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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 Tri-specific T cell engager immunotherapy targeting tumor initiating cells

By

Wei Ma

A Thesis Presented to the
FACULTY OF THE KECK MEDICINE SCHOOL OF USC
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(Molecular Microbiology and Immunology)

May 2021

Copyright 2021 Wei Ma

ii
Acknowledgments
I want to first appreciate Dr. Keigo Machida for accepting me as a member into his and
laboratory and taught me everything of the lab; he provided continuous support throughout all my
master program. I also want to appreciate Dr. Da-Wei yeh and Dr. Hye Choi for teaching me the
operations of the lab’s equipment and training me in various experiments. During the process of
assays, Carlos Hernandez helped me a lot in data collection and material orders. Lastly, I would
like to appreciate Dr. Alan Epstein, Dr. Weiming Yuan and Dr. Ling Shao for their guidance and
supports.

iii

Table of Contents

ACKNOWLEDGMENTS……………………………………………………….….……………ii
LIST OF TABLES……………………………………………………….…….……...…….…iii
LIST OF FIGURES………………………………………………….……………..……………iv
ABSTRACT………………………………………………………………………..……...……v
INTRODUCTION AND LITERATURE REVIEW…………………………………………….1
MATERIAL AND METHODS…………………………………………………………………5
SPECIFIC AIMS……………………………………………………………………………….15
RESULTS AND DISCUSSION………………..………………………………………………16
SUMMARY…………………………………………………………………………………….25
FUTURE DIRECTION…..…………………………………………...…………………………26
REFERENCE…………………………………………………………………………………...32
APPENDIX……………………………………………………………………………………..34

iv
List of Tables

Table 1: The components volume in RE digestion……........…………………….…..……..… 8
Table 2: The components volume in ligation process .…………………….….……….……… 9
Table 3: The groups design of anti-TIC ScFv in FACS assay……….…………………..…… 11
Table 4: PCR reaction master mix for ligation product verification………………………..… 14
Table 5: The input and output volume of phage library screening of group one……………....18
Table 6: The input and output volume of phage library screening of group two………………18
Table 7: The effector / target ratios in cytotoxicity assay…………………………………....…27
Table 8: The percentage of CD133 expression in different tumor types……………………….31

v
List of Figures

Figure 1: The overview of BiTE and TRITE protein structures………………………….…….. 4
Figure 2: The process of phage display library and principle of TRITE………………………. 7
Figure 3: The gene map of anti-TIC ScFv plus pSecTag2 A vector………………….……....... 8
Figure 4: The gene map of TRITE sequence plus pSecTag2 A vector…………………………13
Figure 5: The immunohistochemistry of CD3 and CD14 in “cold tumor” condition..…………16
Figure 6: The immunohistochemistry of CD3 and CD14 in “hot tumor” condition……..…..…17
Figure 7: The positive percentages of CD3 and CD14 in different tumor conditions………......17
Figure 8. The clone growth conditions of reconstructed plasmids..........................................….. 19
Figure 9. The clone numbers in per square centimeter of different ratios………………..…….. 19
Figure 10. The RE digestion results of recombinant plasmid…………....……………………… 20
Figure 11. The Coomassie Brilliant Blue Staining and Western-blot results of anti-TIC ScFv.. 21
Figure 12. The FACS assay results…………………………………………………………….. 22
Figure 13. The PCR result……………………………………………………………………… 23
Figure 14. The Western-blot result of TRITE………………………………………………….. 24
Figure 15. The overview of Chromium-51 labeling cytotoxicity assay…………………………27
Figure 16. The overview of preclinical PDX mouse model building………….……………….. 29
Figure 17. The overview of ligand genes knockout assay……………………………………… 29

vi
Abstract
Hepatocellular Carcinoma (HCC) is widely acknowledged as one of the biggest threats to public
health. More than 10 million people are estimated to die from various forms of cancer every year.
For recent decades, mainstream Chemotherapy does not sufficiently eliminate tumor-initiating
cells (TICs). Immunotherapy, as a burgeoning technology that focuses on applying the principle
of immune reaction to clear up cancer cells, has come to scientists' notice. Emerging data suggest
that nanoparticle-based siRNA delivery targeting stemness transcription factor NANOG
(pluripotency transcription factor) potentiates anti-tumor immunity. Here, we hypothesized that
designing a specific engager protein targeted tumor-initiating cells, T-lymphocytes and antigen
presenting cells (APCs) would sensitize the efficacy of immunotherapy on liver cancers. Through
phage-display library screening, a type of novel TIC-targeting immunoglobulin variable chain
fragments (anti-TIC ScFv) were identified. After screening out the DNA sequence of anti-TIC
ScFv, it was ligated with pSecTag2 A vector and transfected into the HEK293T cell line for
targeting protein producing. After the purification process, the specific binding ability of the TIC-
targeting scFv to the CD133(+) Huh7 TIC is evaluated by the FACS assay; Besides, the candidate
ligands of anti-TIC ScFv was investigated via mass-spectrometry assay. Furthermore, the
recombinant sequence consists of anti-TIC ScFv sequence, anti-human CD14 ScFv, and anti-
human CD3 ScFv sequence was synthesized by GBlock method. Through transfecting to
HEK293T cell lines and purification, the tri-specific binding engager protein TRITE was obtained,
which owns the specific simultaneously binding ability to CD133(+) Huh7 TICs, dendritic cells
(DCs), and T-lymphocytes. TRITE will promote immune recognition and activation to help clean
up the tumor cells. To test the specific killing ability of TRITE, human PBMC is purified and co-
cultured with TRITE in CD133(+)/(-) TICs cell culture and traditional human hepatocyte cultures.

vii
By comparing the detection of radiation released from Chromium
51
of pre-marked CD133(+)/(-)
TICs and normal human hepatocytes, its specific killing ability is identified in vitro. The results of
our study have identified the specific binding ability of anti-TIC ScFv to the CD133(+) Huh7 TICs
in vitro. In future research, the cytotoxicity of the engager protein TRITE will be tested in the
tumor-bearing immuno-compromised PDX mice model in vivo. The relevant genes of potential
ligands of TIC-targeting ScFv will be knockout/enhanced-expression in mice models for further
identifying its specific binding ability. The purpose of the study is to potentiate the anti-tumor
immunotherapy function of the T cell engager protein TRITE in clinic application.

1
Chapter 1. Introduction
1.1 The hepatocellular carcinoma (HCC) and human tumor initiating cells (TICs)
Hepatocellular carcinoma (HCC) is a primary malignancy of the liver. There are several trigger
factors, including obesity, Hepatitis B and C, excessive alcohol intake, etc
[1]
. Deaths due to
hepatocellular carcinoma (HCC) continue to mount due to a low success rate of clinical
intervention. It is now the third most deadly cancer in the world (660,000 deaths per year)
[2]
.

