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Reprogramming exosomes for immunotherapy of acute myeloid leukemia

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Reprogramming exosomes for immunotherapy of acute myeloid leukemia

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i
Reprogramming Exosomes for Immunotherapy of Acute Myeloid
Leukemia
By
Tianling Hou

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA

In partial Fulfillment of the
Requirement for the Degree
MASTER OF SCIENCE
(Pharmaceutical Sciences)
May 2019

ii
ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to my advisor Dr. Yong Zhang for his guidance, and
encouragement towards my graduate work. You always support me in both research and career
and give me many opportunities to work and learn in lab.

Beside my advisor, I would like to thank the rest of my thesis committee: Dr. Angel Tabancay and
Dr. Curtis Okamoto for their advice and support on my work and thesis.

Moreover, I would like to thank all members of Dr. Yong Zhang’s Lab for their help in my research:
Xiaojing Shi, Qinqin Cheng, Jeff Dai, Xiao-Nan Zhang, Julianna Chen, Xinping Duan and Wilson
Lee. Also, I would like to thank my friends and all other members in USC who give me help and
support me during the time at USC.

iii
TABLE OF CONTENTS
ABBREVIATIONS ................................................................................................................................. v
ABSTRACT ............................................................................................................................................ vi
INTRODUCTION ................................................................................................................................... 1
1. Leukemia ......................................................................................................................................................... 1
2. Current therapy for Acute Myeloid Leukemia ............................................................................................ 4
2.1 Chemotherapy ............................................................................................................................................ 4
2.2 Hematopoietic stem cell transplantation .................................................................................................... 4
2.3 Nanoparticle-based therapy ........................................................................................................................ 5
2.4 Immunotherapy .......................................................................................................................................... 6
3. Exosomes for cancer therapy ......................................................................................................................... 7
3.1 Biochemical composition of exosomes ...................................................................................................... 7
3.2 Exosomes as next-generation cancer therapy .......................................................................................... 12
OBJECTIVES OF PROJECT ............................................................................................................. 13
MATERIALS AND METHODS ......................................................................................................... 14
1. Materials .................................................................................................................................................. 14
2. Cell lines ................................................................................................................................................... 14
3. Molecular cloning of SMART-Exos ....................................................................................................... 14
3.1 PCR .......................................................................................................................................................... 17
3.2 Restriction enzyme digestion ................................................................................................................... 20
3.3 Plasmid transformation ............................................................................................................................ 20
3.4 Colony PCR ............................................................................................................................................. 20
3.5 Agarose gel electrophoresis ..................................................................................................................... 20
3.7 Plasmid purification ................................................................................................................................. 21
4. Expression of SMART-Exos ................................................................................................................... 21

iv
5. Exosomes purification ............................................................................................................................. 22
6. Nanoparticle tracking analysis (NTA) ................................................................................................... 22
7. Transmission electron microscopy (TEM) ............................................................................................ 22
8. Western blot analysis. ............................................................................................................................. 22
9. Flow cytometric analysis ......................................................................................................................... 23
RESULTS ............................................................................................................................................... 25
1. Plasmid construction ............................................................................................................................... 25
2. Expression and identification of SMART-Exos .................................................................................... 27
3. Characterization of SMART-Exos ........................................................................................................ 30
4. Antigen expression analyses of AML cell lines ..................................................................................... 33
5. Binding assays .......................................................................................................................................... 35
6. In vitro cytotoxicity assays ..................................................................................................................... 40
DISCUSSION ........................................................................................................................................ 45
REFERENCES ...................................................................................................................................... 48

v
ABBREVIATIONS

AML: Acute Myeloid Leukemia
SMART-Exos: Synthetic Multivalent Antibodies Retargeted Exosomes
CLL-1: C-type Lectin-like Molecule-1
HSCs: Hematopoietic Stem Cells
CLL: Chronic Lymphocytic Leukemia
CML: Chronic Myeloid Leukemia
ALL: Acute Lymphocytic Leukemia
PBS: Phosphate-buffered saline
FBS: Fetal Bovine Serum
PBMCs: Peripheral Blood Mononuclear Cells
PCR: Polymerase Chain Reaction
PDGFR: Platelet-derived Growth Factor Receptor
scFv: Single-chain variable Fragment
HA: Hemagglutinin

vi
ABSTRACT

Exosomes are nano-sized membranous vesicles and are widely distributed in various body fluids.
These cell-derived nanoparticles are less immunogenic than artificial delivery vehicles, and
engineered forms of exosomes hold great potential as novel therapeutic modalities. Acute myeloid
leukemia (AML) is a common type of leukemia with poor prognosis in adults. In this thesis study,
synthetic multivalent antibodies retargeted exosomes (SMART-Exos) were genetically
engineered, and two distinct types of monoclonal antibodies were displayed on the exosomal
surface. Human C type lectin like molecule 1 (CLL-1) is overexpressed in AML blasts and
leukemic stem cells. By targeting CLL-1 and T-cell CD3 receptor, the generated SMART-Exos
were designed to redirect T cells against AML cells for killings. It was demonstrated in this study
that the resulting anti-CD3×CLL-1 SMART-Exos not only bind tightly to both T-cells and CLL-
1-positive AML cells but also elicit potent antitumor immunity in a dose-dependent manner. This
work provides a novel approach for the development of exosomes as immunotherapeutics for AML
treatment.

