were selected, cloned into
chimeric antigen receptor lentivirus, and transduced into T cells in order to test target cell lysis
assays. Two groups were designed with the same amount of phages as input. After six rounds of
screening, a cluster of phages containing the desired DNA sequences was obtained. The data
indicate that our lab successfully screened a volume of 1.5x1013 phages for phages carrying the
target DNA sequence. The DNA sequences that code for a protein with high-affinity binding to
human tumor-initiating cells are carried by phages derived from the fifth round.

Table 3 The input volume and outcome volume of every round of group one
Table 2 the input volume and outcome volume of every round of group two

16

The result of the FACS assay shows that anti-TIC ScFv binds specifically to Huh7 CD133(+)
TICs.

Figure 7. The FACS assay results. (A) anti-TIC ScFv binds to Huh 7 CD133(+) TICs and mouse anti-his tag antibody
conjugated with FITC was utilized to detect the positive binding percentage. (B) anti-TIC ScFv binds to CD133(-) TICs and
mouse anti-his tag antibody conjugated with FITC was utilized to detect the positive binding percentage. (C) Normal human IgG

17

antibody conjugated with Fluor® 458 binds to Huh7 CD133(-) TICs was utilized to detect the positive binding percentage. (D)
Goat antihuman CD133 antibody binds to CD133(+) TICs conjugated with FITC was utilized to detect the positive binding
percentage.
Figure 7A depicts the FACS results demonstrating the binding of anti-TIC ScFv to Huh7 CD133(+)
TICs. The gate was set in the R1 region, and the FITC signal peak is between 101 and 102 with a
cell account number > 170. Figure 10B demonstrates that anti-TIC ScFv binds to Huh7 CD133(-),
with the FITC signal peak located within 101 cells with an account number >175. Figure 7C
demonstrates the binding of human IgG immunoglobulin to Huh7 CD133(-) TICs. The signal peak
for Fluor® 458 is centered within 101, with the highest cell number being close to 200. Goat
antihuman CD133 antibody binds to CD133(+) TICs as depicted in Figure 7D, where the FITC
signal peak is placed beyond 102 and the greatest cell count is approximately 190. The results of
the FACS assay demonstrated that antihuman CD133 binds most strongly to Huh7 CD133(+) TICs.
The binding affinity of anti-TIC SCFv to Huh7 CD133(+) TICs is much higher than that of anti-
TIC ScFv and human IgG to Huh7 CD133(+) TICs. This research demonstrated that anti-TICs
ScFv exhibited a binding affinity for Huh7 CD133(+) TICs.
The PCR result confirmed the ligation process of the TRITE and pSecTag2 A vector

Figure 8. The PCR result (the band indicated by the red arrow is the PCR product)

18

After ligation, the insert was tested using polymerase chain reaction (PCR) for verification of the
ligation procedure. The forward and reverse primers were produced by the Idt DNA firm. The
band revealed a PCR product length between 2kbp and 3kbp, which corresponds to the length of
the TRITE sequence (2.3kbp). This result confirmed the ligation between the TRITE sequence
and the pSecTag2 A vector.
Western blot analysis revealed the expression of engager protein TRITE.

Figure 9. The Western-blot result of TRITE
The mouse anti-His tag antibody was used as the primary antibody in the western blot assay, and
the donkey anti-mouse antibody conjugated with FITC was used as the secondary antibody.
There is a band between 100KD and 150KD that matches the estimated molecular weight of
TRITE (143KD). Western blot results reveal that the target protein TRITE is expressed.