The
incidence of HCC continues to rise with an estimated 33,660 new cases and 24,550 deaths in the
US for 2015
[3]
. The 3-year survival rate of HCC is 13-21% without any specific treatment
[4]
. Several
factors have been identified as the HCC trigger factors, including chronic infection with HBV or
HCV, excessive alcohol consumption and accumulation of fat in the liver, etc.
Cancer stem cells (CSCs) are tumor-initiating cells (TICs) that are characterized by abnormal self-
renewal ability and producing various differentiated progeny that can develop into a bulk of tumor
rapidly
[5]
. The origin of the CSCs is still unknown, and up to now, there are two major persuasive
hypotheses, the clonal evolution model and the CSC model. The clonal model posits some
irreversible genetic mutations or hereditable epigenetic changes in the typical new clones, and the
accumulation of those relative genetic changes leads the oncogenesis
[6]
.

The alternative hypothesis
CSC model believes the cancer stem cells are derived from the normal stem cells because they
share many of the same biomarkers on their surfaces
[7]
.

It points out the tumor cells have high
hereditary-similarity, and only a small subset of tumor stem cells keep the ability to reproliferate
and form the tumor
[8]
. The previous studies showed that the tumor's heterogeneity is closely
associated with the TIC. Thus its eradication may be critical to cure aggressive malignancies.

2
Few common hallmarks between different CSC phenotypes, and CD133 is one of those markers.
Besides, the high-level expressions of the CD133 are also found in several tumor types, including
prostate, lung, breast, and HCC. Furthermore, the scientists used to compare CD133(+) cell
fractions and CD1333(-) cell fractions and found that CD133(+) tumor cells were more likely to
promote the tumor growth and metastasis process
[9]
.

There are currently several FDA-approved
anti-CD133(+) ScFv conjugated anti-tumor drugs in clinic application, which shows high-
efficiency performance in anti-tumor treatment
[10]
.

1.2 The cellular immune response to tumor cells and immunotherapy.
The cellular immune system is composed of T-lymphocyte, APCs, macrophage, and nature killing
cells; it makes critical function against cells infected with viruses, intracellular bacteria, fungi and
protozoans, and cancerous cells
[11]
. In the process of tumor cell-associated cellular immune
response, the cellular immune system's initiation is APCs recognizing and capturing the target Ags.
After the binding of Ags, the APC will present its MHC complex on the cellular surface to mature
T-lymphocytes that will direct the T-lymphocytes' activation and proliferation. Activated T-
lymphocytes will differentiate into a cytotoxic T cell and helper T cell. In the cellular immune
process, the cytotoxic T cells will secrete perforin, which will induce the tumor cell's apoptosis.
The helper T cells will secrete several types of cytokines, which increase the killing activity of
macrophages and nature killing cells
[12]
.

When against the foreign antigens, the cellular immune also needs to avoid causing damages to
normal cells. As we know, the tumor cell is also derived from normal cells through some
complicated cellular mechanisms mutations. The homology of the tumor cell may be one reason
that in clinical cases, the antitumor immune response is not always effective and sometimes gets
tolerance
[13]
. Immunotherapy is regarded as the most effective and potential treatment to activate

3
the immune activity to destroy tumor cells. At present, several immune mechanisms have been
found that provide supportive ideas for immunotherapy methods, including immune checkpoint
blockade, CAR T-cell therapy, bi-specific T cell engager antibody construct, and other forms
[14]
.
The immunotherapy has been approved in the United States and elsewhere to treat a variety of
cancer and are prescribed to patients by oncologists.
[13]
1.3 Anti-TIC ScFv and Tri-specific T cell engager protein
Bispecific T cell engager antibody (BiTE) is constructed of two single-chain variable fragments
(ScFv)
[14]
. Typically, one of the ScFv with specific binding ability to T lymphocyte is connected
with another tumor-associated antigen (TAA) binding ScFv
[15]
. The anti-tumor effect of BiTE has
been identified in many types of solid cancers, and the FDA has approved the anti-CD3/CD19
BiTE antibody in some types of malignancies treatments
[16]
. However, the BiTE treatment does not
benefit all patients. One of core mechanisms of the BiTE is capturing an activated T lymphocyte
which process is unexpected in immunosuppressed patients and this condition is describes as “cold
tumor”. For a better anti-tumor effect, an efficient T lymphocytes activation method is wanted
[17]
.
Our lab's previous research applied the phage-display library screening and identified a type of
novel TIC-targeting immunoglobulin variable chain fragments (anti-TIC ScFv). After obtaining
the DNA sequence of anti-TIC ScFv, we incoporated anti-TIC ScFv into pSecTag2 A vector and
transfected it into the HEK293T cell line for target protein generation
[18]
. After the purification
process of anti-TIC ScFv, the FACS assay and cell lysis mass-spectrometry assay were applied to
verify its specific binding ability to Huh7 CD133(+)TIC and figure out its potential binding ligand
on the surface of the tumor cell
[19]
.

4
CD3 is initially expressed in the cytoplasm of primary T-lymphocytes; in the later stage of
differentiation, the CD3 antigen will migrate to the cell membrane. The antigen is found bound to
all matured T lymphocytes, and it also involves activation of cytotoxic T cell and T helper cells
[20]
.

CD14 (cluster of differentiation 14) is described as monocytes differentiation antigen on the
surface of dendritic cells (DCs), macrophages, and some monocytes.
[15]
Dr. Fol Marek and his
group used to generate human moDCs in vitro from peripheral blood CD14
+
monocytes or CD34
+

progenitors
[21]
.
In this research, the anti-human CD3 ScFv sequence was linked with the anti-TIC ScFv sequence
and anti-human CD14 ScFv sequence via the gBlocks method. After the ligation process with
pSecTag2 A vector, the reconstructed plasmid was used to transfect the HEKT293 cell line for the
generation of tri-specific engager protein TRITE. We hypothesized that the tri-specific T cell
engager protein TRITE would make the function as a “bridge” to link human DCs, T-lymphocytes,
and TICs simultaneously. The captured TIC will be recognized by the captured dendritic cell,
which will present the MHC molecule to the captured T-lymphocyte and activated the T-
lymphocyte efficiently. Then activated T-cell will differentiate and proliferate to trigger the tumor-
immunity response for inhibition of tumor progress and metastasis.