1
INTRODUCTION

1. Leukemia
For a healthy adult, approximately 10
11
-10
12
new blood cells are produced daily to maintain a
steady state in the peripheral circulation
[1]
. This remarkable cell renewal process is supported by
a small population of bone marrow cells termed hematopoietic stem cells (HSCs). Bone marrow
is the home to HSCs that can differentiate into any mature blood cell types. Figure 1. shows a
diagram of the formation of different blood cells from HSCs to mature cells
[2]
.
Leukemia is any myeloid or lymphoid malignancy that develops in the peripheral blood and bone
marrow
[3]
. Patients with leukemia have bone marrow that produces abnormal and poorly
functioning white blood cells that divide out of control. The broad classification of leukemia is
based on the rapidity of the clinical course, either chronic or acute leukemia. And these two types
of leukemia can be further grouped based on the lineage of affected white blood cells. Leukemia
that affects lymphoid cells is called lymphoid, lymphocytic, or lymphoblastic leukemia, while the
one derived from myeloid cells is classified as myeloid, myelogenous, or myeloblastic leukemia.
Thus, depending on whether the leukemia is chronic or acute, myeloid or lymphocytic, there are
four main classifications of leukemia
[4]
:
• Chronic lymphocytic leukemia (CLL) occurs when the bone marrow produces a high abundance
of lymphocytes. In most cases, CLL progresses slowly and exhibits no symptoms at early stages.
This type of leukemia most commonly occurs in adults, and patients diagnosed with this disease
most often are over the age of 55. It accounts for nearly 15,000 new cases of leukemia each year.
• Chronic myeloid leukemia (CML) is a slow, progressive bone marrow and blood type cancer
caused by an increased number of granulocytes. CML is generally diagnosed by detection of a

2
malfunction in two chromosomes resulting in the hybrid creation of another chromosome called
the Philadelphia Chromosome. It accounts for around 5,000 new cases of leukemia each year and
mainly affects older adults.
• Acute lymphocytic leukemia (ALL) is a type of progressive bone marrow and blood cancer
caused by the rapid proliferation of immature lymphocytes. The excessive abnormal lymphoid
cells eventually crowd out healthy cells in the bone marrow, and metastasize to other organs, which
can be fatal in weeks to months if left untreated. The symptoms of AML include anemia, infection,
fever, and unexpected bleeding. ALL is most commonly found in childhood and young adulthood.
It accounts for around 5,000 new cases of leukemia each year.
• Acute myeloid leukemia (AML) is a form of bone marrow and blood cancer that is characterized
by an increased number of undifferentiated myeloblasts. AML occurs when leukemia affects the
myeloid cells in the bone marrow which under normal conditions, turn into red blood cells, white
blood cells, and platelets
[5]
. As a result, leukemia cells rapidly proliferate in the bone marrow and
blood and will migrate to other parts of the body, including the central nervous system (brain and
spinal cord) and skin. AML progresses quickly and can eventually lead to fatal complications of
infection, bleeding, or organ infiltration within weeks or months. It occurs in both adults and
children. The five-year survival rate of AML is less than 25% for adults. It accounts for over
13,000 new cases of leukemia each year.

3

Figure 1. The process of hematopoiesis
[2]
.

4

2. Current therapy for Acute Myeloid Leukemia
2.1 Chemotherapy
Chemotherapy is the primary treatment for people with AML and characterized by a cure rate
between 20-75% for patients younger than 60 years old
[6]
. This wide range depends primarily on
leukemia-cell cytogenetics, which is related to the structure and function of the cell, especially the
chromosomes
[6]
. Usually, once a patient has been in remission for three years, the likelihood of
relapse declines sharply to less than 10%
[7]
. At best, standard approaches of AML treatment can
achieve ultimate curative value in 40% of patients. For certain subtypes, however, the curative
potential is far from satisfactory level. In patients older than 60 years, chemotherapy results in a
cure rate of less than 10%, due to the inability of elderly patients to survive the treatment and they
are likely to exhibit therapeutic resistance or have medical impediments to the successful
completion of such regimens.
Although chemotherapy can improve overall survival in patients with AML, the prognosis is still
poor with a five-year survival rate of 30%, regardless of receiving hematopoietic stem cell
transplantation (HSCT). Thus, there is an urgent need to develop new therapeutic approaches for
AML treatment.
2.2 Hematopoietic stem cell transplantation
Hematopoietic stem cell transplantation (HSCT) has been used for the treatment of AML for
decades
[8]
. It involves the infusion of hematopoietic stem cells to reestablish bone marrow function
in cancer patients whose bone marrow is removed by receiving bone-marrow-toxic doses of
cytotoxic drugs. In AML disease, there is a high incidence of relapse, which has prompted the

5
application of post-remission strategies using either patients' own stem cells (autologous HSCT)
or stem cells from another acceptable donor (allogeneic HSCT). Although HSCT has cured
patients with hematologic malignancies and bone-marrow damages, it is much more toxic than
chemotherapy and immunosuppressive therapy. Moreover, relapse after allogeneic HSCT does
occur, and the vast majority of elderly patients are not eligible for HSCT. Therefore, HSCT is only
suggested for cases in which the survival time and quality of life exceed that of treatments other
than HSCT and should be carefully evaluated in terms of the latest guidelines and transplantation
outcomes for each patient.
2.3 Nanoparticle-based therapy
Traditional treatments for AML involve chemotherapy and radiation, which often cause long-term
side effects and multidrug resistance. The nanotechnology has become a powerful approach to
overcome limitations associated with conventional drugs. Nanoparticles can enhance the
therapeutic efficacy of anticancer agents, improve biocompatibility and delivery, and help
overcome treatment resistance
[9], [10], [11]
. The nanosize range of these particles allows them to
cross biological barriers more effectively that may be further improved by functionalizing the
nanoconstructs’ surface with specific ligands for precise delivery to the disease targets
[12]
.
In general, the nanosized particles allow for efficient uptake by a variety of cell types and
selectively deliver anticancer agents to target sites
[13]
. There is a wide variety of nanoparticles,
including organic, inorganic, and hybrid nanoparticles. Organic nanocarriers have been
extensively explored in cancer, including dendrimers, lipid-based nanoparticles, and polymeric
nanoparticles. Dendrimers are highly branched, exhibiting high versatility and functionality in
drug delivery with a maximum of 10 nm of size. Lipid-based nanoparticles, such as liposomes,
micelles, and hybrid systems are prominent drug delivery vehicles with improved biocompatibility