19

The result of Bioinformatic Analysis identifying the binding target of anti-TIC on the
tumor surface
Table 6: summary location, alternation, and number of alternation of candidate gene in TCGA data
Gene
Name
location Alternation # of alter total ratio
TBCE Intracellular, Required for correct
organization of microtubule
cytoskeleton and mitotic spindle, and
maintenance of the neuronal microtubule
network
amplification 59 749 7.877169
56
mutation 2 749 0.267022
7
XPO5 Nucleoplasm, Prognostic marker in liver
cancer (unfavorable) and endometrial
cancer (unfavorable),transportation
amplification 39 749 5.206942
59
mutation 9 749 1.201602
14
multiple
alternation
2 749 0.267022
7
MRPL15 Mitochondria, Ribonucleoprotein,
Ribosomal protein
amplification 43 749 5.740987
98
CEP170 Intracellular, Membrane (different
isoforms), Mitotic spindle, Centrosome,
Cytosol (Single cell variability, CCD
Protein),Plays a role in microtubule
organization 1. Required for centriole
subdistal appendage assembly
amplification 57 749 7.610146
86
mutation 9 749 1.201602
14

20

10.A

21

10.B

22

10.C

23

10.D

24

Figure 10A-D: multi-gene summary of target candidate of TriTE on surface of Huh7 CD133+ TIC. Diff: the number of
differentially expressed datasets; Red/Blue for consensus upregulated/down-regulated. Prognosis: the number of significant
datasets by survival analysis; Red/Blue for UNFavorable/Favorable. HCC/AllTumor: Red/Blue for positive/negative fold change
in log2 scale by comparing HCC with all tumors (TCGA data) HCC/AllAdjacent: Red/Blue for positive/negative fold change in
log2 scale by comparing HCC with all adjacent samples (TCGA data) HCC/Adjacent: Red/Blue for positive/negative fold
change in log2 scale by comparing HCC with adjacent samples (HCCDB data) Liver/OtherNormal: Red/Blue for
positive/negative fold change in log2 scale by comparing liver with normal tissues (GTEx&TCGA data)

Previously, to identify the binding target of anti-TIC on the tumor surface, we performed mass-
spectra technology analyzed by Creative Proteomic. In the report, relative abundance of protein
in the CD133- lysis bind to anti-TIC ScFv, protein in CD133+ lysis bind to anti-TIC ScFv, and
protein in CD133+ lysis bind to IgG. The difference of relative abundance of protein in the
CD133- lysis bind to anti-TIC ScFv to protein in CD133+ lysis binds to anti-TIC ScFv, and the
difference of relative abundance of protein in the CD133+ lysis bind to anti-TIC ScFv to protein
in CD133+ lysis bind to IgG was calculated. There are 7 candidate genes which have differences
in both comparisons larger than 3, 42 candidate genes with differences between 1-2 in both
comparisons, and 161 candidate genes with differences of 1 in both comparisons. To select the
most promising candidates, we identified gene alternation of these candidate genes in the TCGA
cohort and ranked the gene by the number of patients with amplification of candidate genes. In
addition, we study the RNA expression of specific gene in HCCDB comparing relative fold
change by gene expression of the HCC comparing to adjacent tissue(Figure 10). By combining
all these results, we finally concluded 4 candidates as tubulin folding cofactor E (TBCE),
exportin 5 (XPO5), centrosomal protein 170 (CEP170), and mitochondrial ribosomal protein
L15(MRPL15). The summary of four candidate genes is shown in Table 6.

25

The upregulation of candidate gene in Huh7 CD133+ TIC identified by qRT-PCR

Figure 11: qRT-PCR data showing four ca gene expression at the mRNA level in CD133+ Huh7 cell line compared to
CD133- Huh7 cell line. Quantitative data of candidate genes obtained by qRT-PCR method from CD133+ Huh7 cell compared
with CD133- Huh7 cells. GAPDH gene was used for normalization. qRT-PCR data were obtained from three independent
each cell group in independent
-test was used to analyze statistics.

Using bioinformatics analysis, we understand that our four candidate genes have upregulation
expression in HCC comparing to adjacent tissue. However, our TriTE is specifically targeted on
TICs. Therefore, to investigate whether the candidate genes have higher expression in TICs, we
performed a qRT-PCR to quantified our candidate genes expression at the mRNA level in
CD133+ Huh7 TIC cells compared to CD133- Huh7 cells. TBCE, XPO5, MRPL15, CEP170
have significantly higher expression in CD133+ Huh7 cell comparing to in CD133- Huh7 cell
(p=0.0026, p=0.0004, and p=0.0003, and p=0.0444). These results indicate that our candidate
genes have higher expression in TICs comparing to non-TIC tumor cells.