Figure 1. The overview of BiTE and TIRTE protein structures

5
Chapter 2. Material and methods
2.1 Immunohistochemistry
The immunohistochemistry was performed on paraffin-embedded tissues, which is provided by
the USC liver diseases research canter. In this study, the human tissue samples were supplied by
HCR Los Robles Hospital and from an HCC patient volunteer who had signed the informed
consent form for our lab’s research. The tissue samples in this study are HCC tumor tissue and
normal hepatic tissue.
After the melt of paraffin, all the slides were baked in a 37℃ incubator, and three Coplin jars filled
with 90% xylene and six jars filled with alcohol in gradient concentration (pure alcohol, 95%, 75%,
50%, 25%, and DW) were prepared. After the bake, the slides were placed in 90% xylene solutions
for 10 minutes in each jar. Next, the slides were re-hydrated by placing them in different alcohol
concentrations for 5 minutes in each jar and placed in the water for 15 minutes wash. Afterward,
the slides were placed in the pot filled with Citrate buffer (2.94 g sodium citrate, 500µl of Tween
2.0 diluted in 1000ml DW) for half-hour heating in the microwave. Then the slides were taken out
and wiped up cautiously by the dry tissue. Next, the slides were placed in the blocking buffer (5%
of goat serum+10% BSA in PBS) at room temperature for 2 hours and washed by DW. Primary
antibodies rat anti-human CD14 antibody, and goat anti-human CD3 antibody (both were
purchased through Santa Cruz Biotechnologies) were twenty times diluted in 10%PBS and
1%BSA solution as the recommendation. The well-prepared primary antibodies were dropped on
the slide till the whole tissue was covered by the answer. All the slides were kept in the cold room
overnight. On the second day, the slides were washed by DW. Secondary antibodies mouse anti-
rat antibody (Alexa Fluor 568
®
) and chicken anti-goat antibody (FITC) were dropped on the slides

6
cover the whole tissue for 2 hours incubation in a lighttight box at room temperate. Finally, slides
were washed by DW, and a drop of DAPI was added for nucleus staining. The microscopy
observation was performed by the Cell and Tissue Imaging Core of the USC Liver diseases
research center. The positive percentage will be calculated and compared by student T-test’s and
Chi-square test method. Asterisks (*) indicates a p-value <0.05.
2.2 Cell culture
Huh7 cells are used in the study as HCC TICs model, and CD133 MicroBead Kit (produced by
MACS Biotec company) was used to isolate the CD133(+) Huh7 cell line. HEK293T cell line was
used as transfected-target cells to help produce the target protein anti-TIC ScFv and engager
protein TRITE. DMEM (Dulbecco’s Modified Medium) supplied with 10%FBS, 1% Streptomycin
antibiotics, 1% Penicillin, and 1% Glutamine and 1% non-essential amino acids are used as the
culture medium.
Cell stock from -80 freezer in cell banker medium is seeded in T25 flasks, and culture in the
parameters well-set incubator. The cell passage is applied to add 1ml trypsin for 3 min incubation
to detach the cells from the flask. The collected cell culture is centrifuged in 1300rmp for 5 min,
then discarding the supernatant. 1ml prewarmed medium is used to resuspend cell pellets, then
seed 0.5ml resuspended cell into every new T25 flask to finish the passage.
2.3 Phage Display Library screening
In our lab's previous research, an array of filamentous phage M13 carrying multiple type single-
chain antibody sequences was obtained. The host strain for transfection is E. coli (Rosetta cells),
and I inoculated the host strain in LB medium in a shaking bed until the mid-log phase. Before the
transfection, the phage was pre-warmed (37℃) and tenfold diluted into 1x10
10
, then 200µl E. coli

7
culture was added into 10 µl phage dilution in a quick vortex and incubated for 5 min. In the end,
the above mixture was separated on the LB plate evenly and kept overnight.
Purified Huh7 CD133(+) TICs are grown in the sterile polystyrene 12-well plate. 20 µl transfected
E. coli was added every well for binding 2 hours and washed unbinding E. Coli cell by culture
medium. The binding E. Coli was collected and added into a new purified Huh7 CD133(+) TICs
grown well, then the unbinding E. Coli has washed away, and this process was repeated six times.

Figure 2. The process of phage display library and principle of TRITE.
About 10
2
scales of E. Coli with specific binding ability were collected and sent to the Genome
company for high-throughput DNA sequencing. The company decoded the sequence and sent back
the anti-TIC ScFv sequence, which is used later in this study.
2.4 Plasmid construction and generation of anti-TIC ScFv
After received the anti-TIC ScFv plasmid (after dissolution, the concentration is 0.35ng/µl), an
appropriate volume of restriction enzymes Hind III and Not I (both produced by BioLabs company)
were used to digest the anti-TIC ScFv plasmid and pSecTag2 A vector (produced by TaKaRa
company). The mixtures were incubated in 37℃ for 2 hours, and the purified by DNA purification
Kit (produced by QIAGEN company).

8
Table 1. The components volume in RE digestion
Deionized Water 33.8𝜇l
10x Cut smart buffer 5𝜇l
Hind III (20U/ 𝜇l) 0.5 𝜇l
Not I (20U/ 𝜇l) 0.5 𝜇l
pSecTag2 A vector 10 𝜇l
Total 50 𝜇l

The Gel Extraction Kit (produced by QIAGEN company) was applied after the inserter and vector
digestion processes. The digested products were loaded with dye in 1% agarose gel for 20 min
electrophoresis. Before the gel extraction, the empty 1.5 ml Eppendorf tube was weighted. The
bands were observed via UV light (the lengths of anti-TIC ScFv sequence and pSecTag2 A vector
are 0.7kbp and 5.6kbp respectively) and excised carefully by the blade. The extracted gels were
loaded into the tubes, and increased weight was gel volumes.

Figure 3. The gene map of anti-TIC ScFv plus pSecTag2 A vector
DNA concentrations were estimated according to reference table of the Generuler 1kb plus
(produced by Thermo Fisher Scientific company). The inserter fragment and vector fragment in
three molar ratios in 1:0, 1:3, and 1:10 (inserter/vecto). For example, 3 µl of vector fragment and
4.8 µl of anti-TIC ScFv fragment are added into 1.5 ml tube with 2 µl T4 ligase (produced by

9
BioLab company) and 20 µl DW for ratio in 1:3. In order to verify the ligation process, agarose
gel electrophoresis was applied after the HindIII / NotI and both digestion process.
Those three tubes above were incubated in 16℃ overnight and transformed into competent cells
(produced by BioLab company) with ligation mixtures. The cells were incubated on ice for 10 min,
then incubating in 42℃ for 30 sec, and then put the tube back on the ice for 2 min. 200 µl of SOC
solution was added into the tube and incubated in the warm shacking bed for 30 min. After 30 min
incubation, the mixtures were dropped evenly on the LB + Ampicillin plates and incubated in 37℃
overnight.
Table 2. The components volume in ligation process
Linearized pSecTag2 A vector (50ng/ 𝜇l) 1.0𝜇l
Annealed anti-TIC ScFv template 0.5𝜇l
10x T4 DNA ligase buffer 1.0 𝜇l
Deionized water 6.5 𝜇l
T4 DNA ligase (40U/ 𝜇l) 1.0 𝜇l
Total 10.0 𝜇l