6
and prolonged blood circulation, and typically have 50–100 nm of size. Polymeric nanoparticles,
ranging from 10 to 400 nm, are produced from natural, synthetic, hydrolytically, or enzymatically
degradable polymers onto which a cytotoxic drug can be covalently attached, dissolved,
encapsulated, or entrapped
[14], [15]
.
2.4 Immunotherapy
Immunotherapy has radically revolutionized cancer therapy over the past decade. Though HSCT
is one of the most successful immunotherapeutic strategies for postremission therapy in AML
[16],
[17]
, relapse after allogeneic HSCT does occur, and it is not eligible for most elderly patients.
Alternative AML immunotherapies have been studied in the past few years. However, the slow
progression of translating immunotherapeutics for AML to the clinic is hindered by the complexity
of the disease, including heterogeneous antigen expression of diverse AML cell populations, low
endogenous immune responses, and intrinsic immune response-driven resistance mechanisms of
the leukemic blasts. Therefore, new immunotherapeutic strategies for AML are urgently needed
to improve patients’ survival of this aggressive disease.
At the current stage, various therapeutic modalities have been developed for AML immunotherapy,
including targeted immunotherapy, checkpoint inhibitors, therapeutics vaccines, antibody-drug
conjugates (ADCs), and chimeric antigen receptor-T cells (CAR-T) therapies. For targeted
immunotherapy, it relies on a suitable target antigen to minimize unwanted on-target off-tumor
toxicity. In AML, it is difficult to find a lineage-restricted target antigen with a minimal expression
on healthy hematopoiesis cells. It is expected that targeting AML-associated antigens will result
in boosting the ability of immune cells to kill cancer cells.
Checkpoint inhibitors rely on the improvement of endogenous immune responses by blocking
signaling pathways that stop the immune system from attacking the cancer cells. They have been

7
successfully approved in several solid organ malignancies and are now entering the treatment of
hematological diseases
[18]
.
Priming the immune system with therapeutic vaccines, particularly studies based on dendritic cells,
have also been shown to induce anti-leukemic immune responses reliably
[19]
. Immune checkpoint
blockade therapy and dendritic cells vaccines appear to be safe but have yet to demonstrate their
clinical potency when used as a monotherapy for the treatment of AML.
Antibody-drug conjugates (ADCs), consist of monoclonal antibodies conjugated to small-
molecule cytotoxic drugs, show great therapeutic potential in AML. Several clinical trials have
been performed to evaluate the risk-benefit ratio
[20]
. In contrast, T cell recruiting antibodies and
CAR-T cell constructs are still in the early clinical development for the therapy of AML. Their
feasibility of applications and potential side effects have been studied under currently ongoing
phase I trials. Future efforts have to be taken to integrate best immunotherapeutic approaches into
individualized curative treatment for AML patient.
3. Exosomes for cancer therapy
3.1 Biochemical composition of exosomes
There are various types of extracellular vesicles (EVs) and can be mainly categorized in to three
classes based on their size and biogenesis pathways: apoptotic bodies, microvesicles, and
exosomes. Apoptotic bodies are generally larger in size (500-2000 nm) and are derived from cell
undergoing programmed cell death. Microvesicles are membranous vesicles (100-1000 nm) that
bud directly from plasma membrane. Exosomes are lipid bilayer-enclosed nano-sized EVs, ranging
from 30 to 100 nm in diameter, are secreted throughout all stages of the cell cycle (Figure 2.). They
are produced from inward budding of endosomal compartments called multivesicular bodies
(MVBs) and are released into the extracellular space upon fusion of the MVBs with the plasma

8
membrane. As endogenous nanocarriers, exosomes play important roles in mediating intercellular
communication
[21]
.

9

Figure 2. The categories of extracellular vesicles
[22, 23,24]
. Apoptotic bodies (500-2000 nm) are
derived from cell undergoing programmed cell death. Microvesicles (100-1000 nm) are produced
directly from plasma membrane. Exosomes (30 -100 nm) are generated from late endosomal
compartments through fusion of multivesicular bodies with the plasma membrane.

10
Owing to their endosomal origin, several members of the tetraspanin family including CD9, CD63,
and CD81 are enriched on exosomal membranes and serve as unique marker proteins (Figure 3.)

[25]
. Exosomes contain proteins required for membrane transport and fusion (Rab proteins,
annexins, flotillin), proteins associated with MVB biogenesis (Alix, TSG101), and heat shock
proteins (Hsc70, Hsp90) as previously reported
[26]
. This type of vesicles also carries a variety of
cellular proteins, RNA and miRNAs, cytoskeletal proteins and metabolic enzymes. In addition,
the exosomal membrane is enriched with lipid-rafts including cholesterol, sphingolipids, and
ceramide. Interestingly, exosomes secreted from antigen-presenting cells such as dendritic cells
express functional major histocompatibility complexes (MHC I and II) on their surface
[27, 28]
.

11

Figure 3. Biochemical composition of exosomes. Exosomes carry a variety of cellular components
including mRNAs, miRNAs and proteins that are often selectively packaged from the host cells.
The exosome membrane contains various proteins, including major histocompatibility complexes
(MHC I and II), targeting and adhesion (integrins and tetraspanins) molecules, membrane
trafficking regulators (annexins and Rab proteins) as well as lipid-rafts.

12
3.2 Exosomes as next-generation cancer therapy
Exosomes hold great therapeutic potential for cancer therapy. The tetraspanins on the exosomal
surface can promote direct membrane fusion and facilitate the release of their soluble cargoes into
the cytosol. Furthermore, CD47 found on exosomes is shown to prevent exosomes from
phagocytosis by circulating monocytes and macrophages and prolong exosomes half-life in the
blood circulation
[29]
. Current nanoparticle delivery systems are confronting with many issues such
as off-target cytotoxicity, poor biocompatibility, and low efficacy
[30], [31]
. Compared with
traditionally synthesized nanoparticles and viral vectors, cell-derived exosomes may exhibit
significantly reduced immunogenicity while possessing intrinsic targeting properties. Exosomes
may cross biological barriers and deliver their cargoes to recipient cells with high selectivity
[32]
.
Various anticancer drugs and functional proteins have been packed into exosomes for cancer
treatment. There are several ways to load cargoes into exosomes. After incubation small
membrane-permeable agents together with exosomes, the agents can be passively loaded into
exosomes
[33]
. However, to load membrane-impermeable drugs and functional proteins, such as
miRNAs, siRNAs, and small DNAs, electroporation is required to create pores on exosome lipid
bilayer membrane to allow them to be incorporated within exosomes
[34], [35], [36]
. Another approach
is surface engineering of exosomes via membrane proteins. The transmembrane protein on the
surface of exosomes can be used as a fusion partner
[37], [38], [39]
.
To deliver protein cargoes, researchers have fused soluble cargoes, such as transcription factors or
cytosolic proteins, with membrane proteins of exosomes, aiming to alter or supplement the
biological pathways of recipient cells. And engineering tissue-specific ligands on the exosomal
surface can enable targeted delivery of drugs and RNA therapeutics to specific target cells
[40], [41],
[42], [43], [44]
.

