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Optimization of nanomedicine based drug delivery systems for the treatment of solid tumors

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Optimization of nanomedicine based drug delivery systems for the treatment of solid tumors

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OPTIMIZATION
OF NANOMEDICINE BASED DRUG
DELIVERY SYSTEMS FOR THE
TREATMENT OF SOLID TUMORS

Doctor of Philosophy (Pharmaceutical Sciences)

By

Yu Jeong Kim
2018-05-11

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
1985 Zonal Avenue
Los Angeles, CA 90089

1

COMMITTEE MEMBERS

Dr. Pin Wang
Faculty of the USC graduate school
Professor & Kaprielian Fellow in Engineering
MFD, BME and PPSI of USC

Dr. Wei-Chiang Shen
Faculty of the USC graduate school
John A. Biles Professor in Pharmaceutical Sciences of USC

Dr. Curtis T. Okamoto
Faculty of the USC graduate school
Associate Professor in Pharmaceutical Sciences of USC
2

ACKNOWLEDGEMENTS ........................................................................................................................ 4
CHAPTER 1: INTRODUCTION .............................................................................................................. 5
CANCER THERAPY ............................................................................................................................. 5
Tumor Microenvironment ..................................................................................................................... 5
Cancer stem cells .................................................................................................................................. 7
NANOMEDICINE IN CANCER THERAPY ...................................................................................... 11
Overview............................................................................................................................................. 11
Nanomedicine for combination therapy ............................................................................................... 12
Cross-linked Multilamellar Liposomes (cMLVs) ................................................................................. 14
ADOPTIVE CELL THERAPY ............................................................................................................ 15
CAR-T cell Therapy ............................................................................................................................ 15
CAR-Natural Killer (NK) cell Therapy ................................................................................................ 17
Immune cell-mediated drug delivery ................................................................................................... 18
CHAPTER 2: CO-ERADICATION OF BREAST CANCER CELLS AND CANCER STEM CELLS BY
CROSS-LINKED MULTILAMELLAR LIPOSOMES ENHANCES TUMOR TREATMENT .......... 20
ABSTRACT .......................................................................................................................................... 20
INTRODUCTION ................................................................................................................................ 21
RESULTS .............................................................................................................................................. 23
Characterization of drug-loaded crosslinked multilayer liposomes...................................................... 23
In vitro efficacy study by XTT assay. ................................................................................................... 25
The inhibitory effect of combination nanoparticles on cancer stem cells. ............................................. 26
In vivo anti-tumor efficacy of Dox and Sal co-loaded cMLVs. ............................................................. 32
The inhibitory effect of cMLV(Dox+Sal) on cancer stem cells in vivo. ................................................. 32
Inhibition of the expression of CA-9 in tumor-derived tissue. .............................................................. 33
DISCUSSION ........................................................................................................................................ 34
METHODS ........................................................................................................................................... 37
CHAPTER 3: COMBINATION CANCER THERAPY USING CHIMERIC ANTIGEN RECEPTOR-
ENGINEERED NATURAL KILLER CELLS AS DRUG CARRIERS ................................................ 43
ABSTRACT .......................................................................................................................................... 43
INTRODUCTION ................................................................................................................................ 43
RESULTS .............................................................................................................................................. 46
Anti-CD19 and anti-Her2 CARs are expressed in NK92 cells .............................................................. 46
cMLVs are stably conjugated to the NK cell surface ............................................................................ 46
CAR.NK cells have greater cytotoxic effects against antigen-expressing target cells in vitro and are less
sensitive to PTX .................................................................................................................................. 48
CAR.NK function is unaffected by cMLV conjugation and enhanced with cMLV(PTX) conjugation in vitro
........................................................................................................................................................... 50
CAR.NK.cMLV enhances delivery of cMLVs to the tumor site ............................................................. 53
3

CAR.NK.cMLV(PTX) enhances antitumor efficacy in vivo................................................................... 55
CAR.NK.cMLV(PTX) enhances PTX delivery into tumor site .............................................................. 56
DISCUSSION ........................................................................................................................................ 57
METHODS ........................................................................................................................................... 59
CHAPTER 4: CAR-T CELLS SURFACE-ENGINEERED WITH DRUG-ENCAPSULATED
NANOPARTICLES CAN AMELIORATE INTRATUMORAL T CELL HYPOFUNCTION ............ 67
ABSTRACT .......................................................................................................................................... 67
INTRODUCTION ................................................................................................................................ 67
RESULTS .............................................................................................................................................. 70
Nanoparticles stably attached to the surface of CAR-T cells ................................................................ 70
CAR-T cells conjugated with nanoparticles maintain T effector functions ........................................... 71
Conjugation to CAR-T cells increases tumor localization and systemic circulation of cMLVs ............. 73
Surface-conjugated cMLVs colocalize with CAR-T cells inside the tumor mass ................................... 74
CAR-T cells conjugated with nanoparticles encapsulated with A2aR antagonist shows improved antitumor
responses in vivo ................................................................................................................................. 76
CAR-T cells conjugated with nanoparticles encapsulated with A2aR antagonist is able to rescue
hypofunctional tumor-residing T cells in vivo ...................................................................................... 78
DISCUSSION ........................................................................................................................................ 80
METHODS ........................................................................................................................................... 84
CHAPTER 5: CONCLUSION AND FUTURE PERSPECTIVES ......................................................... 90
REFERENCES ......................................................................................................................................... 93

4

ACKNOWLEDGEMENTS

It is a great pleasure to acknowledge my deepest appreciation and gratitude to my advisor, Professor Pin Wang
for his guidance and support. I truly respect his motivation and enthusiasm for research. I feel deeply honored to
work under his research team.
I would like to express my deep appreciation to my committee members, Dr.Shen and Dr.Okamoto for being
on my dissertation committee members. I truly thank for their time and support through this process.
Also, I would like thank Dr. Yarong Liu for being a great mentor in my junior years. I truly appreciate her help
and guidance. One of the most important lessons I learned from working with her is the importance of work
productivity and efficiency.
I would like to give special thanks to my sincere friend, Natnaree Siriwon for being a great collaborator. I share
a lot of special moments, especially the sleepless nights we were working together on our in vivo study. Through
those moments, we have become stronger and more mature. I truly enjoyed working with you.
Also, I really want to thank my lab member, Elizabeth Siegler for being a great collaborator. I think we made a
great team together and achieved a lot of things together in last two years. I truly appreciate your diligence.
Additionally, I want to thank my rest of lab members, Xianhui Chen, Jennifer Rohrs, Dr. Si Li, John Mac
Dr.Paul Bryson, Dr.Xiaolu Han, Dr.Xiaoyang Zhang, Gunce Cinay and Yun Qu for their support and help.
Last of all, I would like to give my sincere love and thanks to my family. To my parents, Thank you for giving me
everything you could have given, and being such wonderful parents. My appreciation also goes to my sister, Na
Jeong for her help and support throughout my PhD. Above all, without the love and encouragement from my
husband Jun and my daughter Anna, I wouldn't be the person I am today.

5

CHAPTER 1: INTRODUCTION

CANCER THERAPY

Tumor Microenvironment

Cancer treatments typically include surgical removal, chemotherapy, radiation, or some combination of these
therapies
1
. While these methods can remove the majority of the tumor cells, tumor recurrence and metastasis
remain a major obstacle in cancer treatment
2
. It is apparent that cancer growth and metastasis are not solely
dependent on the tumor cells themselves, but involve pathologies in the surrounding tumor microenvironment
(TME) as well. In the 19th century, Stephen Paget's “seed and soil” hypothesis postulated that cancer “seeds” or
metastases preferentially established secondary tumors at specific sites (the“soil”)
3
. Only within the past few
decades have researchers focused on anticancer treatments which target the TME rather than the actual cancer
cells. The TME contains various cell types, such as fibroblasts, myofibroblasts, adipocytes, and immune cells, as
well as extracellular matrix (ECM), and blood and lymphatic vasculature
4
.Increasing evidence suggests that the
TME is a crucial part of cancer development, proliferation, and metastasis
5
. The TME also contributes to the
failure of many conventional cancer therapies to completely eradicate the tumor.
Abnormal tumor vasculature and interference with drug delivery
Blood vessel development is critical for the continued growth and progression of solid tumors. Unlike normal
blood vessels, structurally and functionally abnormal tumor vasculature impedes the efficient delivery of both
oxygen and therapeutics at effective concentrations to all cancer cells
6-11
. This phenomenon is mainly caused by
an imbalance of pro- and anti-antiangiogenic factors, which leads to endothelial cell proliferation, migration, and
new vessel formation. This contributes to the formation of poorly organized blood vessels, impaired blood flow,
increased hypoxic regions within the tumor, and higher interstitial fluid pressure (IFP)
11, 12
. Due to vascular
hyperpermeability, the gradients between vascular and interstitial pressure are not maintained, resulting
6

in elevated interstitial pressure within the tumor. Reduced lymphatic drainage from the tumor further exacerbates
elevated IFP within the tumor. High IFP hinders effective penetration of anticancer agents into the deeper core of
solid tumors
12
.
Abnormalities of tumor ECM and effects on tumor progression
Major ECM components include collagen, glycoproteins, proteoglycans, elastin, and hyaluronan as well as ECM
associated enzymes and growth factors which direct cell proliferation and differentiation
13
. However, in a
hypoxic TME, biophysical and biological characteristics of the ECM are altered, contributing to tumor
progression, invasion, and metastasis. Stromal stiffness is significantly increased in breast
tumor tissue stroma as compared to normal tissue stroma
8, 9
. Lysyl oxidase (LOX) is a major player that
upregulates crosslinking of collagen fibers with other ECM components
14
. Furthermore, the interstitial space is
mostly composed of collagen. In the TME, the collagen content is higher than that of normal tissue and is a major
barrier of interstitial drug penetration
13
. Interactions between cancer cells and the various components of the ECM
serve as one of the main obstacles that prevent effective penetration of chemotherapeutic drugs into tumor tissue.
Immune dysfunction in the TME
The TME contributes to chronic inflammation and dysregulation of immune cells. In a healthy individual, both
innate and adaptive immune cells partake in cancer immunosurveillance. In the TME, chronic exposure to
inflammatory cytokines often results in immune cell anergy and a lack of antitumor response. Over time, these
immune cells themselves start to secrete pro-tumorigenic mediators and contribute to maintaining the TME.
Immune suppressor cells also accumulate within the TME. T cells are part of the adaptive immune system that
have antitumor effects, but are suppressed in the TME.Foxp3+ CD25+CD4+ regulatory T cells (Tregs) are
recruited to the tumor site and secrete inhibitory cytokines such as IL-10 and transforming growth factor-b (TGF-
b)
15
.Myeloidderived suppressor cells dampen immune responses by hindering antigen presentation by dendritic
cells (DCs) and impairing CD8þ T cell cytotoxicity
16
. The TME also impacts the function of innate immune cells,
including DCs and natural killer (NK) cells. DCs are antigenpresenting cells which stimulate T cell response to
7

tumor antigens. DCs pulsed with tumor peptides are being researched as a cancer vaccine, but have seen limited
clinical efficacy due to the inhibitory effects of the TME
17
. Rather than being activated by an antigen-specific
receptor, NK cells are stimulated by a complex balance of inhibitory and stimulatory signals. Under normal
conditions, their cytotoxic abilities are inhibited by engaging with HLA class I present on healthy
autologous cells. Tumors tend to downregulate HLA class I, leaving NK cells uninhibited and able to target the
cancer cells. In the TME, cancer cells are often able to escape NK cytotoxicity by suppressing NK cells with IL-
10 and TGF-beta
18
. Macrophages play roles in both the innate and adaptive immune systems. Macrophages with
the M1 phenotype have enhanced cytotoxicity and contribute to an inflammatory environment by upregulating
the release of pro-inflammatory cytokines and reactive oxygen species. Within the TME, macrophages can be
polarized from the M1 to the immunosuppressive M2 phenotype
17
. Tumor-associated macrophages (TAMs) can
also modulate the ECM by signaling to increase collagen secretion or by releasing matrix metalloproteases
(MMPs), which break down ECM and can lead to metastasis. Additionally, TAMs can secrete proangiogenic
factors and contribute to tumor vascularization
19
. All of these functions make TAMs an important target in cancer
immunotherapy.
Cancer stem cells