In the second day, the clone numbers in per square centimeter on those three plates were accounted.
After verification of the ligation process, clones were picked up from plates in ration 1:3 and 1:10
by tips and incubated in 5 ml LB ampicillin solution for overnight shaking incubation. QlAprep
Spin Miniprep Kit was used to elute (produced by QlAGEN company) plasmid, and then detected
plasmid concentration via Nanodrop facility.
HEK293T cell line was used to produce the target protein. One hour before the transfection, the
medium was refreshed to keep the cell in good condition. The mixture components were: 10 µg
plasmid / pSecTag2 A vector only (function as the control group), 500 µl free medium, 15 µl Bio-

10
T reagent (produced by Bioland Scientific company). The mixture was spread in the culture plate
evenly and incubated for 48 hours. The supernatants were harvested and filtered by 0.22 µm filter.
For every supernatant harvested above, 200 µl Ni-NTA agarose beads were added (produced by
QlAGEN company) and fixed on the shaker in 4℃ for 2 hours. After the completely binding, the
tubes were settled on the rack for 20 min for the target protein and Ni-NTA bead complex falling
to the bottom. The supernatant was removed from the tube carefully, and then 1 ml wash buffer
was added (produced by Pharmacia Biotech company) into every tube. The tubes were inverted
deliberately, and then the samples were transferred into 1.5 ml microtubes to centrifuge (1700g for
5 min 3 times). The supernatant of microtubes was removed every time after the centrifugation.
10 µl 2M imidazole (produced by Pharmacia Biotech company) and 200 µl elution buffer
(produced by Pharmacia Biotech company) were added into the mixtures. In the end, all the
mixtures were kept in -20℃ overnight. The supernatant was extracted on the second day and kept
in the freezer. Bradford assay was applied for purified proteins concentration estimation.
2.5 FACS Analysis for specific binding ability of anti-TIC ScFv
There were four groups designed for the FACS assay. In the experimental groups, the purified
anti-TIC ScFv antibody was used to bind to Huh7 CD133(+) TICs / Huh7 CD133(-) TICs. The
rabbit anti-6X His tag antibody conjugate with DyLight®488 (produced by Abcam company) was
used as the second antibody for fluorescent detection. For the negative control group, the human
normal IgG antibody conjugated with Alexa Fluor® 458 (produced by Abcam company) was used
to bind to human Huh7 CD133(+) TICs. For the positive control, goat anti-human CD133 antibody
conjugated with FITC (produced by Abcam company) was used to bind to Huh7 CD133(+) TICs.

11
Huh7 CD133(+) TICs were grown in the DMEM medium. 10
7
cells were co-cultured in a 12-well
plate for 24 hours incubation. Next, the medium was removed, and fresh medium was added into
every well 24 hours before in the incubation. After 24 hours, PBS was used to wash the co-cultured
cells in the scraper, and the concentration of the cell was adjusted at 10
6
/ml in cold FACS buffer
(1%BSA, 0.1% NaN3, PBS). The cells were transferred into 10 ml conical vial and centrifuged
(1500rmp for 5min at 4℃). The supernatant was discarded, and the remained cells were washed
by PBS and aliquoted into three Eppendorf tubes.
In every group, 30µg of the primary antibody was added, then we used PBS replenished the
mixture to 300µl. All the samples were incubated for a half-hour in 4℃ in the dark. Next, the
unbound antibodies were washed away through centrifugation (1500rpm, 5 min, three times) and
re-suspended by 200µl cold FACS buffer. The Flow cytometry was performed in the USC flow
cytometry facility in the Liver Disease Research Center.

Group design Antibody Cell line
Positive control Goat anti-human CD133 antibody
conjugated with FITC
Hu7 CD133(+) cell line
Negative control Normal human IgG antibody
conjugated with Fluor
®
458
Hu7 CD133(+) cell line
Experimental groups Anti-TIC ScFv Hu7 CD133(+) cell line
Anti-TIC ScFv Hu7 CD133(-) cell line
Table 3. The groups design of anti-TIC ScFv in FACS assay

12
2.6 Mass-spectrometry analysis
There were three groups designed: anti-TIC ScFv mixed with the Huh7 CD133(+) TIC lysis, anti-
TIC ScFv mixed with the normal liver cell human and IgG antibody mixed with the Huh7
CD133(+) TIC lysis.
The reagent-based method was used to lyse the target cell. Before the cell lysis, the media of the
cell lines was removed, and PBS was used to wash for three times. TrypLE reagent was used to
dissociate the cells. After 3 min of incubation, the mixtures were transferred into the centrifuge
tubes and centrifuged in 1300rmp for 3 min, and the supernatant was discarded. 1 ml of RIPA lysis
buffer (produced by Thermo Fisher company) with 10µl 100x protease inhibitor reagent was added
into the samples. All the samples were kept in the ice for 1-hour incubation and centrifuged at
13000 rpm for 10 min. The supernatant was discarded, and remained part was sent to Creative
Proteomics company for the Co-IP experiment and protein identification analysis.
2.7 Recombinant Plasmid construction and engager protein TRITE generation
The TRITE sequence contains three parts including anti-TIC ScFv sequence, anti-human CD3
ScFv sequence, and anti-human CD14 ScFv. Anti-human CD3 ScFv sequence, and anti-human
CD14 ScFv were obtained from published resources (Yong-Min Tang et al.,) which were linked
with each other via (GGGS)3 linker sequence. After the revise of the sequence, the sequence of
TRITE was sent to Integrated DNA Technologies company for synthesis by GBlock method.
TRITE sequence shares same RE cutting sites (NotI and HindIII) with pSecTag2 A vector. After
received the synthesized sequence, the TRITE sequence was incorporated into pSecTag2 A vector
through RE digestion and ligation processes. The QIAquick Gel Extraction Kit (produced by
QIAGEN) was used for purification process, and the molar volume ratio of the inserter and vector

13
was 1:3. After the ligation process, PCR by using plasmid primer across the insert was used for
ligation product verification.
After the transformation process via competent cells, Miniprep Kit was used for plasmid extraction.
Next, the concentration of TRITE + pSecTag2 A plasmid was detected by Nanodrop facility. The
appropriate volume of reconstructed plasmid was mixed with 500 µl free medium and 15 µl Bio-
T reagent, then transfected into the medium refreshed HEK293T cell line. After 48 hours
incubation, the supernatant was harvested and filtered with 0.22 µm filter. 200 µl Ni-NTA Agarose
beads were added into every supernatant tube for 2 hours incubation in the cold room. After twenty
minutes of precipitation, the supernatant was removed. The reminding part was centrifugated three
times(1700g for 5 min), and the supernatant was discarded every time. 10 µl 2M imidazole and
200 µl elution buffer were used to detach the magnetic beads from TRITE. After the purification
process, the Bradford assay was used to detect the concentration of the TRITE.