26

Chapter 5: Summary
Previous study indicated the large number of immune cells surrounding inflammatory liver
tumor tissue, providing a foundation for our idea that designing a tri-specific binding engager
protein can enhance immune recognition and activation for tumor cell therapy. In later studies,
the top candidates for the anti-TIC ScFv sequence were selected from the human
immunoglobulins phage display library. For these studies, the FACS assay was used to
determine the specific binding ability of Huh7 CD133(+) TICs. Co-IP and Mass spectrometry
assays were utilized to characterize the potential ligands for anti-TIC ScFv detection on the
surface of Huh7 CD133(+) cells.
After performing this extensive study, candidate ligands of anti-TIC ScFv on tumor surface were
identified through a series of bioinformatic analysis. The results of these studies provided the
prequisities for one of our aims, namely that TriTE function better by engaging these cell tyle in
the tumor environment. In the later assays, we confirmed that all the candidates had higher
expression in CD133+ TIC Huh7 cells compared to CD133- non-TIC Huh7 cells which ensured
that they are eligibility to be good candidates verifying TriTE engagement if their specificity to
TIC cells.

27

Chapter 6: Future Direction
To test binding ability of TriTE on candidate knockdown Huh7 cells.
For experiment a groups, the purified TriTE will be used to bind to Huh7 cell lines, shRNA
Huh7 cells line, and primary human hepatocytes. The rabbit anti-6x his tag antibody conjugate
with Dylight 488 and goat anti-human CD133 antibody conjugate with FITC will be used as the
secondary antibody for fluorescent detection.

To test the surface expression of candidate ligands on Huh7 cell lines
Normal Huh7 cells will be double-stained for flow cytometry with antibodies that detect the
potential ligand and goat anti-human CD133 antibody conjugated with FITC. The integrated
mean fluorescence per cell before and after permeabilization will be calculated as proportional to
surface and total receptors, respectively, after correcting for values with control IgG.

T cell and myeloid cell activation assay
Tumor cells (2x10
4
/well) will be seeded into microtiter 96-well plates 24 hours before. At 37
degrees Celsius, the wells will be incubated with CM or increasing concentrations of purified
antibodies for 30 minutes. After washing, Jurkat cells or myeloid cells will be isolated from
healthy volunteers using density gradient centrifugation and added at a ratio of 5:1 effector:target
(E:T). After 24 hours of incubation, the expression profile of activation marker CD will be
evaluated by FACS using a PE-conjugated anti-CD69 mAb and a FITC-conjugated anti-CD3
mAb that have been incubated on ice for 30 minutes. MACS Quant Analyzer 10 will be used to
analyze the samples (Miltenyi Biotec GmbH).

28

Myeloid depletion cytotoxicity assay
Immunomagnetic selection will be used to deplete CD3+, CD14+, and NK cells from PBMC
separately. Huh 7 cells and shRNA Huh7 cells will be cultured in a 12-well bottom plate with
rature. The
concentration of cells per well will be changed to 10
4
cells. After three washings, the PBS will be
used to remove any unattained 51Cr. Different ratios of well-prepared PBMCs or isolated CD3
or CD14 cells with engager protein TRITE will be added and incubated at room temperature for
3 hours. The supernatant of each well is collected and 51Cr is counted by a gamma detector. In
one of the wells, 1% NP40 is added to measure the maximal death, while another well of cell
culture treated without PBMCs is used to quantify the spontaneous release. For counting lysis
percentage, the following equation will be used: the percentage of specific lysis = 100 [(test 51Cr
release) - (spontaneous 51Cr release)] /[(maximal 51Cr release) (spontaneous 51Cr release)].

29

In vivo experiment comparing TriTe killing ability with BiTE and single anti-CD3 ScFv
NSG-SGM3 mice, which are immune deficient and readily engrafted with fetal liver and PDX
tissue, can serve as a model for investigating the in vivo effects of TRITE and controls.
Beginning on day seven, mice will get five daily injections of TRITE, BiTE(anti-TIC ScFv and
anti-CD3 ScFv), or anti-CD3 ScFV only. Mice will be administered PBS as a negative control.
Kaplan-Meier plots will document curves for each group of mice to demonstrate the anti-tumor
efficacy of TRITE in vivo compared to BiTEs and the control group.