Cancer stem cells (CSCs) are a small subset of cancer cells with the ability to self-renew and initiate tumor growth.
They were first discovered in acute myeloid leukemia (AML) in the late 1990s
20
. Since then, CSCs have been
discovered in many solid tumors
21-25
. Within the last two decades, CSCs have become a subject of intense research
as a potential target for cancer therapeutics. The discovery of CSCs led to a major shift in cancer modeling.
Previously, cancers were thought to be made up of equipotent malignant cells which either renewed or
differentiated stochastically, giving rises to a heterogeneous tumor. In contrast, the CSC model suggests that a
hierarchy exists among tumor cells, with CSCs at the top, producing the bulk of the tumor cells while maintaining
their own renewal
22
. A third model, clonal evolution, states that heterogeneity comes from genetic or epigenetic
changes that arise during cancer progression. The CSC and clonal evolution models are not mutually exclusive,
8

as CSCs can also evolve over time, generating different clonal subpopulations within the tumor
25
. CSCs share
a number of properties with normal stem cells (SCs). Both typically make up a small percentage of the total
number of cells in a tissue, they are largely quiescent, and, most notably, they are multipotent and can self-renew
indefinitely. Many pathways vital to SC function, such as Wnt, Hedgehog, Notch
26
, and PI3K/Akt
27
, are
dysregulated in CSCs, potentially contributing to neoplastic transformation. For example, cases of multiple
myeloma have displayed abnormal signaling in response to elevated levels of Hedgehog ligand secreted by tumor
stromal cells
28
, and upregulated Notch4 signaling has been implicated in drug-resistant breast CSC activity
29
.
Like SCs, CSCs are able to repair damaged DNA more quickly and overexpress drug-efflux pumps such as ATP-
binding cassette (ABC) transporters. In a glioblastoma model, aberrant Akt signaling contributed to overactivation
of the ABC transporter ABCG2 in CSCs, leading to increased drug expulsion and rendering them resistant to
mitoxantrone
30
. CSCs may also contribute to metastasis. During normal wound healing, cells are able to migrate
to the wound site through the epithelial to mesenchymal transition (EMT) process. CSCs may also undergo EMT
when migrating from the primary tumor site. Another theory hypothesizes that the CSC microenvironment --
including the surrounding vasculature facilitates metastasis
31
. While the exact mechanisms have not been
discovered, there are many reports of CSC-driven metastasis. In fact, numerous studies have used breast CSC-
rich cell lines such as MDA-MB-231 to first produce primary tumors and then seed lung metastases. Studies of
CSC-targeted therapy depend on the isolation and enrichment of CSCs. They can be identified, isolated, and
characterized by several methodologies, including flow cytometric analysis of CSC-specific cell surface markers,
detection of side-population (SP) phenotypes by Hoechst 33342 dye exclusion, changes in aldehyde
dehydrogenase (ALDH) enzymatic activities using an aldeflour assay, ability to grow as suspension spheres in
serum-free medium, SC-related gene expression, and auto-fluorescence
25, 32-34
.There are no widely accepted
techniques solely developed to isolate CSCs, necessitating the use of combination markers and methods rather
than single strategies.
9

Table1. Cancer stem cell surface markers in human cancers

Surface marker-based assays have become the mostly commonly used method
35
.Table 1 summarizes the list of
cell surface phenotypes of CSCs in different tumors. The detection can be performed with specific antibodies in
flow cytometry, competitive ELISA, or magnetic beads
36
. Dick and coworkers showed the first evidence of the
presence of CSCs in human AML by the flow cytometric display of the CD34+CD38- surface marker phenotype
37
.
A breast CSC subpopulation was identified and isolated by the combination of CD44 and CD24 markers
36
.
Functional CSC properties like intracellular ALDH enzymatic activities and ABC transporter efflux activities of
vital DNA dyes such as Hoechst 33342 have been used for CSC isolation
21,22
. Increased aldehyde dehydrogenase
isoform 1 (ALDH1) activity has been used to identify and analyze different types of CSCs. Furthermore, CSCs
have a distinct efflux mechanism, stemming from their high expression of ABC transporter proteins. These cells,
referred to as the “side population” (SP), are able to actively transport fluorescent dyes such as Hoechst 33342
out of the cells. Flow cytometric SP analysis has been performed with numerous cancer cell lines and the SP has
shown enriched CSC activities
38
. A subpopulation of CSCs exhibit intrinsic autofluorescence and were shown to
be exclusively linked to a functional CSC phenotype in different epithelial tumors. These autofluorescent cells
had CSC characteristics such as high self-renewal, long-term tumorigenic capacity, invasiveness, and
chemoresistance. These cells have intrinsic autofluorescence with excitation wavelengths at 488 nm and emission
at about 520 nm. This new marker has been proven to be a more reliable and accurate way to identify and
characterize CSCs
33, 39
. Another important functional property of CSCs, as well as normal stem SCs, is the
ability to produce sphere-forming colonies from a single cell in serum-free medium or in soft agar medium, as
differentiated cells cannot survive and proliferate in this environment
40
. Thus, several studies have used the sphere
formation assay as an efficient method for isolating, enriching and maintaining CSCs from various primary tumors.
10

Generally, these CSC-driven spheres are greater in both number and size as compared to ones generated from
non-CSCs
35, 41
.These spheres clearly demonstrated stem-like properties and expressed characteristics of CSCs
33
.

Figure 1. Novel therapeutic strategies for targeting CSCs.

It is clear now that conventional chemotherapy is not enough to overcome the abilities of CSCs to self-renew
and metastasize. Various strategies that can act effectively against CSCs have been developed as shown Figure
1. A combination of surface markers and their functional properties have been used to identify and isolate
CSCs. Despite this progress, there is still a lack of reliable and accurate CSC markers. This must be overcome
in order to develop therapeutic strategies with higher specificity and fewer side effects. Using either small
molecule inhibitors or RNAi to target CSC-associated oncogenes and signaling pathways have resulted in
decreased functionality and numbers of CSCs and tumor regression in several pre-clinical models. CSCs
develop resistance to conventional chemotherapeutics, but targeting ABC transporters resensitizes CSCs to
those same drugs. Several studies have shown greater CSC targeting effects by employing antibodies against
CSC-specific biomarkers. Anti-CSC approaches such as CD44 and EpCAM antibodies could selectively induce
differentiation and inhibit proliferation
21, 42
.
11

NANOMEDICINE IN CANCER THERAPY

Overview

Although cancer remains one of the most devastating diseases in the world, recent advances in nanomedicine
have offered better ways for cancer detection, prevention and treatment. Due to the leaky tumor vasculature,
nanoparticles can accumulate within the tumor and its surrounding environment more effectively than free drugs.
This phenomenon, referred to as the enhanced permeability and retention (EPR) effect, is further enhanced by
poor lymphatic drainage around the tumor, effectively trapping the nanoparticles at the site
14
.As shown in Figure
2, nanoparticles can escape into the tumor tissues via the leaky blood vessels and accumulate in poor lymphatic
drainage in tumor regions via the EPR effect, allowing the release of chemotherapeutics into tumor cells.

Figure 2. Schematic representation of nanomedicine mediated drug delivery via the EPR effect.

Other advantages of nanoparticles in drug delivery include protecting the drugs from premature degradation,
controlling the in vivo pharmacokinetics and distribution profiles, and improving intracellular penetration by
conjugating with targeting ligands, peptides or cells. Moreover, there have been numerous studies on developing
nanocarriers with an extended circulating half-life in serum and controlled drug release profile. Nanoparticles
12

targeting different properties of the TME have been presented in both preclinical and clinical studies. Polymeric
nanocarriers were the earliest reported materials for cancer therapy. A nanoparticle formulation of paclitaxel in
which albumin is utilized as a carrier has been applied in the clinic for the treatment of metastatic breast cancer.
However, its therapeutic potential was hindered by its inherent structural heterogeneity. Later, lipid-based carrier
has gained more attention as their composition are biocompatible and biodegradable
43
. Moreover, studies have
shown that lipid-based carriers are capable of delivering drugs with different physicochemical properties.
Currently, liposomal formulations of doxorubicin (Doxil,Myocet) and daunorubicin (DaunoXome) have been
approved for the treatment of metastatic breast, ovarian cancer and Kaposi’s sarcoma
43
.
Nanomedicine for combination therapy

Target-based drug design has been successfully used to develop many drugs that can act on novel molecular
targets; however, these drugs have shown poor efficacy in clinical trials. This can be attributed to the
compensatory mechanism, or drug-mitigating response, enacted by complex diseases such as cancer
44
.
Overcoming this drug-mitigating response often requires high drug doses, which can induce drug resistance in
target cells or side effects in other tissues,
45
thus limiting the efficacy of many potential drugs in cancer therapy.
These limitations of monotherapy can be overcome by synergistic combination of two or more agents, which can
kill cells at lower drug doses by affecting multiple disease targets
46
. However, current combination methods,
through cocktail administration, have shown limited improvement over single drugs in clinical studies due to the
distinctive pharmacokinetics of individual drugs, which lead to noncoordinated distribution after systemic
administration
47
. Moreover, unexpected adverse effects were reported in clinical trials using these cocktail
combinations, raising concerns about the induction of synergistic systemic toxicities by combination
therapies. For instance, although a combination of doxorubicin (Dox) and paclitaxel (PTX) has been widely used
in the treatment of tumors, particularly in metastatic breast cancer, the clinical results were limited by increased
cardiotoxicity
48, 49
. Clinical pharmacokinetic studies also revealed a noncoordinated plasma distribution of Dox
and PTX when given in combination
50
, rendering in vitro data ineffective in predicting in vivo therapeutic
13