Figure 4. The gene map of TRITE sequence plus pSecTag2 A vector

14
The reagent Volume
Q5
®
Reaction Buffer (5X) 20𝜇l
10mM dNTP 2 𝜇l
20mM forward primer 2.5 𝜇l
20mM reverse primer 2.5 𝜇l
Recombinant plasmid templet 1 𝜇l
Q5
®
DNA Polymerase 4 𝜇l
Sterile water 68 𝜇l
Total 100 𝜇l

2.9 Western Blot assay
25 𝜇l of TRITE was mixed with 5 𝜇l 6x laemmli buffer. The mixtures were incubated at 95℃ for
10 min. 20 µl of sample mixture was loaded into 10% Acrylamide gel for electrophoresis. Nearly
2.5 hours later, the separated proteins are transferred from the Acrylamide gel to PVDF membrane
(produced by Bio-Rad company). After 2 hours membrane transfer, the transferred PVDF
membrane was incubated with 5% non-fat milk PBST overnight in the cold room. On the second
day, the membrane was placed in 2 𝜇l of primary antibody (mouse anti-His tag antibody produced
by Abacam company) diluted in TBST with 5% BSA for 2 hours incubation. After washed by
PBST, the membrane was incubated with anti-mouse HRP antibody for 1 hour in 1:10000 diluted
concentration. After the wash process, the fluorescent result can be observed with
chemiluminescence.

Table 4. PCR reaction master mix for ligation product verification

15
Chapter 3. Specific Aim
Aim 1. Identification of the specific binding ability of anti-TIC ScFv to CD133(+) Huh7
tumor-initiating cells and the specific binding domain of the surface of TICs.
Through phage display library screening, a sequence maned anti-TIC ScFv encoding TIC-specific
binding single-chain antibody was found. In this study, the anti-TIC ScFv sequence was ligated
with the pSecTag2 A vector and transfected into CHO cell lines for interest single-chain antibody
expression. The FACS assay and Mass-spectrometry were used to evaluate its binding activity and
identify the binding domain on the surface of TICs.
Aim 2. Designing a reconstructed plasmid encoding the tri-specific T cell engager protein
TRITE which can specifically bind to human T-lymphocyte, dendritic cells and TICs.
The linker sequences were used to link three interest sequences, including anti-human CD3 ScFv
sequence, anti-human CD14 ScFv sequence, and anti-TIC ScFv sequence, to build a reconstructed
plasmid. Theoretically, the engager protein expressed by this reconstructed plasmid owns the
ability to bind to human T-lymphocytes, dendritic cells, and TICs specifically.
Aim 3. Evaluating the killing activity of engager protein TRITE in vitro.
The cytotoxicity assay through radiation material chromium
51
label was used to identify the killing
activity of recombinant protein TRITE. PBMCs worked as the effector cells that were added into
Huh7 CD133(+) cell line. Theoretically, the TRITE will work as the “bridge” to link tumor cells
and immune cells spontaneously, increasing the efficiency of the immune recognition and immune
response.

16
Chapter 4. Results and Discussion
4.1 The quantity and distribution of dendritic cells and T cells in “hot tumor” condition are
significantly higher than that in “cold tumor” condition in HCC patients.

Figure 5. The immunohistochemistry of CD3 and CD14 in “cold tumor”. Confocal fluorescent images showing CD3 (green) and
CD14 (red) in HCC tissue. Scale car 50µm.

Anti-CD3
Anti-CD14
DAPI Anti-CD14 Anti-CD3
DAPI Anti-CD14
DAPI

17

Figure 6. The immunohistochemistry of CD3 and CD14 in “hot tumor”. Confocal fluorescent images showing CD3 (green) and
CD14 (red) in HCC tissue. Scale car 50µm.

Figure 7. The positive percentages of CD3 and CD14 in different tumor conditions
The CD14 and CD3 positive percentages surrounding the inflammatory liver tumor tissue and non-
tumor liver tissues are compared in the immunohistochemistry assay. The quantification of both
makers revealed that in “hot tumor” HCC tissue the percentages of positive staining of both CD14
(34.6%) and CD3 (26.2%) are significantly higher than CD14 (18.2%) and CD3 (13.7%) in “cold
tumor” HCC tissue. Those results indicated that the immune cells have the limited ability and
Anti-CD3

18
affinity to capture and recognize the tumor cell efficiently in “cold tumor” condition, leading to
the dysfunction of the immune activation and immune response. The low quantity of the immune
cells in the “cold tumor” condition indicates the limitation of current BiTE treatment and supports
our hypothesis of promoting the immune response by simultaneously linking TICs and T
lymphocytes and antigen present cells via the engager protein TRITE.
4.2 Novel TIC-targeting immunoglobulin variable chain fragments were identified by
phage-display library screening

In our lab's previous research, a phage display library of human immunoglobulins to identify and
isolate TIC-specific antibodies. The top ScFv candidates were identified and individually cloned
into chimeric antigen receptor lentivirus and transduced into T lymphocytes to test target cell lysis
assays. There were two groups designed with the same input volume of the phages and took six
rounds of the screening. In the end, a cluster of the phages carrying the interest DNA sequence
was obtained.
Table 6 The input and outcome volumes of phage library screening of group two
Table 5 The input and output volumes of phage library screening group one

19
The data shows that our lab screened out the phages carrying the target DNA sequence from
1.5x10
13
volume of phages. The phages of the output of the 5th round carry the interest DNA
sequence, which express a high-affinity binding protein to the human tumor-initiating cells.
4.3 The insertion and ligation of the anti-TIC ScFv sequence with pSecTag2 A vector
The restriction enzymes (NotI and HindIII) were used to cut the same cutting sites on the anti-TIC
ScFv sequence and pSecTag2 A vector. The Gel Extraction Kit (produced by QIAGEN) was used
to extract the target fragments. Then inserter and vector fragments in an appropriate volume was
mixed to achieve the three different inserter/vector volume ratios at 1:0, 1:5, and 1:10. Those three
mixtures were transferred into competent cells then transformed on the LB plate. The the clone
numers in per square centimeter on those plates were calculated on the second day.
Figure 8. The clone growth conditions of reconstructed plasmids. The plat A is the one with the ration of inserter/vector in 1:0.
The B is the LB plate with ration of 1:5. The C is the one with the ration in 1:10.

Figure 9. The clone numbers in per square centimeter of different ratios
The plates in the figure. 8 indicated clonal growths of recombinant plasmids at 1:0, 1:5, and 1:10
inserter/vector volume ratios. A is the plate with the reconstructed plasmid at 1:0 inserter/vector
A B
C

20
volume ratio, which showed no clone growth on this LB plate. B and C were the plates with the
reconstructed plasmid at 1:5 and 1:10 inserter/vector volume ratios, respectively. The clones on
plate B at a ratio of 1:5 grew evenly on the LB plate and in a healthy dense. The clone on plate C
with the ratio of 1:10 grew denser than that on A and B plates. The per square clone numbers of
different ratios were accounted for in the graph bar in Figure 9. There were 35 clones per square
centimeter in the 1:10 inserter/vector ratio averagely, significantly higher than that in the 1:5
inserter/vector ratio averagely (22). It further verified the reconstruction and ligation process of
anti-TIC ScFv with pSecTag2 A, and random clones in the plates with inserter/vector volume ratios
of 1:5 and 1:10 are chosen for the transformation process.