Figure 12: In vivo experiment on comparing TriTe killing ability with BiTE and single anti-CD3 ScFv

30

Knockdown candidate gene on PDX tissue implanted in mouse model
Once the possible ligand candidates of anti-TIC ScFv have been discovered, their specific
binding ability will be determined by knocking down critical genes in the PDX mice model. To
clarify the role of target genes and anti-TIC ScFv ligand, the anti-tumor effects of TriTE will be
evaluated in WT and relevant gene knockdown phenotypes. Survival time and tumor growth rate
will be collected and analyzed for the control groups, which will consist of PBS therapy alone
and scramble shRNA treatment. The immunostaining of CD3 and CD14 on the tumor can be
used to determine the amount of tumor inflammation before and after TriTe injection.

Figure 13: Protocol of knockdown candidate gene on PDX tissues implanted in mouse model

31

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Abstract (if available)

Abstract Hepatocellular carcinoma (HCC) is a malignancy of hepatocytes that develops in the liver. Patients with a history of liver illness, such as liver fibrosis and cirrhosis, develop HCC. About 80% of all liver cancers are HCC, making it one of the most prevalent malignancies worldwide. Liver tumor initiating cells (TICs) are responsible for liver tumor formation, metastasis, medication resistance, and relapse. As a result of their capacity for self-renewal and differentiation, liver TICs can recur and produce new tumors once medication treatment is stopped. CD133, wnt/beta-catenin, and nanog are stemness genes expressed by both TICs and pluripotent stem cells. CD133, a glycoprotein with a 5-transmembrane domain, is expressed in both normal and malignant tissues. In recent years, cancer immunotherapy has expanded significantly in terms of agents that give a prognosis benefit by stimulating the immune system to mount a response against growing tumors, but have been unsuccessfully explored for decades in HCC. Tri specific T cell engager protein (TriTE) is created based on BITE in this study. When attached to a tumor cell, TriTE has the ability to attract two distinct types of immune cells to enhance cytotoxicity. The previous work of our lab screened out a series of DNA sequences generating single-chain antibodies with strong binding affinity to tumor initiating cells (TIC) and confirmed the antibody's high affinity for CD133+ Huh7 cells. The TriTE was designed to incorporate sequences encoding single-chain anti-human CD14, anti-human CD3, and anti-TIC antibodies. It connects human tumor initiating cell, myeloid cell, and T-lymphocyte by acting as a "bridge." We hypothesized that the design of Tri-specific T cell engager protein (TRITE) with specific binding capacity of myeloid cell, T cells, and TICs will increase the efficiency of immunotherapy against HCC. In our study, we identified four candidates for the binding target of anti-TIC ScFv of Trite on TICs. These candidates were validated by multiple bioinformatics analyses and quantitative reverse transcription polymerase chain reaction (qRT-PCR), which demonstrated that their expression was higher in tumor initiating cells than in non-tumor initiating tumor cells. In subsequent investigations, we will evaluate the specific binding target of anti-TIC ScFv of TriTE on TIC using FACS analysis on knockdown cell lines and knockdown mice models. Using a TUNEL assay, we'll then determine the TriTE killing mechanism. Then, we will evaluate the cytotoxicity of TriTE using an in vitro FACS analysis and a myeloid cell depletion test, as well as an in vivo mice model including BiTE and a single anti-CD3 ScFv. Finally, in vivo and in vitro toxicity tests will be conducted on TriTE.

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Asset Metadata

Creator Wang, Nuan (author)

Core Title Tri-specific T cell engager immunotherapy targeting tumor initiating cells

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Degree Conferral Date 2022-08

Publication Date 07/22/2022

Defense Date 06/02/2022

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

Tag cancer immunotherapy,OAI-PMH Harvest,Tri-specific T cell engager,tumor initiating cells

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Machida, Keigo (committee chair), Epstein, Alan (committee member), Yuan, Weiming (committee member)

Creator Email angela_nuan@hotmail.com,nuanwang@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC111373921

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