efficacy of combination therapy. A more effective combination strategy with the ability to coordinate the
pharmacokinetics and biodistribution of various drug molecules is highly desirable to maximize the combinatorial
effects without significant toxicity
51
. The development of nanotechnology has provided a novel combination
strategy by enabling the simultaneous delivery of multiple chemo-drugs to a site of interest via a single
vehicle
47
. Nanoparticles are considered promising drug delivery vehicles for cancer therapy based on their ability
to prolong drug circulation time, reduce systemic toxicity, and increase drug accumulation at tumor sites through
the enhanced permeation and retention (EPR) effect
51, 52
. The pharmacokinetic behavior of the co-formulated
drugs can be determined by the pharmacokinetic behavior of the drug carriers. Thus, nanoparticle delivery
systems offer the potential to coordinate the plasma elimination and biodistribution of multiple drugs, enabling
dosage optimization to maximize cytotoxicity while minimizing the chances to develop drug resistance. In
addition to nanoparticle mediated delivery of multi-chemotherapeutics, a more advanced approach utilizing
nanoparticles have been investigating such as targeting a combination of the vascular, ECM, CSCs and immune
cells within the TME, as well as the actual tumor cells. For instance, the efficacy of immunotherapy alone may
be inadequate to produce clinical results. Therefore, combination therapy with conventional modalities as well as
with immunomodulatory agents may be of future interest to enhance therapeutic effects. Moreover, nanocarriers
enhanced the delivery and cytotoxic activity of CSC-inhibitors. It has been shown that using a CSC-targeted
inhibitor alone is not very effective in reducing the tumor bulk due to the fact that these inhibitors are not highly
cytotoxic as compared with conventional chemotherapeutics. Thereby, dual targeting nanoparticles loaded with
CSC inhibitors and conventional cytotoxic agents can improve clinical outcomes by effectively eradicating both
CSCs and bulk tumor cells at the same time. When compared with the free drugs, the nanoparticle formulated
drugs were significantly more effective and less toxic both in vitro and in vivo
53-55
. CSCs are characterized by
certain surface markers; this allows specific targeting of CSCs as a therapeutic strategy for drug delivery.
Swaminathan et al.
56
demonstrated that their targeted nanoparticles induced a significant tumor volume reduction
compared to untreated control and non-targeted groups in an in vivo MDA-MB 231 xenograft tumor model by
14

developing paclitaxel-loaded polymeric PLGA nanoparticles conjugated with CD133 mAb. Another advantage
of using nanoparticles is the additional capability to modify their surfaces with targeting agents such as mAbs and
peptides. High target selectivity and internalization can be achieved by surface modification of nanoparticles with
targeting moieties. Numerous active vasculature targeting approaches using nanoparticles have emerged to
enhance the intracellular concentration of drugs in tumor cells. Sengupta et al. developed a PLGA polymeric
nanoscale delivery system targeting tumor cells and the tumor vasculature. Doxorubicin was covalently attached
to the inner PLGA core, and the anti-angiogenic agent combrestatin was encapsulated within the outside lipid
envelope. Both doxorubicin and combrestatin were successfully encapsulated into their ‘nanocell’ delivery system,
enabling the temporal release of two drugs more effectively than with free drugs or simple liposomal formulations.
As a result, combrestatin released from the outer envelope was able to induce a rapid vascular shutdown inside
the tumor by disrupting the cytoskeletal structures, which first took advantage of the EPR effect and then further
trapped the nanoparticles within the tumor after disrupting tumor vasculature. Afterward, doxorubicin from the
inner nanoparticle was efficiently taken up by the tumor, improving overall therapeutic index with reduced
toxicity
57
.
Cross-linked Multilamellar Liposomes (cMLVs)

Phospholipid vesicles (liposomes) constitute a class of nanoparticle drug carriers generally characterized by
composition of one of more phospholipid bilayer membranes, and capability of delivering aqueous or lipid drugs.
It has been reported that liposomes are capable to enhance the pharmacokinetics profile, and/or to allow for site-
specific drug delivery to solid tumors. Despite the advantages, the inherent instability of conventional unilamellar
liposomes in the presence of serum components, which is apparently related to their drawback of a rapid drug
release profile, has limited their utility for delivering anticancer agents. It has been shown that the release rate of
liposomal Dox is intrinsically linked to the toxicity level and therapeutic activity. Briefly, slower release rates
usually result in lower toxicity and higher therapeutic efficiency. Therefore, it is desirable to develop more robust
liposomal formulation wit sustainable release kinetics and improved vesicle stability. Here, we have investigated
15

a new strategy to develop a multilayer liposomal system that shows promising in delivery of cancer therapeutics
with a more controlled and sustained release kinetics. The multilamellar vesicles were generated through
covalently crosslinking functionalized headgroups of adjacent lipid bilayers, as illustrated in Figure 3. This
design was adapted from a recently reported multistep procedure based on the conventional dehydration-
rehydration method : (1) the incorporation of a thiol-reactive maleimide headgroup lipid (N-(3-Maleimide-1-
oxopropyl)-L-α-phosphatidylethanolamine, MPB-PE) onto the surface of unilamellar liposome (UL); (2) divalent
cation-triggered vesicle fusion that yields a multilamellar structure; and (3) interbilayer crosslinking across the
opposing sides of lipid bilayers through the reactive headgroups with dithiothreitol (DTT) to generate robust and
stable vesicles. As a final step, the surface of the crosslinked multilamellar liposome (CML) was PEGylated with
thiol-terminated PEG, which could further improve vesicle stability and blood circulation half-life
58
.

Figure 3. Stable liposomal formulation and physical characterization of cMLVs. (a) Schematic
representation of the crosslinked multilamellar liposome.

ADOPTIVE CELL THERAPY

CAR-T cell Therapy

Cancer immunotherapy has the potential to achieve long term remissions by harnessing the patient’s own immune
system to attack cancer cells. Chimeric antigen receptors (CARs) are engineered proteins that offer a method of
16

reprogramming human immune cells to recognize and target cancer cells
59
. CAR-engineered T (CAR-T) cells are
not major histocompatibility complex (MHC)-restricted and can be engineered to recognize specific tumor-
associated surface antigens (TAA). Typically, this recognition is due to a single chain variable fragment (scFv)
derived from the desired antibody, which comprises the extracellular portion of the CAR and redirects the immune
cell to corresponding antigens on the target cell’s surface. The scFv is linked to the intracellular signaling domains
of the CAR, which originate from endogenous T cells. These intracellular components include CD3ζ, which
provides the main activating signal, as well as one or more costimulatory domains that further enhance immune
cell function. Antigen binding to the scFv triggers signaling cascades which activate the CAR-T cell against the
TAA-expressing cancer cell
60
(Figure 4).

Figure 4. CAR-T schematic. An antibody-derived scFvbinds to the TAA on the tumor cell, triggering an
intracelluar signaling cascade that activates the T cell against the tumor cell.

In addition to directly killing the target cells via perforin and granzyme release, activated CAR-T cells can secrete
pro-inflammatory cytokines such as IL-2 or IFNγ to signal to other immune cells for a broader immune response
61
.
Since their inception in the late 1980s4, the number of CAR-T cell studies has exploded. In particular, CD19-
targeting CAR-T cells have been successful at treating many patients with advanced B cell malignancies
60
.
Additional CAR-T therapies are being tested against many different TAAs in both preclinical and clinical settings.
CAR-T cells offer advantages over unmodified T cells: they are not limited by MHC- and dendritic cell-presented
antigen peptides, and both CD4+ and CD8+ T cells can be reprogrammed to have direct cytotoxic effects against
17

target cancer cells
61
. CAR-based therapies have proven thus far to be an important branch of cancer
immunotherapy. Despite its success in treating hematological cancers
62, 63
, it has faced many setbacks in treating
solid tumors
64, 65
. Often this is because CAR-T cells become exhausted and lose their cytotoxic capabilities after
long exposures to TAA
66
. The limitation of adoptive CAR T cell therapy in solid tumors is attributed to multiple
factors, such as the absence of unique tumor associated antigens, inefficient homing of T cells and the ability to
persist in an immunosuppressive tumor microenvironment. The immunosuppressive environment of the tumor is
one of the ultimate obstacles for immunosurveillance, and especially adoptive CAR T cell therapy. The
immunosuppressive tumor microenvironment consists of multiple elements –for instance, regulatory T cells, NKT
cells, and subsets of immature and mature dendritic cells. These regulatory cells in combination with tumors
themselves foster a tolerant microenvironment that hinders any successful attempt of an effective immune
response to eradicate the tumor mass
65
. In addition to regulatory cells, the tumor microenvironment is
characterized by hypoxia and immunosuppressive soluble factors that inhibit T cell function such as adenosine
67
.
CAR-Natural Killer (NK) cell Therapy

Natural killer (NK) cells are a subset of cytotoxic lymphocytes that play critical role in cancer immunosurveillance.
NK cells are part of the innate immune system and mediate rapid, short-lived responses by releasing cytokines
that directly lyse infected or abnormal cells including tumor cells. The NK92 cell line is identical to the parental
NK line isolated from a leukemia patient
68
. NK92 cells are well-documented and have antitumor effects against
various types of cancer including melanoma, leukemia and breast cancer in both preclinical and clinical settings
69
.
Engineering of NK92 cells to express chimeric antigen receptors (CARs) to redirect their antitumor specificity
has shown significant promise. NK cells are not specifically cytotoxic to certain antigen-expressing target cells,
but their specificity and efficacy can be enhanced with CARs. Typically, CARs are expressed in T cells, but recent
studies show CARs are effective tumor-targeting domains in NK cells as well. The inclusion of CARs in NK92
cells increases homing, specificity, and efficacy of tumor killing
70, 71
.
18

Immune cell-mediated drug delivery

Many variations of antitumor drug-loaded nanoparticles have been created to exploit the enhanced permeability
and retention (EPR) effect observed around solid tumors. Leaky and irregular tumor vasculature and poor
lymphatic drainage results in a passive accumulation of nanoparticles at the tumor site
72
. However, this effect is
often insufficient in poorly vascularized tumors. Additionally, only a small percentage of injected drug-
nanoparticle conjugates reach the target site due to particle clearance and a lack of targeted delivery
73
. Cancer
immunotherapy has garnered much attention within the last decade and more recently, much attention has been
given to human immune cell-directed nanoparticle drug delivery, as immune cells can traffic to the tumor and
inflammatory sites
74
. Instead of relying on passive delivery, immune cells can be used as active carriers for
nanoparticles that are either directly cytotoxic to the tumor or nanoparticles which carry chemotherapeutic agents.
Nanoparticles can be conjugated to the cell surface by directly utilizing functional groups such as amino or thiol
groups, through hydrophobic insertion into the lipid bilayer, by electrostatic interactions, or by attaching to
specific cell surface receptors
75
.
Nanoparticles can be conjugated to cells without a drug payload and still have antitumor potential. Within the
past decade, research groups have demonstrated that nanoparticles delivered by cells can enhance antitumor
effects. One study conjugated iron-based nanoparticles to the surfaces of NK cells
76
, which showed increased
homing to the tumor site in vivo when guided by an external magnet. Another group has also shown that T cells
can be conjugated to gold nanoparticles and migrate to tumor sites
77
, whereupon the nanoparticles can be heated
and induce photoablation of tumor cells. In these cases, the nanoparticles themselves assist in killing cancerous
cells without the use of a chemotherapeutic drug or immune adjuvant. In many of the current cell-based
nanotherapies, the cell serves as a carrier for nanoparticles and their cargo
78
. TAA-targeted T cells have shown to
carry adsorbed oncolytic viruses to tumor sites in several studies
79
, and this concept has been extended to synthetic
nanoparticle delivery. One group described a method of conjugating IL-15 and IL-21-loaded maleimide-
functionalized liposomes to free thiols found on T cell surfaces. In this way, T cells were sustained by cytokines
19

from nanoparticles attached to their cell surfaces instead of relying on systemic cytokine doses
78
. Similarly,
palmitoylated B7, a T cell costimulatory protein, conjugated to mouse T cells enhanced the antitumor function of
the cells in vivo36. Immune cells are an attractive option for active cancer therapeutic delivery, due to their natural
ability to home to the tumor site, and more studies of lymphocytes conjugated to synthetic drug-loaded
nanoparticles are needed.