13
OBJECTIVES OF PROJECT

Cell-derived exosomes have shown to be promising nanotechnology platform in recent years.
Their ideal size range and intrinsic biocompatibility appeal great research interests in developing
exosomes as nanomedicine for various diseases. While the majority of studies have focused on the
applications of exosomes as therapeutic drug carriers, there is also growing interests in studying if
exosomes can be genetically modified for immune modulation. Here, we performed a proof of
concept study in which we designed and genetically engineered a new class of exosomes, termed
as synthetic multivalent antibodies retargeted exosomes (SMART-Exos). Exosomes were
genetically engineered by displaying two distinct antibodies on their surface. The resulting dual
targeting SMART-Exos can bind the T cell CD3 and CLL-1, a myeloid lineage-restricted cell
surface marker, with the aim of stimulating the immune responses by redirecting and activating T
cells to induce T cell-mediated tumor cell killing. Our study on engineering exosomes for AML
cancer immunotherapy could lend further support to the state-of-the-art concept of exosomes as
attractive immunotherapy candidates in preclinical and clinical development.
.

14
MATERIALS AND METHODS

1. Materials
Dulbecco’s modified Eagle’s medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640
medium, were purchased from Corning Inc. (Corning, NY). Fetal bovine serum (FBS) was
purchased from VWR International (Radnor, PA). Opti-modified Eagle’s medium (Opti-MEM),
Expi293 expression medium and ExpiFectamine 293 transfection reagent were purchased from
Thermo Fisher Scientific (Waltham, MA). Carboxyfluorescein succinimidyl ester (CFSE) and
FITC anti-human CD371 (CLEC12A) antibody were purchased from BioLegend (San Diego, CA).
Pierce Coomassie Plus (Bradford) assay kit was purchased from Thermo Fisher Scientific
(Waltham, MA).
2. Cell lines
Expi293F cells are suspension cells derived from human HEK293 cell line and were purchased
from Thermo Fisher Scientific (Waltham, MA). The Expi293 expression medium is a chemically
defined serum-free medium, developed for the growth and transfection of Expi293F cells. This
cell line is maintained in the cell medium with shaking at a speed of 125 rpm/min at 37°C in 8%
CO2. U937, HL-60, KG-1A, and Jurkat cell lines were all obtained from the American Type
Culture Collection (ATCC) (Manassas, VA) and cultured in RPMI 1640 medium supplemented
with 10% FBS at 37°C in 5% CO2. Human peripheral blood mononuclear cells (PBMCs) were
purchased from HemaCare (Van Nuys, CA).
3. Molecular cloning of SMART-Exos
The genes encoding scFv fragments of αCLL-1 (1075.7
[45]
) and αCD3 (UCHT-1
[46]
) antibodies
were inserted into the pDisplay vector (Thermo Fisher Scientific, Waltham, MA). (GGGS)4 and

15
(GGGGS)3 linkers were inserted between VL and VH regions of αCD3 scFv and αCLL-1 scFv,
respectively. To make αCD3-αCLL-1 scFv fusion protein, a (GGGGS)3 flexible linker was
designed between two distinct scFv fragments by overlap extension polymerase chain reactions
(PCR). The αCD3-αCLL-1 scFv fusion protein is genetically linked to platelet-derived growth
factor receptor (PDGFR) transmembrane (TM) domain on the surface of exosomes. The
orientation of variable region for designed construct is arranged as VL-αCD3 -VH-αCD3 -VL-αCLL-1-
VH-αCLL-1. In addition, αCD3 scFv antibodies and αCLL-1 scFv antibodies were separately fused
with PDGFR TM domain for generation of monoclonal exosomes as controls. An N-terminal HA
tag was added for all the antibody-PDGFR TM domain fusions.
Primers used for PCRs to amplify these gene fragments are listed in Table 1. with highlighted
restriction enzyme sites of BglII and SalI.

16
Table 1. List of primer sequences used for molecular cloning (BglII restriction site underlined;
SalI restriction site in bold).
Name Sequence
αCD3 scFv Forward 5′-GGCCAGATCTGATATCCAGATGACACAGACAACCTCAAGTCTTAG-3′
αCD3 scFv Reverse 5′-CCTGAGCCTCCCCCGCCTGATCCGCCACCGCCGCTGCTAACGGTAACGGTGGTACC-3′
αCLL-1 scFv
Forward
5′-AGGCGGGGGAGGCTCAGGCGGAGGTGGCAGCGAGAACGTGCTCACCCAATCCCC-3′
αCLL-1 scFv
Reverse
5′-GTTCGTCGACGCTTCCGCCACCCCCAGACACGGTCACGCTGGTGC-3′
αCD3-αCLL-1 scFv
overlap Forward
5′-AGGCGGGGGAGGCTCAGGCGGAGGTGGCAGCGAGAACGTGCTCACCCAATCCCC-3′
αCD3-αCLL-1 scFv
overlap Reverse
5′-CCTGAGCCTCCCCCGCCTGATCCGCCACCGCCGCTGCTAACGGTAACGGTGGTACC-3′

17
3.1 PCR
To generate gene fragments for cloning, AccuPrime ™ Taq DNA Polymerase (Thermo Fisher
Scientific, Waltham, MA) was used for the PCR amplification. The condition for a 50 μl PCR
reaction were as follows:
Template: 1 μl (50 ng)
Forward primer (10 μM): 1.5 μl
Reverse primer (10 μM): 1.5 μl
10X AccuPrime ™ PCR Buffer: 5 μl
AccuPrime ™ Taq DNA Polymerase: 0.5 μl
ddH2O: 38 μl
PCR and overlap extension PCR were followed by steps according to Table 2. and Table 3.