A B
Figure 10. The RE digestion results of recombinant plasmid. (A) The inserter targeted PCR result in 1% DNA agarose
electrophoresis. (B) Reconstructed plasmid RE digested results in 1% agarose DNA electrophoresis. The two bands indicate by
the red arrows are the locations of pSecTag2 A vector (5.6kbp), anti-TICs ScFv (728bp) sequence respectively.
After the ligation process, inserter targeted PCR was used to verify the process of the ligation. The
product of PCR was loaded in the 1% DNA agarose for electrophoresis analysis. The clear band
nearby 700bp was consistent with the expected length of the inserter part. Besides, the
transformation of the reconstructed plasmid was processed, and the product was eluted. NotI and
HindII were used to digest the transformation product for 2 hours and then loaded in 1% DNA
Anti-TIC ScFv sequence
Linear pSecTag2 A vector
bp
500
700
20000
5000
2000
bp
20000
5000
1500
700

21
agarose gel for 20 minutes electrophoresis. The result indicated there were two clear bands which
were consistent with expected lengths of anti-TIC ScFv Sequence and linear pSecTag2 A vector.
Those two electrophoresis results further verified the digestion and ligation process.
4.3 Coomassie blue staining and Western blot results verify the anti-TIC ScFv expression

M: maker
A: Free Medium
B: Medium (DMEM Ham’s F12+FBS+NEAA+P/S)
C: Supernatant of transfect pSecTag2 A vector only
D: Supernatant of transfect of reconstructed anti-TIC ScFv plasmid
To verify the long and apparent bands near and above to the 80KD, the free medium group (A)
was added. There was no band in this group, which indicated that those bands represented protein
components in the culture media. In group D, there is an apparent band (the positions are indicated
by the red arrows near to 63KD) indicated the expression of the interest protein. The group D is
the raw supernatant sample, and group G is the sample of eluted supernatant. The target band in
the group D is more evident than that in group G. One of the possible reasons is the protein loss
during the purification process. In future research, the purification and elution methods will be
modified for decreasing the protein loss during the process. The protein concentration of anti-TIC
ScFv was detected by Bradford Assay which is estimated as 1.4mg/𝜇𝑙 after the purification process.
M A B C D
50
150
100
75
250
Figure 11. The Coomassie Brilliant blue staining (left) and Western-blot results (right)
of anti-TIC ScFv
37
25
KD
50
150
100
75
250
37
25
KD
20
M
Anti-TIC
ScFv

22
4.4 The result of FACS assay indicates the specifical binding ability of anti-TIC ScFv to Huh7
CD133(+) TICs.

A

B

C

D
Figure 12. The FACS assay results. (A) anti-TIC ScFv binds to Huh 7 CD133(+) TICs and mouse anti-his tag antibody
conjugated with FITC was used to detect the positive binding percentage. (B) anti-TIC ScFv binds to CD133(-) TICs and mouse
anti-his tag antibody conjugated with FITC was used to detect the positive binding percentage. (C) Normal human IgG antibody
conjugated with Fluor
®
458 binds to Huh7 CD133(-) TICs. (D) Goat anti-human CD133 antibody conjugated with FITC binds to
Huh 7 CD133(+) TICs.
Figure 12A. is the FACS result of anti-TIC ScFv binds to Huh7 CD133(+) TICs. The gate was set
in the R1 region, and the FITC signal peak is located between 10
1
to 10
2
with a cell account number
more than 170. Figure 10B results from anti-TIC ScFv binds to Huh7 CD133(-), and the FITC
37.8%
%%
21.5%
%%
8.7%
%%
59.6%
%%
R1

23
signal peak is located within 10
1
with cell account number more than 175. Figure 10C. is the result
of Human IgG immunoglobulin bind to Huh7 CD133(-) TICs. The Fluor
®
458 signal peak is
located within 10
1
, with the highest cell number nearly 200. Figure 10D. is the result of Goat anti-
human CD133 antibody binds to CD133(+) TICs and the FITC signal peak located beyond 10
2
with the highest cell number nearly 190. The FACS assay results provided evidence that anti-
human CD133 has the highest binding percentage to the Huh7 CD133(+) TICs. The binding
percentage of anti-TIC SCFv to the Huh7 CD133(+) TICs is significantly higher than that of
human IgG to the Huh7 CD133(+) TICs and that of anti-TIC ScFv to Huh7 CD133(-) TICs. This
result showed that anti-TICs ScFv had a specific binding ability to the Huh7 CD133(+) TICs.
4.5 The PCR result verified the ligation process of the TRITE and pSecTag2 A vector

Figure 13. The PCR result (the band indicated by the red arrow is the PCR product)
After the ligation process, PCR targeting the insert part was used for verification of the ligation
process. The well designed forward and reverse primers were produced by IdtDNA company. The
band indicated length of the PCR product located between the 2kbp and 3kbp which is consistent
with the length of the TRITE sequence (2.3kbp). This result verified the ligation of TRITE sequence
and pSecTag2 A vector.
bp
20000
10000
7000
5000
1500
2000
500
The PCR product

24
4.6 Western-blot result indicated the expression of engager protein TRITE

Figure 14. The Western-blot result of TRITE
In the western-blot assay, the mouse anti-His tag antibody was used as the primary antibody, and
donkey anti-mouse conjugated with FITC antibody was used as the second antibody. There is a
band observed between 100KD-150KD which is also matched with estimated molecule value of
TRITE (143KD). The result of western blot indicates the expression of the target protein TRITE.

TRITE

25
Chapter 5. Summary
After performing this extensive study, the high-quantity of immune cells surrounding the
inflammatory tumor liver tissue was identified, which provides the prerequisites for our hypothesis
that designing tri-specific binding engager protein to promote immune recognition and activation
for tumor cell clean up. In the later assays, the anti-TIC ScFv sequence's top candidates are
screened out by the phage display library of human immunoglobulins. After the ligation with
pSecTag2 A vector, the reconstructed plasmid was transfected into HEK293T cell lines for the
generation of anti-TIC ScFv. The purified anti-TIC ScFv was detected by Western-blot assay. The
FACS assay identifies its Huh7 CD133(+) TICs specific binding ability. The Co-IP and Mass-
spectrometry assay were used for the potential ligands of anti-TIC ScFv detection on the surface
of Huh7 CD133(+).
Furthermore, the sequence of tri-specific T cell engager protein TRITE was designed and
synthesized by the GBlock method. Through the digestion and ligation process with pSecTag2 A
vector, the recombinant plasmid was constructed. The engager protein TRITE was produced by
HEK293T cell line and purified by Ni-NTA Agarose beads. The Coomassie Brilliant Blue staining
and Western-blot assay were used to verify the target protein expression. In the future, the
Chromium
51
label cytotoxicity assay will be used to identify its cytotoxicity by promoting function
of cellular immunity activation in vivo, and humanized PDX mouse models will be built for the
animal trial. All the current data indicated the specific binding ability of engager protein TRITE
to the T-lymphocytes, DCs and TICs which owns prospective and potential application in anti-
tumor immunotherapy.