20

CHAPTER 2: CO-ERADICATION OF BREAST CANCER
CELLS AND CANCER STEM CELLS BY CROSS-
LINKED MULTILAMELLAR LIPOSOMES ENHANCES
TUMOR TREATMENT

ABSTRACT

The therapeutic limitations of conventional chemotherapeutic drugs have emerged as a challenge for breast cancer
therapy; these shortcomings are likely due, at least in part, to the presence of the cancer stem cells (CSCs).
Salinomycin, a polyether antibiotic isolated from Streptomyces albus, has been shown to selectively inhibit cancer
stem cells; however, its clinical application has been hindered by the drug’s hydrophobility, which limits the
available administration routes. In this paper, a novel drug delivery system, cross-linked multilamellar liposomal
vesicles (cMLVs), was optimized to allow for the co-delivery salinomycin (Sal) and doxorubicin (Dox), targeting
both CSCs and breast cancer cells. The results show that the cMLV particles encapsulating different drugs have
similar sizes with high encapsulation efficiencies (>80%) for both Dox and Sal. Dox and Sal were released from
the particles in a sustained manner, indicating the stability of the cMLVs. Moreover, the inhibition of
cMLV(Dox+Sal) against breast cancer cells was stronger than either single drug treatment. The efficient targeting
of cMLV(Dox+Sal) to CSCs was validated through in vitro experiments using breast cancer stem cell makers. In
accordance with the in vitro combination treatment, in vivo breast tumor suppression by cMLV(Dox+Sal) was 2
fold more effective than single drug cMLV treatment or treatment with the combination of cMLV(Dox) and
cMLV(Sal). Thus, this study demonstrates that cMLVs represent a novel drug delivery system that can serve as
a potential platform for combination therapy, allowing co-delivery of an anti-cancer agent and a CSC inhibitor
for the elimination of both breast cancer cells and cancer stem cells.

21

INTRODUCTION

Breast cancer is one of the most commonly diagnosed cancers in women
80-83
, but, despite the emergence of early
detection methods and improved treatment options, it is still the second leading cause of death in women
80, 84
.
Chemotherapy has been widely used to treat breast cancer at various stages of progression; however, current
chemotherapy has therapeutic limitations, especially for highly invasive or recurrent tumors, two diagnoses which
are associated with a high mortality rate. Hence, new methods are needed to target and treat recurrent or
chemoresistant breast cancer to help improve the outcome for these patient populations
82, 83, 85, 86
.
Cancer stem cells (CSCs) have been studied as a potential target for overcoming drug resistant breast cancer as
they may play a significant role in tumor initiation, recurrence, metastasis, and the building of resistance to
standard chemotherapy
84, 87, 88
. CSCs are defined as a small population of cancer cells that possess characteristics
associated with normal stem cells. Unlike normal cancer cells, CSCs are able to self-renew and differentiate into
multiple cell types within a particular cancer sample
87-91
. Studies have shown that standard chemotherapy can
enrich the CSC population, leading to increased resistance to drug treatments
83, 92, 93
. Thus, increasing efforts have
been made to investigate the role of CSCs in cancer progression and relapse in order to achieve more successful
outcomes in cancer therapy. In fact, among the various treatments being studied in clinical trials for breast cancer,
CSC-targeted therapy is considered one of the most promising strategies
94
. Since cancer stem cells do not respond
to conventional chemotherapeutics, an inhibitor specific to CSCs must be used to defend against potential tumor
recurrence. One potential inhibitor of CSCs is salinomycin, a polyether antibiotic isolated from Streptomyces
albus
89, 95-97
. Salinomycin has been shown to selectively deplete the CSC population, leading to breast tumor
growth inhibition
89
. During recent years, salinomycin has gained recognition for its therapeutic potential;
however, its in vivo application has been hindered by its hydrophobicity, which limits the available administration
routes
89
.
Recent developments in nanoparticle delivery systems have provided new strategies to efficiently deliver
therapeutics with different hydrophobicities
98-100
. Due to the enhanced permeability and retention (EPR) effect,
22

nanoparticle delivery systems are much more efficient than free drugs at delivering therapeutics with prolonged
blood circulation time, sustainable drug release, reduced systemic toxicity, and increased drug accumulation at
tumor site
101-106
. Conjugating these nanoparticles with cell penetrating peptides enables them to actively penetrate
tumors to further enhance antitumor efficacy
107-109
. In addition, several studies have demonstrated the potential
of nanoparticle delivery systems to act as novel platforms for drug combination therapy
55, 102, 105, 110-112
. This
combination therapy approach can potentially be applied to the CSC field to improve clinical outcomes by
combining CSC-targeted inhibitors with conventional cytotoxic agents to effectively eradicate CSCs and bulk
tumor cells at the same time. This added therapeutic effect, combining free salinomycin with other
chemotherapeutics, has been demonstrated in vitro
54, 113, 114
. More recently, some studies reported that, in vitro,
treatment with a mixture of anticancer agent-formulated liposomes and CSC inhibitor-formulated polymeric
micelles induced a significantly higher anticancer effect than individual administration of single-drug formulated
nanoparticles, confirming the intriguing potential of CSC targeted combination therapy to out perform
monotherapy
54, 82, 83
However, the in vivo anti-tumor effect of a mixture of single drug-formulated nanoparticles
failed to show significantly greater effects than single nanoparticle drug treatment or free drug combinations
82, 83
.
These shortcomings are due to the inability to manage the site specific delivery of two drugs with the different
physico-chemical properties, pharmacokinetics profiles, mechanisms of action, toxicity, and drug resistance. In
this delivery setting, it is also difficult to control the delivery of synergistic drug ratios in vivo, indicating that it
may be important to co-localize the delivery of the two drugs in a single nano-carrier to achieve enhanced in vivo
anti-tumor effects
86, 102, 110, 111, 115
. To our knowledge, no study has successfully developed a nanoparticle delivery
system for the simultaneous co-delivery of conventional chemotherapeutics and a CSC inhibitor.
Previously, we have demonstrated a cross-linked multilamellar liposomal vesicle (cMLV) drug delivery system
for the sustained delivery of doxorubicin both in vitro and in vivo
101, 103
. Furthermore, we reported the successful
application of cMLVs for combinatory drug delivery by showing that these multi-drug cMLV nanoparticles
induce a synergistic anti-tumor effect in vitro and in vivo by maintaining a predetermined synergistic drug ratio
23

at the tumor site for over 24 hours
102,

116
. In our current work, we validate the ability of cMLVs to act as a novel
method to target multiple cell populations in a single treatment by co-delivering doxorubicin and salinomycin to
concurrently target both cancer cells and CSCs. Doxorubicin (Dox) was chosen as a hydrophilic model drug since
it is widely used for the chemotherapeutic treatment of various types of cancer. Salinomycin (Sal), the
hydrophobic CSC inhibitor described earlier, was co-encapsulated into cMLVs with Dox at the synergistic drug
ratio pre-determined in our previous study
102
, resulting in multi-drug cMLV(Dox+Sal) particles. Both Dox and
Sal can achieve a high level of encapsulation efficiency in cMLVs. The flow cytometry and cell cytotoxicity
results further demonstrate that cMLV(Dox+Sal) is able to efficiently eliminate breast cancer cells and CSCs,
simultaneously. Furthermore, our in vivo studies with a 4T1 mouse breast tumor model revealed that the co-
delivery of combined drugs by cMLVs could induce a higher anti-tumor efficacy compared to single drug
treatments or co-administration of single drug-encapsulated cMLVs. These results confirm the potential of
cMLVs as a novel nanoparticle-based system for co-localized delivery of combination drug treatments that can
target two different cell populations, leading to advanced outcomes in cancer therapy.

RESULTS

Characterization of drug-loaded crosslinked multilayer liposomes.

Previous studies have shown that both hydrophobic and hydrophilic drugs can successfully be incorporated into
cross-linked multilayer liposomal vesicles (cMLVs)
102
. In the current study, we simultaneously target cancer cells
and cancer stem cells by incorporating Salinomycin (Sal), a hydrophobic drug, into the lipid layers of the
liposomes and Doxorubicin (Dox), a hydrophilic drug, into the aqueous core of the liposomal vesicles. We
compared this dual drug loaded formulation, cMLV(DOX+Sal), to single drug loaded formuations, cMLV(Dox)
and cMLV(Sal). A surface modification was made on each of the cMLV formulations by adding thiol-termineated
polyethylene glycol (PEG) chains, as previously described
101,102
. The conjugation of polyethylene glycol
24

(PEGylation) to nanoparticles is an effective way to enhance drug delivery to the site of interest by further
enhancing the plasma half-life and serum stability of the vesicles
117-119
.
To characterize the cMLVs, the particle size was measured by dynamic light scattering (DLS). The measurements
showed that the mean diameters for all three liposomal formulations, cMLV(Dox+Sal), cMLV(Dox) and
cMLV(Sal), were within a similar size range (Figure 5 A-C). Moreover, all three liposome vesicles showed a
narrow side distribution with a similar polydispersity, indicating that there was no significant aggregation of
particles during the process of synthesis. To further confirm the capability of cMLVs to simultaneously deliver
two drugs, we tested the encapsulation efficiencies of Sal and Dox in cMLVs using a spectrofluorometer (Dox)
and/or an HPLC (Sal). The encapsulation efficiencies of all three formulations were in close proximity,
confirming that loading the two drugs in a single vesicle does not affect their encapsulation efficiencies (Figure
5 D).

Figure 5. Characteristics of cMLVs. (A-C) The hydrodynamic size distribution of cMLV(Dox), cMLV(Sal),
and cMLV(Dox+Sal) was measured by dynamic light scattering. The mean hydrodynamic diameter (HD)
and polydispersity index (PI) of CMLV (Dox), CMLV(Sal), and cMLV(Dox+Sal) are indicated on the
graph. (D) Encapsulation efficiency of Dox and Sal in cMLV(Dox), cMLV(Sal), and cMLV(Dox+Sal). Error
bars represent the standard deviation of the mean from triplicate experiments.

25

Additionally, an in vitro drug release assay was performed to study the individual drug release profiles of Sal and
Dox from the cMLVs. Our results confirmed the stability of the vesicles, showing sustained release of both drugs
over two weeks (Figure 6 A-C); this effect has also been validated in previous studies
101, 102
.

Figure 6. Release kinetics proflies (%) and in vitro cytotoxicity of drugs loaded in cMLVs. In vitro release
rates (%) of doxorubicin in cMLV(Dox) (A) , salinomycin in cMLV(Sal) (B) and doxorubicin and
salinomycin in cMLV(Dox+Sal) (C). In vitro cell inhibitory effects of cMLV(Dox), cMLV(Sal) and
cMLV(Dox+Sal) at 1:5 molar ratio in 4T1 (D), 4T1D (Dox-resistant) (E) and MDA-MB-231 (F) cell lines.