18

Table 2. PCR
Step Temp Time Number of cycles
Initial Denaturation 95°C 30 seconds

Denaturation 95°C 30 seconds
35 cycles
Primer Annealing 62°C 45 seconds
Extension (1 minute per
kb)
68°C 1 minute
Final Extension 68°C 5 minutes
Hold 4-10°C -

19
Table 3. Overlap Extension PCR

Step 1 (Without Primers) Temp Time Number of cycles
Initial Denaturation 95°C 30 seconds

Denaturation 95°C 30 seconds
15 cycles
Primer Annealing 64°C 45 seconds
Extension (1 minute per kb) 68°C 1 minute
Final Extension 68°C 5 minutes
Hold 4-10°C -

Step 2 (Add Primers) Temp Time Number of cycles
Initial Denaturation 95°C 30 seconds

Denaturation 95°C 30 seconds
25 cycles
Primer Annealing 62°C 45 seconds
Extension (1 minute per kb) 68°C 2 minutes
Final Extension 68°C 5 minutes
Hold 4-10°C -

20
3.2 Restriction enzyme digestion
The amplified inserts and pDisplay vector were digested by restriction enzymes BglII and SalI
(New England Biolabs, Ipswich, MA). The digestion of DNA fragments was carried out under the
conditions recommended by manufacturers. The digested products were ligated between the N-
terminal signal peptide and the transmembrane domain of human platelet-derived growth factor
receptor (PDGFR) in pDisplay vector by using T4 DNA ligase (New England Biolabs, Ipswich,
MA). A mixture of the digested pDisplay vector and insert gene fragments were incubated at 16℃
overnight.
3.3 Plasmid transformation
The generated expression plasmids were transformed into E. coli (strain DH10B). After
electroporation, DNA mixed with competent cells were recovered in LB for 1 h and was spread
onto a pre-warmed LB agar plate, containing appropriate selective antibiotic (100 μg/ml penicillin),
and was incubated at 37℃ for the colonies to grow.
3.4 Colony PCR
OneTaq DNA Polymerase (New England Biolabs, Ipswich, MA) was used in colony PCR to
screen the target colony. Single colonies were picked and resuspended in 10 μl of Taq polymerase
mixture to perform PCR. The positive recombinant plasmids screened by colony PCR were
confirmed by DNA sequencing provided by GENEWIZ (South Plainfield, NJ).
3.5 Agarose gel electrophoresis
Agarose gel electrophoresis was used for analysis of PCR products. 1.5 % agarose gel was used to
separate DNA fragments. The agarose gel was prepared by mixing agarose powder with 1× TAE
buffer to the desired concentration and then heated until completely melted.

21
3.6 DNA gel recovery
Zymoclean™ Gel DNA Recovery Kit (Zymo Research, Irvine, CA) was used to recover DNA
fragments from agarose gel. The excision and recovery of the DNA fragments from agarose gel
were carried out by the protocols provided by the manufacturer.
3.7 Plasmid purification
Sequence-verified expression plasmids were isolated from bacteria culture either by small-scale
purification or large-scale purification. The ZR Plasmid Miniprep-Classic kit (Zymo Research,
Irvine, CA) was used for small scale plasmid purification. Plasmid-bearing bacteria were
inoculated in LB medium containing antibiotic one day prior to plasmid purification. After 12~16
h of incubation at 37℃ and shaken at 250 rpm, the bacteria were centrifugated at 4000 ×g for
10min at 4℃ to form compacted pellets. The plasmids were then extracted and purified as
described in the instruction provided by the manufacturer.
ZymoPURE™ II Plasmid Maxiprep Kit (Zymo Research, Irvine, CA) was used for large scale
plasmid purification. Plasmid-bearing bacteria were inoculated in LB medium containing
antibiotic and incubated at 37℃ for 12~16 h to make a starter culture. 5 ml of starter culture was
added to 150 ml of LB medium and was incubated at 37℃, shaken at 250 rpm for another 12 h.
The bacteria were harvested by centrifugation at 4000 ×g for 10 min at 4℃. The plasmids were
then purified by following the manufacturer’s instruction.
4. Expression of SMART-Exos
The expression constructs of SMART-Exos were transfected into Expi293F cells cultured in
chemically-defined Expi293 expression medium by using ExpiFectamine 293 transfection kits
under the manufacturer’s protocol. Media were harvested on day 3 and day 6 post-transfection
through centrifugation.

22
5. Exosomes purification
Engineered exosomes were purified from the harvested culture media through differential
centrifugation. Cell culture media were centrifuged at a low speed of 500 ×g at 4°C for 10 min to
remove detached cells and then 30 min at 4000 ×g, followed by 15,000 ×g for 50 min to remove
cell debris and large vesicles. The obtained supernatants were then centrifugated at 371,000 ×g in
a Type 70 Ti rotor (Beckman Instruments, Indianapolis, IN) for 2 h at 4°C. After removing
supernatant, exosomes were washed and resuspended in PBS, followed by filtration through a 0.22
μm syringe filter. The protein concentrations of purified exosomes were determined by Bradford
assays.
6. Nanoparticle tracking analysis (NTA)
Particle concentration and size distribution of the purified exosomes were determined by NTA
using a Nanosight LM10 (Malvern Instruments, U.K.) according to the manufacturer’s instruction.
7. Transmission electron microscopy (TEM)
The exosomes were prepared and imaged by a JEOL 2010F TEM (JEOL, Peabody, MA). The
TEM grids were preincubated with 20 μL of the 0.1% poly-lysine solution for 10 min. Excess
liquid was removed with filter paper. 20 μL of the exosomes sample was placed on 200 μm mesh
grids and incubated for 15 min. Residual liquid was removed and dried again from the grids with
filter paper, followed by staining with 20 μL of 2% uranyl acetate solution for 5 min. The grid was
left to air dry.
8. Western blot analysis.
Exosome aliquots containing 3 μg protein were reduced with 10 mM dithiothreitol and boiled at
98°C for 10 min. For western blot without protein reduction (tetraspanins CD81 and CD63), DTT
was omitted and its volume was replaced by PBS. The samples were then separated in 4-20%