26
Chapter 6. Future direction
6.1 The Cytotoxicity Assay of engager protein TRITE in vitro
All the volunteers who participated in this research had signed the informed consent form for
human liver tissue samples. This study's process was also approved by the ethics committees of
the Keck medicine school of USC.
The peripheral blood mononuclear cells donated by HCC patients were isolated through Ficoll-
Hypaque density centrifugation. They were grown in RPMI medium (supplied with 100U/ml
penicillin, 200µg/L streptomycin, 2mM L-glutamine, and 100µg/ml 2-mercaptoethanol) with the
concentration of 107 cells/ml. The solution consisted of anti-CD3 (50ng/ml), IL-2 (300U/ml), and
IFN-γ(1000U/ml) was used for the activation and expansion of effector cells for 72 hours. The
concentration of the effector cells was adjusted to 10
6
cells/ml and incubated for 14 days. The
medium was refreshed every 48 hours
[22]
.
The homological human CD133(+) tumor-initiating cells were also collected from the same HCC
patients and grown in the DMEM medium in a 12-well bottom plate. 300µCi
51
Cr was used for 2
hours labeling at room temperate. The cell concentration was adjusted to 10
4
cells per well. Next,
the PBS was used to wash away unattained
51
Cr for three times. The well-prepared PBMCs and
engager protein TRITE were added in different rations for 3 hours of incubation at room
temperature. The supernatant of every well was collected for released
51
Cr counting by a gamma
counter. 1% NP40 was added into one of the wells for measuring the maximal killing, and there
was another well of cell culture treated without PBMCs for counting the spontaneous release. The
equation: the percentage of specific lysis = 100× [(test
51
Cr release) – (spontaneous
51
Cr release)] /
[(maximal
51
Cr release) − (spontaneous
51
Cr release)] was used for counting lysis percentage.

27
Table 7. The effector/target ratios in cytotoxicity assay
PBMCs (x10
4
cell number)
Engager protein TRITE
(microgram)
Huh7 CD133(+) tumor-initiating
cell (x10
4
cell number)
40
5
1 10
20
10
5
1 10
20
5
5
1 10
20

Besides, there are two groups designed for the detection of spontaneous release and maximum
release. In the spontaneous release detecting group, Huh7 CD133(+) TICs are incubated without
the addition of PBMCs. In the maximum release detecting group, 2.5% Triton X-100 buffer was
used for Huh7 CD133(+) TICs lysis, which leads to the complete release of
51
Cr radiation.

Figure 15. The overview of the Chromium-51 labeling cytotoxicity assay. Huh7 CD133(+)/(-) cell line or normal
human hepatocytes would be cultured in the DEME medium which would be refreshed before the assay. The
chromium-51 will be used to mark the cell nuclei for 2 hours. After completely wash away the unattached chromium-
51, the TRITE and well-prepared PBMC will be added in different volume ratios for 3 hours incubation. The released
radiation signal will be detected by Beckman LS 6000TA.

28
6.2 The humanized tumor-bearing NSG Mouse Model building for animal trials
Currently, immunotherapy is regarded as a hot field of cancer treatment. Although the specific
binding ability of the anti-TIC to the human tumor-initiating cell and the killing activity of fusion
protein TRITE has been verified to improve its potential for clinical treatments further, there is
still much research done. To identify the feasibility of the TRITE for clinical use, we are going to
design an animal trial for pre-clinical treatment evaluation.
The patient-derived xenograft (PDX) model is now widely used for cancer researches. The
scientist can observe original tumor characteristics such as heterogeneity, complexity, and
molecular diversity through the PDX model
[23]
. What we are going to do is using immune-
compromised mice, which are four-week-old nude mice (order from the Central Lab company), as
the receptor of patient-derived hepatic carcinoma to build the PDX model. The primary sample of
the HCC will be obtained by the patient in the age range of 40-59 years old mixed gender and
inoculated in the right rear flank of the mice. After the PDX models are built successfully, we will
treat the human-derived HCC mice with the TRITE mixed human PBMC through subcutaneous
injection then observe and record the tumor growth and mice model survivals. The PBS mixed
with human PBMC treatment will be used as the control group. After the seven-week continuous
injections, we will detect the tumor cell growth situation through the histological analysis and
tumor microenvironment analysis to verify the treatment effect of the TRITE.

29

Figure 16. The overview of Preclinical Xenograft NSG Mouse Model building

6.3 Knockout the genes encoding anti-TIC ScFv ligand in the mouse model to identify its
binding efficiency.
After detecting the potential ligand candidates of anti-TIC ScFv by Co-IP and Mass-Spectrometry
assay, its specific binding ability can be further identified by knocking out its relevant genes in the
humanized PDX mouse model.
The comparison of TRITE anti-tumor effect in WT and relevant genes knockout phenotype will
further identify its specific binding site of anti-TIC ScFv. This result may provide evidence of
combined treatment of TRITE conjugated with other anti-tumor drugs in future clinic applications.

30
Figure 17. The overview ligand genes knockout assay
6.4 Investigate the combination treatment effect of TRITE with inhibition of PD1/PD-L1
Programmed cell death protein 1(PD-1) and its ligand PD-1L play an important role in the human
immune system
[24]
. They work as the immune checkpoint presenting on the surface of the host cells,
and one of the major functions is to suppress the immune response, which would help prevent the
killing of the bystander host cells
[25]
.
In cancer diseases, the interaction of PD-1 on the immune cells and PD-1L on the surface of the
cancer cell will block the T-lymphocyte activation and proliferation, leading to the immune
resistance of cancer
[26]
. The drug inhibitors of PD-1/PD-1L are currently novel and effective anti-
tumor treatments approved by the FDA
[27]
. After the identification of the anti-tumor effect of tri-
specific engager protein TRITE in humanized PDX mice model. The tumor, the combination
treatment of TRITE and PD-1/PD-1L, will be tested in humanized PDX mice, and the tumor
growth, immune cytokines level, and survival time of the mice will be observed and recorded.
Theoretically, the PD-1/PD-1L inhibitor will “switch off” the immune escape pathway of tumor
cells
[28]
; besides, the “bridge” function of the TRITE will significantly strengthen the recognition
and activation of the T-lymphocytes to the tumor cells. The combination treatment of PD-1/PD-
1L inhibitors and TRITE may potentiate anti-tumor therapy in clinic cases.
6.5 Exploring the anti-tumor effect of TRITE in other types solid tumor
After identifying the specific binding ability and killing ability of TRITE to the CD133(+) Huh7
TICs, its anti-tumor effect in different kinds of solid tumors is promising.
The studies found the widely-expression of CD133 in several types of solids tumor, including
ovarian cancer stem cells, head, and neck cancer (HNSCC)
[29]
. According to the research made by