In vitro efficacy study by XTT assay.

To evaluate the potential role of applying conventional drugs and a CSC inhibitor to a viable cancer cell
population, an in vitro cytotoxicity assay was performed at a variety of concentrations of Dox and Sal loaded into
the cMLVs at a predetermined 1:5 synergistic drug molar ratio
102
. Three cell lines (4T1, 4T1D and MDA-MB-
231) were treated with three formulations of cMLVs (cMLV(Dox+Sal), cMLV(Dox) and cMLV(Sal)) for 48 h.
As shown in Figure 6 D-F, in all three cell lines, cMLV(Dox+Sal) had the greatest cytotoxic effect compared to
those of cMLV(Dox) and cMLV(Sal), highlighting the significance of the dual-targeting effect of Dox and Sal
on the overall breast cancer population. The lowest cytotoxicity was observed with cMLV(Sal), as expected,
26

because, unlike Dox, Sal alone is not able to kill the vast majority of the cancer cell population. These results
suggest that co-delivery of conventional anti-cancer agents and a CSCs inhibitor in cMLVs could potentially be
much more effective at inhibiting cell growth than single drug loaded nanoparticles. The results also highlight the
drug synergy at this combination ratio, which was further selected for in vivo efficacy validation.

Figure 7. Flow cytometric profiles a Hoechst effluxing side population and autofluorescent cancer stem
cells and Examination of the RNA expression level of Sox2. After different formulation treatments, (A) 4T1
and (B)4T1D cells are stained with Hoechst 33342 dye with or without the Ca-channel blocker, verapamil
hydrochloride to verify the specificity of the SP population. The percentage of effluxed side population is
indicated on each FACS plot. The side population in each group is gated by its negative control
(+verapamil). (C) Flow cytometry analysis of autofluorescence after different cMLV formulation
treatments. Autofluorescent cells are excited with a 488-nm blue laser and best selected as the intersection
with filters 525/50 and 575/26. (D) Change in Sox2 RNA expression level was measured using RT/Q-PCR
after treated as in ALDH staining assay. Sox2 expression level of each treatment group in both cell lines
was normalized by an expression level of control group in 4T1 cell line. Significance was determined
between each treatment group and a control group in each cell line. (D) Data represented as mean±SD
(n=3). (*p < 0.05, **p < 0.01)

The inhibitory effect of combination nanoparticles on cancer stem cells.

To validate the superiority of the combination therapy of Dox and Sal over Dox alone, we examined its effects
on CSCs, identified using several markers in both murine and human breast cancer cells. First, we stained 4T1
27

and 4T1D murine breast cancer cell lines with Hoechst 33342 dye to observe the inhibitory effect of the drug
combination on CSCs. As previously reported, Hoechst dye 33342 dye exclusion is a traditional method for
determining and isolating CSCs from the bulk tumor cells
33, 92
. FACS analysis of these two cell lines clearly
defined a side population (SP), which indicates the CSC population based on their exclusion of the dye, of 1.04%
(4T1) and 3.33 % (4T1D) (Figure 7A and B). In both cell lines, the cMLV(Dox) treatment increased the
percentage of the SP compared to the control group, consistent with the previous report that CSCs are resistant
to Dox treatment
93, 113
. On the other hand, cMLV(Sal) significantly decreased the percentage of the SP in both
cell lines, proving the specificity of Sal in targeting CSCs. Moreover, a significant reduction of the SP was
observed in both cell lines exposed to cMLV(Dox+Sal), indicating that cMLV(Dox+Sal) can efficiently retain
Sal activity in the presence of Dox (Figure 7A and B). In addition to Hoechst dye staining, aldehyde
dehydrogenase (ALDH) can also be used as a functional marker for cancer stem cells
32, 120, 121
. Therefore, an
ALDEFLOUR assay was performed to investigate changes in ALDH enzymatic activities after treatments of
cMLV(Dox), cMLV(Sal), and cMLV(Sal+Dox). The ALDH staining results were consistent with our Hoechst
staining results, validating the targeting effects of Sal to CSCs in both cMLV(Sal) and cMLV(Dox+Sal) for both
4T1 wild type and Dox resistant cells (Figure 8A and B).

28

Figure 8. Flow cytometric profiles of Aldefluor
pos
cells in 4T1 and 4T1D cell lines. Each plot represents
the staining pattern for ALDH
pos
population after treatments of cMLV(Dox), cMLV(Sal) and
cMLV(Dox+Sal). The percentage in each group was compared to one in control group and gated by its
negative control (DEAB). (B) Quantification graph showing ALDH
pos
population of 4T1 and 4T1D cells
after treated with all three formulations.

A functional CSC phenotype in several different tumor types has been shown to be linked to a distinct
subpopulation of autofluorescent cells
39
. These autofluorescent cells exhibited enhanced CSC functional features
and phenotypes such as high self-renewal, long-term tumorigenic capacity, invasiveness, and chemoresistance.
This new autofluorescent marker has been shown to be a more reliable and accurate way to identify and
characterize CSCs
39
. Therefore, we employed this method to analyze our CSC populations. We detected a small
subpopulation of autofluorescent cells in 4T1 (0.597%) and 4T1D (0.902%) cells (Figure 7C, control panel). In
accordance with previous analyses, cMLV(Dox) treatment resulted in the enrichment of the autofluorescent
population to ~98% of the total number of cells in both cell lines. In contrast, there was a significant reduction of
autofluorescence in cells treated with cMLV(Sal) in both cell lines. Moreover, it was noted that the inhibitory
effect of Sal on the autofluorescent cells was retained in presence of Dox for cells exposed to cMLV(Dox+Sal)
(Figure 7C). It should noted that the extremely high autofluorescent population caused by Dox treatment is not
the fluorescent signals from Dox itself, because the cMLV(Dox+Sal) with the same Dox concentration
significantly abrogated this population.
Sox2 is a stem cell transcription factor crucial for maintaining the self-renewal properties of embryonic stem cells
(ES)
122
. Previous studies have detected high expression levels of Sox 2 in numerous types of tumors, including
breast cancer, and proven its role in tumor initiation and tumor sphere formation
81, 91, 123, 124
. To evaluate the effect
of cMLV treatment on Sox2 expression, changes in the Sox2 RNA expression level in 4T1 and 4T1D cell lines
after treatment with cMLV(Dox), cMLV(Dox+Sal), or cMLV(Sal) for 48 h was determined using quantitative
RT-PCR. As predicted, 4T1D cell had a 2.5 fold higher Sox2 RNA expression level compared to 4T1 cells, shown
in Figure 7D. In both 4T1 and 4T1D cell lines, there was a significant increase in the Sox2 expression level
after cMLV(Dox) treatment compared to that of the control group (p < 0.05). On the other hand, the RNA
29

expression level of Sox2 was significantly decreased in 4T1 and 4T1D cells after the cMLV(Sal) and
cMLV(Dox+Sal) treatments (p < 0.01) compared to the control group. The effect of Sal on Sox2 RNA expression
level further validates its inhibitory effect on CSCs.
To further investigate the combinatory effect of cMLV(Dox+Sal), we expanded these findings to the human breast
cancer cell line, MBA-MB-231, using several CSC markers. First, we examined the changes in ALDH enzymatic
activities upon different cMLV formulation treatments. FACS analysis revealed the presence of 8.27% of
ALDH
pos
population in the control group MDA-MB-231 cells (Figure 9A). Consistent with the results from the
mouse cell lines, the percentage of ALDH
pos
cells significantly increased to 17.1% with cMLV(Dox) treatment. In
contrast, there was only a small percentage of ALDH
pos
cells remaining in the cMLV(Sal) and cMLV(Dox+Sal)
treated samples.

Figure 9. Flow cytometric profiles of Aldefluor
pos
cells and autofluorescent cancer stem cells and Quantification
of tumor-sphere formation and tumor-sphere diameter sizes in MDA-MB-231 cell line treated with Dox and Sal
in three cMLV formulations. (A) FACS analysis of Aldefluor
pos
staining after treatments of different cMLV
formulations. The percentage in each group was gated by its negative control (DEAB). (B) Representative flow
cytometry analysis of autofluorescence after different cMLV formulation treatments. Autofluorescent cells are
excited with a 488-nm blue laser and best selected as the intersection with filters 525/50 and 575/26. (C) Areas
per well were chosen randomly and spheres within the areas were counted and averaged. (D) The diameters of
spheres within the areas that were chosen were measured and the sizes were averaged. (C,D) Data represented as
mean±SD (n=5).
30

In human breast cancer, cells with surface expression of CD44
+
CD24
–
have been reported to display stem cell-
like properties. These proteins are commonly known as putative surface markers for CSCs
33, 34, 84, 89, 125
. It has
been reported that the presence CD44+/CD24- cells is strongly associated with a basal-like or mesenchymal-like
breast cancer phenotype such as MDA-MB-231cell line
126-128
. Flow cytometric analysis showed that 99% of
MDA-MB-231 cells stained positive for CSC markers, with high expression of CD44 and low expression of CD24
(Figure 10A, control). After being treated with cMLV(Sal) and cMLV(Dox+Sal), the percentages of CSCs
decreased to 84% and 79%, respectively, indicating that Sal in both cMLV(Sal) and cMLV(Dox+Sal) was
selectively toxic to breast cancer stem cells (Figure 10A and B). Despite the extensive usage of CD44
+
CD24
–
surface marker to identify CSCs in various cancers, this method might not the most reliable or accurate method
of detection since their expression levels can be subject to environmental conditions
39
.

Figure 10. Percentage of a CD44+/CD24- subpopulation analyzed by flow cytometry in MDA-MB-231 cell
line. (A) MDA-MB-231 cells were stained with FITC-CD24 and APC-CD44 antibodies, and analyzed with
flow cytometer. The plots depict the CD24 and CD44 staining patterns. Each group is gated by its isotype
control. (B)Quantification graph to the flow cytometric profiles of CD44+/CD24- in each treatment group.

31

To exclude such artifacts, we also detected autofluorescent cells in the MBA-MD-231 cell line to verify the
combinatory effect of cMLV(Dox+Sal) on CSCs. FACS analysis revealed that 1.61% of the MBA-MD-231 cells
are autofluorescent (Figure 9B). The cMLV(Dox) treatment dramatically increased the percentage of
autofluorescent cells to 97.5% , whereas the cMLV(Sal) and cMLV(Dox+Sal) treatments resulted in a significant
reduction in the autofluorescent cells to 0.419% and 0.53%, respectively, further confirming the CSC targeting
ability of Sal in human breast tumor cells. The inhibitory effect of combination nanoparticles on CSCs was further
examined by a mammosphere formation assay. Previous studies have reported that the ability to form non-
adherent spherical clusters of cells, termed mammospheres, in a three-dimensional culture is one characteristic of
breast cancer stem cells
87, 129
. As shown in Figure 9C-D, mammospheres exposed to cMLV(Sal) and
cMLV(Dox+Sal) formulations for 48 h had significantly decreased number and diameter compared to the control
group (p < 0.05). It is notable that the number and the diameter size of the mammospheres were markedly
increased by the cMLV(Dox) treatment compared to all other groups. These results indicate that Dox in cMLVs
can only induce cytotoxicity in fast growing cancer cells, whereas Sal in cMLVs can effectively destroy non-
adherent colonies, proving its selective toxicity against CSCs.