23
ExpressPlus-PAGE gels (GeneScript, Piscataway, NJ) at 155 V for 45 min. The gel was carefully
removed, washed with buffer, and subsequently transferred to Immun-Blot PVDF membranes
(BioRad Laboratories, Inc, Hercules, CA) at 16 V for 30 min using a Trans-Blot SD SemiDry
Transfer Cell (Bio-Rad Laboratories, Inc, Hercules, CA). The resulting membranes were blocked
with 5% BSA in PBST for 1 h at room temperature while gently shaking. The membranes were
incubated with the following primary antibodies: mouse monoclonal anti-HA (clone: 2-2.2.14)
from Thermo Fisher Scientific, mouse monoclonal anti-CD63 (clone: H5C6) from BioLegend,
mouse monoclonal anti-CD81 (clone: 1.3.3.22) from Thermo Fisher Scientific, and rabbit
monoclonal anti-CD9 (clone: D3H4P) from Cell Signaling Technology) for 1 h at room
temperature. The membranes were washed (3 × PBST, 5 min) and incubated with secondary
antibodies anti-mouse IgG-HRP (catalog number: 62-6520) from Thermo Fisher Scientific and
anti-rabbit IgG-HRP (catalog number: 65-6120) obtained from Thermo Fisher Scientific, and
further diluted in blocking buffer (1:2000) for 1 h at room temperature. SuperSignal West Pico
PLUS chemiluminescent substrate (Thermo Fisher Scientific) was used to develop blots according
to manufacturer’s instructions and immuno-active bands were detected by a ChemiDoc Touch
Imaging System (Bio-Rad Laboratories, Inc, Hercules, CA).
9. Flow cytometric analysis
Cell-based binding assays, antigen expression analyses, and in vitro cytotoxicity assays were
performed using flow cytometry. The binding of SMART-Exos to AML cell lines (U937, HL60
and KG-1A) and CD3-positive Jurkat cells were analyzed by flow cytometry. Cells were incubated
with 0.1 mg/mL exosomes in ice-cold PBS containing 1% (w/v) BSA and 10% human serum for
30 min on ice and washed with the same medium, followed by incubation with the anti-HA
antibody (clone: 2-2.2.14) from Thermo Fisher Scientific for 30 min on ice. After that, cells were

24
washed again and subsequently incubated with the Alexa Fluor 488-labeled goat anti-mouse IgG
H&L antibody (catalog number: ab150113) from Abcam for 30 min on ice. Then the cells were
washed, and the binding was evaluated by a BD LSR II Flow Cytometer (BD Biosciences, San
Jose, CA). Data were processed by FlowJo_V10 software (Tree Star Inc., Ashland, OR). Cell-
bound fluorescent-labeled antibody was analyzed as the mean fluorescence intensity (MFI) for 10
000 cellular events, and whole cells were analyzed using appropriate scatter gates to exclude
cellular debris and aggregates. Background fluorescence were determined by using target cells
incubated under the same conditions.
For in vitro cytotoxicity assays, target cells stained with CFSE (30,000/well) were mixed with
PBMCs (150,000/well) to afford an E:T ratio of 5:1, and incubated with PBS, different
concentrations of SMART-Exos and mixtures of monoclonal exosomes for 24 h at 37°C and 5%
CO2. Cells were then centrifuged, resuspended in PBS (with 2% FBS), and analyzed with the BD
LSR II flow cytometer. Cells that were FITC
+
(CFSE) were considered as the viable target cells,
and the relative viabilities of all treatment groups were normalized to the PBS group.

25
RESULTS

1. Plasmid construction
SMART-Exos were generated by displaying two individual antibodies on the exosomal surface
with the aim of redirecting the cytotoxic activity of effector T cells to attack cancer cells by
targeting T cell CD3 and CLL-1 simultaneously with high specificity. CD3 is an essential T
cell co-receptor and defines T cell lineage, and CLL-1 is a myeloid lineage-restricted cell surface
marker
[47]
.
Human platelet-derived growth factor receptor (PDGFR) is commonly used for the protein
expression in mammalian cell lines
[48]
. Here, we used the transmembrane (TM) domain of PDGFR
as a fusion partner to display single-chain variable fragment (scFv) antibodies on the exosomal
surface. To generate dual-targeting αCD3-αCLL-1 SMART-Exos, two single-chain variable
fragment (scFv) antibodies were encoded in single polypeptides, which were genetically linked to
the PDGFR TM domain (Figure 4. (C)). Encoding two individual scFvs into single polypeptides
is based on the idea to avoid potential steric hindrance between two antibody scaffolds.
Additionally, αCD3 scFv antibodies and αCLL-1 scFv antibodies were separately fused with
PDGFR TM domain for generation of monoclonal exosomes as controls (Figure 4. (A) and (B)).
A hemagglutinin (HA) epitope tag was fused at the N-terminus of each fusion protein.

26

Figure 4. Molecular designs of fusion protein inserts for (A) αCD3 SMART-Exos, (B) αCLL-1
SMART-Exos, and (C) αCD3-CLL-1 SMART-Exos. Single-chain variable fragment (scFv)
antibodies were genetically linked to PDGFR transmembrane (TM) domain fusions. A
hemagglutinin (HA) epitope tag was fused at the N-terminus of the fusion protein.

27
2. Expression and identification of SMART-Exos
Expression constructs were transfected with Expi293F cells and cultured in Expi293 medium. The
expressed SMART-Exos were harvested and isolated by differential centrifugation. The yield of
30 mL transfection of SMART-Exos is approximately 100 μg, containing ~6.69 × 10
10
exosome
particles.
Western blot analysis indicated that all scFv antibodies were expressed in exosomes (Figure 5-1).
Moreover, SMART-Exos showed expression of exosomal marker CD9, CD81, and CD63, similar
to native exosomes (Figure 5-2).

28

Figure 5-1. The generated fusion proteins were detected by Western blot assay using an αHA
antibody. Lane 1: Native exosomes ；Lane 2: αCD3 exosomes ，Lane 3: αCLL-1 exosomes; Lane
4: αCD3-αCLL-1 SMART-Exos.