31
Dr. Daniel Vallera and his group. Some solid tumor cells and their subpopulation express a high
percentage of CD133 makers
[30]
. In future research, several top CD133 expression solid tumors
may be chosen for testing the TRITE effect, which will provide supportive evidence for wide clinic
applications of TRITE in the future.
Table 8. The percentages of CD133 expression in different tumor types
Tissue source CD133(+) cell group CD133(+) Tumor
cells in tumor tissue
Colon Cancer inducing subpopulation 2.5%
Melanoma Cancer inducing subpopulation 1%
Lung Cancer inducing subpopulation 10%
HNSCC subpopulation 18%
Ovarian Cancer inducing subpopulation 5.6%-16%
Pancreatic subpopulation >15%
Gastric subpopulation >1%
Hepatocellular subpopulation 1%-3%

32
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34
Appendix

Primer for the PCR
Forward Primer TAGCGTCAAGATGTCCTGCA
Reverse Primer GACTTGTCGGTTGGTGTAGC

Sequence of anti-CD3 ScFv (published by Dr. Gilliland and his team)
Anti-CD3 ScFv
sequence
ATG AGG TGC AGC TTC AGC AAT CCG GGG
CAG AAC TGG CTA GAC CTG GGG CTA GCG
TCA AGA TGT CCT GCA AAG CCT CCG GAT
ATA CTT TCA CAC GCT ATA CCA TGC ATT
GGG TCA AAC AAC GAC CAG GTC AAG GCC
TCG AAT GGA TCG GCT ATA TCA ATC CGA
GTA GGG GAT ACA CCA ACT ACA ACC AGA
AGT TCA AGG ATA AGG CTA CAC CAA CCG
ACA AGT CAT CTT CAA CGG CCT ATA TGC
AGC TTT CCA GCC CAA GCG AGG ATT CTG
CCG TTT ACT ACT GTG CGC GGT ACT ATG
ACG ATC ACT ACT GCT TGG ACT ATT GGG
GCC AAG GTA CCA CTT CCG TAT CTA GCA
GGA GGC GGT GGT TCA GGC GGA GGT GGG
AGT GGC GGT GGT GGA TCT ATG CAG ATT
GTG CTC ACT CAG TCC CCT GCA ATC ATG
TCA GCA AGT CCT GGG GAG AAA GTG ACT
ATG ACT TGT TCC GCT AGC AGC AGT GTG
AGC TAC ATG AAC TGG TAC CAG CAG AAG
TCT GGA ACC TCA CCC AAA CGG TGG ATC
TAC GAC ACA TCA AAA CTG GCC AGT GGG
GTT CCA GCG CAT TTT CGT GGG TCT GGC
AGC GGA ACA AGC TAT TCC CTG ACG ATT
TCC GGG ATG GAA GCC GAG GAT GCA GCC
ACC TAT TAC TGC CAG CAG TGG AGC TCA
AAT CCC TTT ACC TTC GGA TCT GGC ACT
AAG CTG GAG ATA AAT

35
Sequence of anti-CD14 ScFv (published by Yong-Min Tang and his team)
Anti-CD14 ScFv
sequence
CAG GTC CAA CTG CAG CAG CCT GGG GCT
GAA CTG GTG ACG CCT GGG GCT TCA GTG
AAG TTG TCC TGC AAG GCT TCT GGC TAC
ACC TTC ACC AGC TAC TGG ATG CAC TGG
GTG AAG CTG AGG CCT GGA CTA GGC TTT
GAG TGG ATT GGA GAG ATT AAT CCT AGC
AAT GGT GGT ACT AAC TAC AAT GAG AAG
TTC AAG AGA AAG GCC ACA CTG ACT GTA
GAC ACA TCC TCC AGC ACA GCC TAC ATG
CAA CTC AGC AGT CTG ACA TCT GAG GAC
TCT GCG GTC TAT TAC TGT ACA ATT GAC
ACC TCG GAC TAC GTG GAC TTT GAC TAC
TGG GGC CAA GGC ACC ACT CTC ACA GTC
TCC TCA GCC GGC GGA GGT GGA AGC GGA
GGC GGA GGA AGC GGC GGT GGA GGC TCT
GAT ATC CAG ATG ACA CAG ACT ACA TCC
TCC CTG TCT GCC TCT CTG GGA GAC AGA
GTC ACC ATC AGT TGC AGT GCA AGT CAG
GGC ATT AGC AAT TAT TTA AAC TGG TAT
CAG CAG AAA CCA GAT GGA ACT GTT AAA
CTC CTG ATC TAT TAC ACA TCA AGT TTA
CAC TCA GGA GTC CCA TCA AGG TTC AGT
GGC AGT GGG TCT GGG ACA GAT TAT TTT
CTC ACC ATC AGC AAC CTG GAA CTT GAA
GAT TTT GCC ACT TAC TTT TGT CAG CAG
TAT AGT AAG CCT CCG TAC ACG TTC GGA
GGG GGG ACC AAG TTG GAA ATA AAA

Abstract (if available)

Abstract Hepatocellular Carcinoma (HCC) is widely acknowledged as one of the biggest threats to public health. More than 10 million people are estimated to die from various forms of cancer every year. For recent decades, mainstream Chemotherapy does not sufficiently eliminate tumor-initiating cells (TICs). Immunotherapy, as a burgeoning technology that focuses on applying the principle of immune reaction to clear up cancer cells, has come to scientists' notice. Emerging data suggest that nanoparticle-based siRNA delivery targeting stem-ness transcription factor NANOG (pluripotency transcription factor) potentiates anti-tumor immunity. Here, we hypothesized that designing a specific engager protein targeted tumor-initiating cells, T-lymphocytes and antigen presenting cells (APCs) would sensitize the efficacy of immunotherapy on liver cancers. Through phage-display library screening, a type of novel TIC-targeting immunoglobulin variable chain fragments (anti-TIC ScFv) were identified. After screening out the DNA sequence of anti-TIC ScFv, it was ligated with pSecTag2 A vector and transfected into the HEK293T cell line for targeting protein producing. After the purification process, the specific binding ability of the TIC-targeting ScFv to the CD133(+) Huh7 TIC is evaluated by the FACS assay

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Asset Metadata

Creator Ma, Wei (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

Publication Date 02/23/2021

Defense Date 12/18/2020

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

Tag immunotherapy,OAI-PMH Harvest,T cell engager protein,tumor initiating cells

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Machida, Keigo (committee chair), Epstein, Alan L. (committee member), Shao, Ling (committee member), Yuan, Weiming (committee member)

Creator Email 326540596@qq.com,wma377@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-423115

Unique identifier UC11667828

Identifier etd-MaWei-9287.pdf (filename),usctheses-c89-423115 (legacy record id)

Legacy Identifier etd-MaWei-9287.pdf

Dmrecord 423115

Document Type Thesis

Rights Ma, Wei

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