Figure 11. Tumor inhibition induced by cMLV(Dox+Sal) treatment. (A) Each group of mice was treated with
PBS, cMLV(1mg/kg Dox), cMLV(5mg/kg Sal), cMLV(1mg/kg Dox) + cMLV(5mg/kg Sal) and cMLV(1mg/kg
Dox+5mg/kg Sal) for every other day. Measurement of tumor growth (A) and body weights (B) were monitored
32

and recorded throughout the period of the experiment. Error bars represent standard error of the mean, n=5 for
each treatment group (*p < 0.05, **p < 0.01).

In vivo anti-tumor efficacy of Dox and Sal co-loaded cMLVs.

In vivo tumor growth experiments were performed to test the central hypothesis that cMLVs are able to improve
cancer therapy by co-delivering Dox and Sal preferentially to the tumor site, eliminating both the bulk tumor and
the CSC cell populations. Dox and Sal co-encapsulated cMLV particles (cMLV(Dox+Sal)), single drug-loaded
cMLVs (cMLV(Dox) or cMLV(Sal)), and the combination mixture of single drug-loaded cMLVs
(cMLV(Dox)+cMLV(Sal)) were administered to mice bearing 4T1 tumors and the tumor growth was monitored.
As shown in Figure 11A, in accordance with the in vitro cell cytotoxicity data, the tumor growth in all of the
treatment groups was inhibited compared to that of the control group. Notably, cMLV(Dox+Sal) had the most
significant tumor inhibition effect among all the groups (p < 0.01) and showed enhanced anti-tumor activity as
compared to cMLV(Dox) (p < 0.05). The mixture of cMLV(Dox)+cMLV(Sal) formulations was not as effective
as co-encapsulated cMLV(Dox+Sal) in a single nanoparticle, revealing the prominent role of co-localized
ratiometric delivery of Dox and Sal through cMLVs. Furthermore, no weight loss was observed in all the
treatment groups throughout the experiment (Figure 11B), suggesting there was no obvious systemic toxicity
from the dose combinations used.
The inhibitory effect of cMLV(Dox+Sal) on cancer stem cells in vivo.

FACS analysis of ALDH
pos
cells was conducted on excised tumors from treated mice to determine the effects of
combination therapy on the CSC population. In agreement with our in vitro ALDH staining results, the percentage
of ALDH
pos
cells was increased by the cMLV(Dox) treatment compared to the control group; conversely, the
ALDH
pos
percentage

was decreased to a great extent by the cMLV(Sal), cMLV(Dox)+cMLV(Sal) and
cMLV(Dox+Sal) treatments (Figure 12A and B). Furthermore, the cMLV(Dox)+cMLV(Sal) treatment was
significantly less effective at reducing the ALDH
pos
population (p < 0.01) than the cMLV(Dox+Sal) treatment.
To further confirm the inhibitory effect of Sal in vivo, the RNA expression level of Sox 2 was evaluated in 4T1
33

tumor-derived cells. Consistent with the in vitro findings, Figure 12D shows that the Sox 2 RNA expression level
in 4T1 tumor-derived cells was noticeably increased by the cMLV(Dox) treatment (p < 0.05) compared to the
control. However, the expression level of Sox2 in 4T1 tumor-derived cells was significantly decreased by
treatments of cMLV(Sal), cMLV(Dox)+cMLV(Sal) and cMLV(Dox+Sal), with cMLV(Dox+Sal) being the most
effective group (p < 0.01). Taken together, co-delivery of Dox and Sal via cMLVs induced a significant cancer
treatment enhancement due to the capability of Dox to eliminate the cancer cells combined with the capability of
Sal to suppress the CSC population.

Figure 12. Flow cytometric profiles of Aldefluor
pos
and Quantification of Sox 2 RNA level change in 4T1
tumor- derived cells and Histology staining of Carbonic anhydrase 9 (CA-9) using 4T1 tumor tissues
obtained from tumors treated in in vivo antitumor study. (A) Each FACS plot represents the staining
pattern for ALDH
pos
population in 4T1 tumor-derived cells after treatments of all five formulations. (B)
Quantification graph of ALDH
pos
population after treatments. (C) Confocal images show the expression
of CA-9 (red) and nuclear staining DAPI (blue) in each treatment group. (D) Change in Sox2 RNA
expression level was measured using RT/Q-PCR in each treatment group of 4T1 tumor-derived cells. Sox2
expression level of each treatment group was normalized by an expression level of control group. (B,D)
Data represented as mean±SD (n=3). (*p < 0.05, **p < 0.01)

Inhibition of the expression of CA-9 in tumor-derived tissue.

34

Carbonic anhydrase 9 (CA-9) is a membrane bound enzyme that catalyzes the reversible hydration of carbon
dioxide
130
. Previous studies have reported that the overexpression of CA-9 correlates with a poor clinical outcome
in breast cancer patients
131
. D’Uva et al. detected the overexpression of CA-9 in mammospheres and highlighted
the crucial role of CA-9 expression as a breast cancer stem cell regulatory gene that promotes survival and
proliferation of cancer stem cells
132
. Lock et al. demonstrated that CA-9 is a crucial regulator of expanding breast
CSCs, thus making CA-9 an important therapeutic target for depleting breast tumor CSCs
133
. Further reports
revealed that CA-9 might be responsible for cancer progression, including cancer resistance, angiogenesis, and
metastasis
130, 134
. Lou et al. also confirmed that 4T1 cells highly express CA-9
134
; hence, we tested the expression
level of CA-9 on tumor tissues obtained from excised 4T1 tumors from treated mice by immunohistology staining.
As predicted, the control group exhibited significant expression of CA-9 (Figure 12C). After the cMLV(Dox)
treatment, the CA-9 expression level was further enhanced; conversely, the expression level of CA-9 was
markedly decreased by the cMLV(Sal), cMLV(Dox)+cMLV(Sal), and cMLV(Dox+Sal) treatments. It is also
evident that the effect of cMLV(Dox)+cMLV(Sal) on CA-9 expression was not as significant as cMLV(Dox+Sal)
treatment, confirming the synergistic efficacy induced by the co-localized delivery of the two drugs.

DISCUSSION
In summary, a potential strategy for eradication of the bulk breast cancer tumor cells and cancer stem cells (CSCs)
was introduced by co-loading doxorubicin (Dox) and salinomycin (Sal) into cross-linked multilamellar liposomes.
The central hypothesis was that the antitumor effect can be significantly enhanced by simultaneously co-
delivering two drugs to tumors via a single nanoparticle, cross-linked multilamellar liposomal vesicles (cMLVs).
In previous studies, we have demonstrated the successful application of cMLVs as a promising combinatorial
drug delivery system, including the ability of cMLVs to co-encapsulate two anti-cancer drugs with different
hydrophilicities, to maintain synergistic drug ratios, and to overcome drug resistance
102, 116
. Our previous work,
particularly, had its focus on the ratio of drugs combined in cMLVs. This study rigorously determined the
35

optimal ratio of combined drugs needed for synergism
102, 116
. In the present study, we optimized the cMLV system
to deliver Dox and Sal to the tumor site in order to simultaneously target two different cell populations: breast
cancer cells and CSCs. We have demonstrated the ability of cMLVs to co-load Dox and Sal at a pre-determined
synergistic drug ratio and to eradicate two different types of cancer cells, leading to enhanced breast tumor
inhibition. Both in vitro and in vivo analysis of CSC populations provided compelling evidence that co-delivery
of Dox and Sal via cMLVs is able to enhance the targeting of both CSCs and breast cancer cells.
The existence of CSCs has been a growing interest in cancer therapy due to the distinct properties they share with
normal stem cells, including increased persistence and self-renewal capabilities, which often lead to the failure of
traditional chemotherapy. Previous studies proposed that CSCs harbor innate resistance mechanisms to traditional
chemotherapeutics, like doxorubicin, resulting in cancer relapse
90, 93, 114
. CSCs some of these survival mechanisms
include stronger DNA repair capability, overexpression of efflux (ABC) transporters, increased anti-apoptotic
factors, and a slower proliferation rate compared to fast dividing cancer cells
84, 87
. A new approach to target these
CSCs has long been desired, and the therapeutic advantage of salinomycin and its potential role as a CSC-targeted
inhibitor has been emphasized by several studies. Although little is known about the exact underlying mechanism
of salinomycin, one proposed mechanistic explanation is that Sal increases DNA damage and the levels of DNA
damage response proteins
83, 113, 114
. This effect is in accordance with the finding that salinomycin has a
sensitizing effect to enhance cellular apoptosis when used with conventional drugs such as paclitaxel,
doxorubicin, and etoposide. Due to the poor aqueous solubility of salinomycin, nanoparticle delivery systems
have been developed to maximize its overall delivery to the tumor in in vivo studies
135, 136
. As previously
introduced, nanoparticle delivery systems have previously been explored as combination therapy delivery systems
to target cancer stem cells and breast cancer cells by combining the single drug-formulated nanoparticles.
However, there was no distinct difference in in vivo tumor suppression induced between the free drug
combinations and formulated single drug combination therapy
82, 83
, revealing the urgent need for a novel system
for co-localized delivery of combined drugs.
36

There is growing evidence that single carriers of multiple drugs allow co-delivery of the combined drugs to the
same site of interest and maximize cytotoxicity by controlling and coordinating the pharmacokinetics and bio-
distribution profiles of drug combinations
102, 111, 115, 137
.
Markovsky et al. found that co-delivery of paclitaxel and Dox conjugated to a single polymeric nanoparticle was
advantageous. Specifically, tumor inhibition by paclitaxel and Dox co-delivered in a single nanoparticle was
superior to that of the mixture of drugs conjugated to separate particles, indicating the ability of the combination
particles to release the two agents simultaneously to tumor site with an identical pharmacokinetic profile
110
. In a
separate study, Blanco et al. emphasized the superior therapeutic efficacy of co-localized delivery of rapamycin
and paclitaxel within nanoparticles. Their studies clearly determined the factors leading to synergistic drug
combination therapy via nanoparticles: encapsulation of optimal ratios of combined drugs, controlled release of
the drugs by maintaining pre-determined synergistic drug ratios, and identical delivery time of combined drugs
to the target site
137
.
The major advantages of single carrier mediated drug delivery systems over delivery of a physical mixture of
individually formulated multiple drugs can be seen in vivo
86, 110, 111
. While our in vitro studies using the human
MDA-MB-231cell line show no significant differences in the therapeutic efficacy of cMLV(Dox+Sal) and
cMLV(Dox)+cMLV(Sal) (Figure 3A and Figure S2A), the in vivo data, from a mouse 4T1 breast cancer model,
clearly shows the benefits of single carrier mediated drug delivery systems over delivery of a physical mixture of
individually formulated drugs. Specifically, cMLV(Dox+Sal) exhibited a greater inhibition on tumor growth and
CSCs population compared to cMLV(Dox)+cMLV(Sal), demonstrating the potential of colocalized delivery via
cMLVs. These results, taken together, reveal the significant therapeutic potential of cMLVs as a single agent-
mediated multiple drug delivery system, allowing codelivery of two drugs, with different mechanisms of action.
By this means, more effective multi-drug treatment of breast cancer can be achieved by simultaneously targeting
both cancer and cancer stem cells.