29

Figure 5-2. The exosome-specific markers CD9, CD63, and CD81 were detected by Western blot
assay. Lane 1: Native exosomes ；Lane 2: αCD3 exosomes ，Lane 3: αCLL-1 exosomes; Lane 4:
αCD3-αCLL-1 SMART-Exos.

30
3. Characterization of SMART-Exos
The αCD3-αCLL-1 SMART-Exos were imaged by transmission electron microscopy (Figure 6.).
Quantification and size determination of αCD3-αCLL-1 SMART-Exos was assessed by
nanoparticle tracking analysis (NTA), indicating a size distribution peaking at 73 nm in diameter
(Figure 7.), which was consistent with previous studies
[29], [40], [49]
.

31

Figure 6. Representative negative staining transmission electron microscopy (TEM) images of
αCD3-αCLL-1 SMART-Exos.

32

Figure 7. NTA measurement of size distribution of αCD3-αCLL-1 SMART-Exos.

33
4. Antigen expression analyses of AML cell lines
To analyze CLL-1 antigen expression levels on different AML cell lines (U937, HL60, and KG-
1A), equal number of cells (500,000 cells/sample) were incubated with 20 nM FITC-conjugated
αCLL1 antibodies in PBS with 2% FBS on ice for 45 min. Cells were washed and analyzed by
flow cytometry. U937 has the highest expression levels of CLL-1, followed by HL60. KG-1A has
the lowest expression levels of CLL-1. (Figure 8.)

34

Figure 8. CLL-1 antigen expression levels on different AML cell lines analyzed by flow cytometry.
Red line: U937 cell line; Green line: HL60 cell line; Orange line: KG-1A cell line.

35
5. Binding assays
Flow cytometric analysis indicated that αCD3-αCLL-1 SMART-Exos have significant binding
affinity to CLL-1 positive cells and Jurkat cells, showing that scFv antibodies displayed on
exosomal surface allow SMART-Exos to target both CLL-1 and CD3-expressing cells (Figure 9-
1, 9-2, and 9-3). For monospecific SMART-Exos, they exhibited selected binding to respective
target cells. αCD3 SMART-Exos can only bind to Jurkat cells (Figure 9-1), while αCLL-1
SMART-Exos only showed high binding affinity to both U937 and HL60 cells (Figure 9-2, Figure
9-3). None of the SMART-Exos displayed strong binding to KG-1A cells (CD3
-
, CLL-1
-
) (Figure
9-4).

36

Figure 9-1. Flow cytometric analysis of the binding of SMART-Exos to Jurkat cells.

37

Figure 9-2. Flow cytometric analysis of the binding of SMART-Exos to U937 cells.

38

Figure 9-3. Flow cytometric analysis of the binding of SMART-Exos to HL60 cells.

39

Figure 9-4. Flow cytometric analysis of the binding of SMART-Exos to KG-1A cells.

40
6. In vitro cytotoxicity assays
To determine whether the cytotoxicity of the SMART-Exos is associated with antigen abundance
on target cells, three AML cell lines with various CLL1 expression levels, including U937 (CLL-
1
+++
), HL60 (CLL-1
++
), and KG-1A (CLL-1
+
) were used to compare the cytotoxicities of SMART-
Exos after an incubation period of 24 hours.
In the presence of human PBMCs (at an E:T ratio of 1:10), αCD3-αCLL-1 SMART-Exos exhibited
highly potent and specific cytotoxicity against U937 cells with an EC50 of 14.21 ± 1.10 ng/mL,
followed by HL60 with an EC50 of 82.84 ± 1.17 ng/mL and significantly decreased cytotoxicity
for KG-1A cells with an EC50 of 685.0 ± 1.31 ng/mL (Figure 10-1, 10-2, 10-3). The cytotoxicity
induced by SMART-Exos to target AML cell lines were positively correlated with levels of CLL-
1 expression (Figure 11).
Our results showed that αCD3-αCLL-1 SMART-Exos possess remarkable potency and specificity
towards CLL-1-positive cells by redirecting T cells to induce immune attacks to target cells in an
antigen-dependent manner, underscoring the promising potential of SMART-Exos as
immunotherapeutics for AML.

41

Figure 10-1. In vitro cytotoxicity of SMART-Exos redirecting healthy PBMCs against U937 after
24 h of incubation at an E:T ratio of 1:10. A mixture of αCD3 and αCLL-1 SMART-Exos was
used as a control. Each data point represents a mean of triplicate samples. Error bars are
representative of standard deviation. N.A. stands for not applicable.

42

Figure 10-2. In vitro cytotoxicity of SMART-Exos redirecting healthy PBMCs against HL60 after
24 h of incubation at an E:T ratio of 1:10. A mixture of αCD3 and αCLL-1 SMART-Exos was
used as a control. Each data point represents a mean of triplicate samples. Error bars are
representative of standard deviation. N.A. stands for not applicable.

43

Figure 10-3. In vitro cytotoxicity of SMART-Exos redirecting healthy PBMCs against KG-1A
after 24 h of incubation at an E:T ratio of 1:10. A mixture of αCD3 and αCLL-1 SMART-Exos
was used as a control. Each data point represents a mean of triplicate samples. Error bars are
representative of standard deviation. N.A. stands for not applicable.

44

Figure 11. In vitro cytotoxicity of αCD3-αCLL-1 SMART-Exos against all three AML cell lines
(U937, HL60, and KG-1A) after 24 h of incubation at an E:T ratio of 1:10. Each data point
represents a mean of triplicate samples. Error bars are representative of standard deviation. N.A.
stands for not applicable.