37

METHODS
Cell lines, reagents, mice
Murine breast cancer cell lines, 4T1 and Dox-resistant 4T1 (4T1D), and human breast cancer cell line MDA-MB-
231, were cultured in a 5% CO2 environment with Dulbecco’s modified Eagle medium (Mediatech, Inc.,
Manassas, VA) or RPMI 1640 (Hyclone, Rockford, IL), respectively, supplemented with 10% FBS (Sigma-
Aldrich, St. Louis, MO) and 2 mM of L-glutamine (Hyclone Laboratories, Inc., Omaha, NE). The doxorubicin-
resistant subculture line (4T1D) was selected by culturing in stepwise increasing concentrations of doxorubicin.
Doxorubicin and Salinomycin were purchased from Sigma-Aldrich (St. Louis, MO) and Cayman Chemical (Ann
Arbor, MI), respectively. All lipids were purchased from NOF Corporation (Japan): 1,2-dioleoyl-sn-glycero-3-
phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phospho-(10-rac-glycerol) (DOPG), and 1,2-dioleoyl-sn-
glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)but-yramide (maleimide-headgroup lipid, MPB-PE).
Female 6–10 weeks-old BALB/c mice were purchased from Charles River Breeding Laboratories (Wilmington,
MA). All mice were held under specific pathogen-reduced conditions in the animal facility of the University of
Southern California (Los Angeles, CA, USA). All experiments were performed in accordance with the guidelines
set by the National Institute of Health and the University of Southern California on the Care and Use of Animals.
Synthesis of cMLVs
Liposomes were prepared based on the conventional dehydration-rehydration method. Lipids (DOPC, DOPG and
MPB-PE; 1.5 µmol in chloroform) were mixed at a molar ratio lipid composition DOPC:DOPG:MPB = 4:1:5,
and the organic solvent in the lipid mixture was evaporated under argon gas and dried under a vacuum overnight
to form dried thin lipid films. To prepare cMLV(Dox+Sal), salinomycin in organic solvent was mixed with the
lipid mixture to form dried thin lipid films. Bis-Tris propane (10 nM, pH 7.0) with doxorubicin was added to
hydrate the resultant dried film and the solution was mixed by vortex every 10 min for 1 h. The solution was then
sonicated (15 s, 4 cycles) (Misonix Microson XL2000, Farmingdale, NY) on ice with 1 min intervals between
each cycle. To induce divalent-triggered vesicle fusion, MgCl2 was added to a final concentration of 10 mM. The
38

resulting multilamellar vesicles were further crosslinked by addition of dithiothreitol (DTT, Sigma-Aldrich) to a
final concentration of 1.5 mM and incubated for 1 h at 37°C. The resulting vesicles were collected by
centrifugation at 14,000 × g for 4 min and then washed twice with PBS. To pegylate cMLVs, the particles were
incubated with 1 µmol of 2 kDa PEG-SH (Laysan Bio Inc. Arab, AL) for 30 min at 37°C. The particles were then
centrifuged and washed twice with PBS. The final products were stored in PBS at 4°C.
Characterization of physical properties
Morphological analysis of multilamellar structure of vesicles were performed and confirmed by cryo-electron
microscopy in previous studies
101, 102
. The hydrodynamic size of cMLVs was measured by dynamic light
scattering (Wyatt Technology, Santa Barbara, CA). The particles were suspended in filtered water, vortexed and
sonicated prior to analysis.
In vitro encapsulation and release profiles
To determine the loading capacity of Dox, cMLV(Dox) and cMLV(Dox+Sal) were collected and washed twice
with PBS, followed by lipid extraction of vesicles with 1% Triton X-100 treatment. Dox fluorescence (excitation
480 nm, emission 590 nm) was then measured by Shimadzu RF-5301PC spectrofluorometer (Japan). The amount
of Sal encapsulated in the cMLV(Sal) and cMLV(Dox+Sal) was determined by C-18 RP-HPLC chromatography
(Backman). The cMLV(Sal) and cMLV(Dox+Sal) suspensions were diluted by adding water and acetonitrile to
a total volume of 0.5 ml. Extraction of Sal was accomplished by adding 5 ml of tert-butyl methyl ether and mixing
the sample by votex for 1 min. The mixtures were then centrifuged and the organic layer was transferred into a
glass tube and evaporated to dryness under argon. Buffer A (95% water, 5% acetonitrile) was used to rehydrate
the dried organic layer. To determine the Sal concentration, 1ml of the solution was injected into a C18 column,
and salinomycin was detected at 392 nm (flow rate 1ml/min). The release kinetics of Dox and Sal from liposomes
were investigated by removing the releasing media from cMLVs incubated in 10% fetal bovine serum (FBS)-
containing media at 37°C and replacing it with fresh media daily. The removed media was quantified for Dox
fluorescence (by spectrofluorometer) and Sal fluorescence (by HPLC) every day.
39

In vitro cytotoxicity study
4T1, 4T1D and MDA-MB-231 cells were seeded in 96-well plates at 5 × 10
3
cells per well in 10% fetal bovine
serum (FBS)-containing media and grown at 37 °C in the presence of 5% CO2 for 6 h. Cells were incubated with
various concentrations of cMLV (single drug) and cMLV (drug combinations) at 1 to 5 molar drug ratios for 48
h as previously described
102, 116
, and cell viability was assessed using the Cell Proliferation Kit II (XTT assay)
from Roche Applied Science (Indianapolis, IN) according to the manufacturer's instructions. Cell viability
percentage was determined by subtracting absorbance values obtained from media-only wells from the treated
wells and then normalized by the control wells containing cells without drugs
138
.
Mammosphere formation assay
MDA-MB-231 cells were seeded into 6-well ultra-low attachment plates (Stem Cell Technologies, Vancouver,
Canada) at 5 × 10
3
cells per well and cultured in complete MammoCult medium (Stem Cell Technologies,
Vancouver, Canada) containing the MammoCult basal medium, MammoCult proliferation supplement, fresh
hydrocortisone and heparin. Cells were cultured for 7 days and treated with cMLV(1µM Dox), cMLV(5µM Sal)
and cMLV(1µM Dox+5µM Sal) for 48 h. Mammospheres were counted and visualized using an EVOS FL
microscope (AMG Micro, Bothell, WA) at 10× magnification. Mammosphere diameters were measured using
Image J software (NIH, USA).
Flow cytometric analysis
FACS analysis was performed using either FACS Aria (Becton Dickinson, Palo Alto, CA) or MACS Quant
analyzer (Miltenyi Biotec Inc., San Diego, CA). 4T1 and 4T1D cells were seeded into 6-well plates at 6 × 10
6

cells per well. When cells reached about 70% confluence, the cells were incubated with PBS control, cMLV(1µM
Dox), cMLV(5µM Sal) and cMLV(1µm Dox+5µM Sal) for 24 h. For Hoechst dye staining, 1 × 10
6
tumor
cells/mL from each well were harvested and stained with 5 µg/mL Hoechst 33342 dye (Sigma, St Louis, MO) or
Hoechst dye plus 100 mM verapamil hydrochloride (Sigma, St Louis, MO) at 37°C for 1 h. For ALDH staining,
4T1, 4T1D and MDA-MB-231 cells (3 × 10
5
) from the same cMLV treatment conditions were harvested and an
40

aldefluor kit was used according to the manufacturer’s instructions (Stem Cell Technologies, Vancouver, Canada).
For analysis of autofluorescent subpopulations, 4T1, 4T1D and MDA-MB-231 cells (2 × 10
5
) from the same
cMLV treatment conditions were harvested and detected Using SORP LSR II (BD Biosciences, San Jose, CA).
Autofluorescent cells are excited with a 488-nm blue laser and best selected as the intersection with filters 525/50
and 575/26. A proper distance between gates for autofluorescent and non-autofluorescent cells is required to
achieve high purity during sorting
39
.
For FACS analysis of in vivo tumor samples, single-cell suspensions were prepared from excised tumor by manual
mincing followed by enzymatic digestion for 40 min at 37°C by 3.0 mg/ml of collagenase A (Roche, Indianapolis,
IN) and DNase I (Roche) dissolved in DMEM (Invitrogen, Grand Island, NY) under stirring conditions. Digestion
mixtures were quenched by adding DMEM containing 10% FBS and then filtered through 0.7 µm nylon strainers
(Falcon, Tewksbury MA). These tumor-derived cells were then stained for ALDH enzymatic activity.
For MDA-MB-231 cell analysis, cells were seeded into 6-well plates at 6 × 10
6
cells per well and allowed to grow
to about 70% confluence, after which the cells were then incubated with PBS control, cMLV(1µM Dox),
cMLV(5µM Sal) and cMLV(1µm Dox+5µM Sal) for 24 hr. Cells from each well were harvested and stained with
anti-CD24-FITC and anti-CD44-APC antibodies (1:20 dilutions) (BD Biosciences, San Jose, CA).
RNA extraction and quantitative reverse transcriptase PCR
4T1, 4T1D, 4T1 tumor-derived cells were seeded into 6-well plates at 6 × 10
6
cells per well. When cells reached
about 70% confluence, the cells were incubated with cMLV(1µM Dox), cMLV(5µM Sal) and cMLV(1µM
Dox+5µM Sal) for 48 hr. Cells (5 × 10
5
)

from each well were harvested and total RNA was isolated using
RNAeasy (Qiagen, Valencia, CA). Quantitative PCR was performed on the Bio-Rad MyiQ real-time system
(Hercules, CA ) using a pair of primers specific for mouse Sox 2 (Forward 5´-TCC CAT CAC CCA CAG CAA
ATG A-3´ and Reverse 5´- TTT CTT GTC GGC ATC GCG GTT T-3´). For an internal control, a primer set of
mouse GAPDH was used (Forward 5´-CGA CTT CAA CAG CAA CTC CCA CTC TTC C-3´and Reverse 5´-
TGG GTG GTC CAG GGT TTC TTA CTC CTT-3´).
41

In vivo antitumor activity
BALB/c female mice (6–10 weeks-old) were inoculated subcutaneously with 0.2 × 10
6
4T1 breast tumor cells.
The tumors were allowed to grow for 8 days to a volume of ~50 mm
3
before treatment. On day 8, the mice were
randomly assigned to 5 groups (5 mice per group) and received the following treatments: group 1, PBS control;
group 2, cMLV(Dox) (1mg/kg Dox); group 3, cMLV(Sal) (5mg/kg Sal); group 4, cMLV(Dox) (1mg/kg Dox) +
cMLV(Sal) (5mg/kg Sal) mixtures; group 5, cMLV(Dox+Sal) (1mg/kg Dox+5mg/kg Sal). All the formulations
were given via tail vein injection every other day, totaling of 5 times. Tumor volumes and body weights of mice
were monitored until the end of an experiment. The length and width of the tumor masses were measured with a
fine caliper every other day after injection. Tumor volume was expressed as 1/2 x (length x width
2
). On Day 18,
mice were sacrificed and tumors were excised for further analysis.