45
DISCUSSION

AML is a common type of leukemia affecting adults. Although standard chemotherapies can
improve overall survival in patients with AML, a majority of patients eventually relapse, with a
five-year survival rate of 30%. Thus, there is an urgent need to develop new therapeutic approaches
for AML treatment.
(CLL-1 is a type II transmembrane glycoprotein, and it is a myeloid lineage-restricted cell surface
marker. Importantly, CLL-1 is overexpressed in both AML blasts and leukemia stem cells, but
extremely low expression in healthy hematopoietic stem cells (HSCs), which presents a promising
therapeutic target for AML treatment.
Exosomes have emerged as attractive nanomedicine platforms in recent years. They offer excellent
advantages as delivery systems, owing to their nano-sized particles, low immunogenicity, and
long-term safety. Moreover, they are highly versatile in terms of their surface engineering and
cargo encapsulation.
In this study, we explored a new concept of using exosomes for immune-modulatory therapy with
the aim of genetically engineering exosomes to elicit antitumor immunity against AML cells. We
genetically engineered exosomes by displaying two individual functional monoclonal antibodies
on the exosomal surface for selectively recruiting cytotoxic T cells to cancer cells. The resulting
aCLL1-aCD3 SMART-Exos showed significant binding CLL-1
+
cell lines and potent and selective
in vitro cytotoxicity against various AML cell lines.
The antitumor efficacies of SMART-Exos are determined by a multitude of parameters, including
E:T ratio, antigen expression level, cytogenetics, and heterogeneity of patients. We are currently
assessing the efficacy of aCLL1-aCD3 SMART-Exos in mouse models. Further works include the

46
genetic design of SMART-Exos, regulation of immune responses by SMART-Exos, their
biodistribution and toxicity, and in vivo mechanism(s) of action.
In general, this study demonstrates that the dual targeting SMART-Exos exhibit excellent
selectivity in inducing potent anticancer immunity against CLL-1-positive cells, highlighting
SMART-Exos as promising candidates for AML immunotherapy.
As stated, the exosomal surface can be genetically engineered to display various functional
membrane proteins. This engineering ability of exosomes provides a versatile platform for the
development of exosomes-based therapeutics. But the potency of functional protein displayed on
the exosomal surface could be affected by various factors, including the identification of a surface
protein to serve as anchoring scaffold, and the configuration of functional peptides. For example,
in our case, different orientation of individual antibodies in fusion protein may have different
potential effects on physicochemical and biological properties of SMART-Exos. Besides our
design of variable regions for the expression construct, other possible orientations of variable
regions are listed as follows: VH-αCLL - 1-VL-αCLL-1 - VL-αCD3 -V H-αCD3 (αCLL-1-αCD3 scFv); VH-
αCLL-1 -VL-αCLL-1 – VH-αCD3 -VL-αCD3 (αCLL-1-αCD3 scFv); VL-αCLL-1 -VH-αCLL-1 – VL-αCD3 -VH-αCD3
(αCLL-1-αCD3 scFv); VL-αCLL-1 -VH-αCLL-1 – VH-αCD3 -VL-αCD3 (αCLL-1-αCD3 scFv); VH-αCD3 -VL-
αCD3 -VL-αCLL-1 -VH-αCLL-1 (αCD3-αCLL-1 scFv); VH-αCD3 -VL-αCD3 -VH-αCLL-1 -VL-αCLL-1 (αCD3-
αCLL-1 scFv); VL-αCD3 -VH-αCD3 -VH-αCLL-1 -VL-αCLL-1 (αCD3-αCLL-1 scFv).
Exosomes can also be used as carriers for the therapeutic delivery of various synthetic and
biological molecules. The efficiencies of current approaches for drug delivery, such as synthesized
nanoparticles, virus-like vectors, and proteoliposomes
[50, 51, 52]
, can still be limited by endosomal
entrapment. In contrast, fusogenic exosomes loaded with therapeutic cargoes can deliver
therapeutic agents by directly enter the cytosol of targeted cells via fusion, which bypasses the

47
potential for becoming entrapped in an endosome. In the future, the SMART-Exos can be loaded
with therapeutic cargos for targeted delivery with enhanced efficacy.
The development of the therapeutic application of exosomes is still in its early stage. It is crucial
to understand the biogenesis and emphasize extensive production of exosomes to succeed in the
exosome-based therapeutic platform. In addition, prediction of the long-term safety and
therapeutic effects of exosomes is hard to conduct due to the lack of knowledge of their
pathophysiological role. Moreover, prior to applying engineered exosomes in clinical applications,
their components need to be characterized in the contexts of exosomes heterogeneity.
Despite these challenges, exosomes represent excellent candidates for cancer therapy. Uncovering
of their pathophysiological roles will be helpful to further develop exosomes-based therapeutics.
And with our in-depth understanding of exosome technology, more exosome-based clinical
applications are expected in the near future.

48
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Abstract (if available)

Abstract Exosomes are nano-sized membranous vesicles and are widely distributed in various body fluids. These cell-derived nanoparticles are less immunogenic than artificial delivery vehicles, and engineered forms of exosomes hold great potential as novel therapeutic modalities. Acute myeloid leukemia (AML) is a common type of leukemia with poor prognosis in adults. In this thesis study, synthetic multivalent antibodies retargeted exosomes (SMART-Exos) were genetically engineered, and two distinct types of monoclonal antibodies were displayed on the exosomal surface. Human C type lectin like molecule 1 (CLL-1) is overexpressed in AML blasts and leukemic stem cells. By targeting CLL-1 and T-cell CD3 receptor, the generated SMART-Exos were designed to redirect T-cells against AML cells for killings. It was demonstrated in this study that the resulting anti-CD3×CLL-1 SMART-Exos not only bind tightly to both T-cells and CLL-1-positive AML cells but also elicit potent antitumor immunity in a dose-dependent manner. This work provides a novel approach for the development of exosomes as immunotherapeutics for AML treatment.

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University of Southern California Dissertations and Theses

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Asset Metadata

Creator Hou, Tianling (author)

Core Title Reprogramming exosomes for immunotherapy of acute myeloid leukemia

Contributor Electronically uploaded by the author (provenance)

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Publication Date 04/25/2020

Defense Date 04/25/2019

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

Tag cancer,exosome,immunotherapy,OAI-PMH Harvest,protein engineering

Format application/pdf (imt)

Language English

Advisor Zhang, Yong (committee chair), Okamoto, Curtis (committee member), Tabancay, Angel (committee member)

Creator Email tianlinghou@gmail.com,tianlinh@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-150162

Unique identifier UC11660644

Identifier etd-HouTianlin-7306.pdf (filename),usctheses-c89-150162 (legacy record id)

Legacy Identifier etd-HouTianlin-7306.pdf

Dmrecord 150162

Document Type Thesis

Format application/pdf (imt)

Rights Hou, Tianling

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