Immunohistochemistry of tumors and confocal imaging
4T1 breast tumors from all treatment groups were excised, fixed, frozen, cryo-sectioned, and mounted onto glass
slides. Frozen sections were then fixed, and rinsed with cold PBS. After blocking and permealization, the slides
were washed with PBS and incubated with rabbit anti-carbonic anhydrase 9 (CA-9) (M-100) (Santa Cruz
Biotechnology Inc, Santa Cruz, CA). Fluorescence images were acquired on a Yokogawa spinning-disk confocal
scanner system (Solamere Technology Group, Salt Lake City, UT) using a Nikon eclipse Ti-E microscope
equipped with a 60 × /1.49 Apo TIRF oil objective and a Cascade II: 512 EMCCD camera (Photometrics, Tucson,
AZ). An AOTF (acousto-optical tunable filter) controlled laser-merge system (Solamere Technology Group Inc.,
Salt Lake City, UT) was used to provide illumination power at each of the following laser lines: 491 nm, 561 nm,
and 640nm solid state lasers (50mW for each laser).
Statistics
42

The differences between two groups were determined with Student’s t test. The differences among three or more
groups were determined with a one-way analysis of variance (ANOVA)

43

CHAPTER 3: COMBINATION CANCER THERAPY
USING CHIMERIC ANTIGEN RECEPTOR-
ENGINEERED NATURAL KILLER CELLS AS DRUG
CARRIERS

ABSTRACT

The therapeutic limitations of conventional chemotherapeutic drugs include chemo-resistance, tumor recurrence,
and metastasis. Numerous nanoparticle-based active targeting approaches have emerged to enhance the
intracellular concentration of drugs in tumor cells; however, efficient delivery of these systems to the tumor site
while sparing healthy tissue remains elusive. Recently, much attention has been given to human immune cell-
directed nanoparticle drug delivery, as immune cells can traffic to the tumor and inflammatory sites. Natural
killer (NK) cells are a subset of cytotoxic lymphocytes that play critical role in cancer immunosurveillance.
Engineering of the human NK cell line, NK92, to express chimeric antigen receptors (CARs) to redirect their
antitumor specificity has shown significant promise. We demonstrate that the efficacy of chemotherapy can be
enhanced in vitro and in vivo while reducing off-target toxicity by using CAR-engineered NK92 cells as carriers
to direct drug-loaded nanoparticles to the target site.

INTRODUCTION

Cancer treatments typically include surgical removal, chemotherapy, radiation, or some combination of these
therapies
139
. While these methods can remove the majority of the tumor cells, tumor recurrence and metastasis
remain a major obstacle in cancer treatment
2
. Solid tumors often do not respond well to traditional anticancer
drugs. After relapse, further treatment options frequently include different chemotherapeutics, and the time to
progression typically shortens after each relapse
140
. At best, it can be managed as a “chronic disease” for many
44

years, but cumulative toxicities of successive chemotherapy treatments are a serious consideration. Cancer
immunotherapy has gained much attention within the last decade, and more recently, the concept of using immune
cells as vehicles to transport anticancer agents directly to the tumor site has gained attention
141-143
. Natural killer
(NK) cells are part of the innate immune system and mediate rapid, short-lived responses by releasing cytokines
that directly lyse infected or abnormal cells including tumor cells
18, 144
. The NK92 cell line is identical to the
parental NK line isolated from a lymphoma patient
145
. NK92 cells are the most extensively characterized and
well-documented with antitumor effects against various types of cancer including melanoma, leukemia and breast
cancer in both preclinical and clinical settings
146-148
. NK cells are not specifically cytotoxic to certain antigen-
expressing target cells
149
, but their specificity and efficacy can be enhanced with the use of chimeric antigen
receptors (CARs). CARs are antigen-specific engineered receptors that can be expressed on human immune
cells
150-152
. They are composed of an extracellular antigen-binding domain derived from an antibody
fragment
153
. This allows the CAR-expressing immune cells to bind to specific surface antigens overexpressed
on cancer cells. Intracellular signaling domains within the CAR provide signals to the immune cell to attack the
antigen-expressing cell
154, 155
. Typically, CARs are expressed in T cells, but recent studies show CARs are
effective tumor-targeting molecules in NK cells as well
156-159
. The inclusion of CARs in NK92 cells (CAR.NK)
increases homing, specificity, and efficacy of tumor killing. Paclitaxel (PTX) is a common chemotherapeutic
that has been used clinically for many years to treat various cancers. However, the therapeutic efficacy and
clinical application of PTX is hindered by its hydrophobicity, high toxicity, and low bioavailability. Many
studies have shown that nanoparticle formulation of PTX has enhanced its delivery to the tumor site with reduced
systemic toxicity as compared to the unmodified free drug
160-162
. For example, Abraxane is an FDA-approved
formulation of PTX and albumin nanoparticles which has been shown to significantly increase overall survival in
clinical trials compared to free drug administration
163
. Nanoparticles are considered promising drug delivery
vehicles for cancer therapy based on their ability to increase drug accumulation at tumor sites via the enhanced
permeability and retention (EPR) effect. The EPR effect describes a phenomenon in which irregular, leaky
45

vasculature and poor lymphatic drainage within a tumor allow larger nanoparticles to extravasate and remain in
the tumor site
164
. Although the EPR effect has been well-characterized in pre-clinical models, this mechanism
is heterogeneous and may be completely lacking in some tumors
165
. Tumor vasculature heterogeneity often
results in nanoparticle accumulation in the extracellular matrix proximal to the tumor vasculature. Tumor cells
that are distant from well-defined vasculature are not effectively targeted by these nanoparticles. Moreover, as
hypoxic tumor regions have dysregulated tumor vasculature, traditional passive drug targeting via nanoparticles
is insufficient
163
. New strategies are desired to guide nanoparticles to poorly vascularized tumor tissue.
Immune cells are an attractive option for active cancer therapeutic delivery, due to their natural ability to home
to tumor sites
141
. Instead of relying on passive delivery, immune cells can be used as active carriers for
nanoparticles that are either directly cytotoxic to the tumor or nanoparticles which carry chemotherapeutic agents.
Nanoparticles can be conjugated to the cell surface by directly utilizing functional groups such as amino or thiol
groups, through hydrophobic insertion into the lipid bilayer, by electrostatic interactions, or by attaching to
specific cell surface receptors
166
. Within the past decade, research groups have demonstrated that nanoparticles
delivered by cells can enhance antitumor effects
167
. In many of the current cell-based nanotherapies, the cell
serves as a carrier for nanoparticles and their cargo. TAA-targeted T cells have shown to carry adsorbed
oncolytic viruses to tumor sites in several studies
168-170
, and this concept has been extended to synthetic
nanoparticle delivery. However, more studies of lymphocytes conjugated to synthetic drug-loaded nanoparticles
are needed. While other immune cell types have been exploited as drug carriers, to our knowledge, NK cells
have not been surface engineered with drug-loaded nanoparticles, despite their ability to home to the tumor site.
To address such a need, we hypothesized that the efficacy of chemotherapy could be enhanced if tumor specific
NK cells were used as carriers to deliver drug-loaded nanoparticles. Based upon the strategies presented in
previous studies
171
, we modified our tumor-specific CAR.NK cells with crosslinked multilamellar liposomal
vesicles (cMLVs) containing PTX. These cMLVs are liposomes functionalized with thiol-reactive maleimide
headgroups
172, 173
, which allow them to be stably conjugated to the thiol-rich NK cell surface
171, 174
. Previously,
46

we have demonstrated that cMLVs can act as a novel agent for combinatory drug delivery by co-localizing two
drugs with distinct physicochemical properties to a single site, inducing a synergistic anti-tumor effect in vitro
and in vivo
175, 176
. Here, our work presents the combination of immunotherapy and chemotherapeutic drug
delivery by utilizing CAR.NK cells as carriers for PTX-loaded crosslinked multilamellar liposomal vesicles
(cMLV (PTX)) to enhance antitumor efficacy in Her2 and CD19 overexpressing cancer models (Figure 13A).

RESULTS

Anti-CD19 and anti-Her2 CARs are expressed in NK92 cells

We confirmed the ability of NK92 cells to express anti-CD19 and anti-Her2 CARs, which consisted of an scFv-
derived antigen binding domain, CD8 hinge and transmembrane region, CD28 and/or 4-1BB costimulatory
domains, and CD3ζ signaling domain. Anti-CD19 CAR.NK cells were generated with retroviral transduction
using the previously documented MP71 vector
177
generously provided by Dr. Wolfgang Uckert. The anti-Her2
CAR.NK cells were generated with lentiviral transduction using a previously described trastuzumab-derived
CAR
178
in a pCCW vector, which is based off the pCCL vector
179-181
with an added WRE posttranscriptional
regulatory region. Transduced cells were sorted using fluorescence activated cell sorting to further increase the
percentage of CAR
+
cells. CAR expression was stable several months after initial transduction and sorting (data
not shown).

cMLVs are stably conjugated to the NK cell surface

Previous studies have shown that cross-linked multilamellar liposomal vesicles (cMLVs) were successfully
incorporated with both hydrophobic and hydrophilic drugs. As previously illustrated
182
, these vesicles were
synthesized through covalently crosslinking functionalized headgroups of adjacent lipid bilayers using the
conventional dehydration-rehydration method. Synthesized cMLV nanoparticles were stably conjugated to the
reduced thiol groups present on the surface of NK cell via the thiol-reactive maleimide headgroups present on the
47

lipid bilayer surface. According to previous studies
142
, high levels of free thiols were detected on the surfaces of
lymphocytes. The conjugation was performed in two steps. First, NK cells and cMLVs were coincubated to induce
particle coupling to free thiols on the cell surface. After the initial reaction, the cMLV-conjugated cells underwent
in situ PEGylation to quench residual thiol reactive groups. To determine the maximum numbers of particles
that could be conjugated per NK cell, we performed a serial dilution of the conjugation at different fluorescent-
labeled cMLVs to cell ratios (2000:1, 1000:1, 500:1, 100:1, and 10:1). Between the conjugation ratio of 2000:1
and 1000:1, the number of conjugated liposomes per cell began to plateau and showed an average conjugation of
approximately 150 nanoparticles per cell (Figure 13B). From this data, we determined that the optimal ratio to
use was 1000:1, as further increasing it did not increase the number of conjugated cMLVs on the cell surface.
We used this ratio for all subsequent experiments. Confocal imaging was used to confirm and visualize the
conjugation of cMLVs to the NK cell surface (Figure 13C).

Figure 13. NK92 cell conjugation to maleimide-functionalized cMLVs. (A) Schematic of CAR.NK cells
conjugated to PTX-loaded cMLVs. CARs are derived from the single chain variable fragment (scFv) of
an antibody and the T cell receptor signaling complex. CARs can be transduced into NK92 cells and
cMLVs can conjugate to the cell surface by interacting with free thiols. (B) cMLVs conjugated to the
NK cell surface at various cMLV: cell ratios. cMLVs containing the fluorescent dye DiD were
coincubated with NK cells over a range of ratios. The number of cMLVs on the surface of each cell was
calculated by analyzing the DiD fluorescence. The ratio of 1000:1 provided the maximum amount of
cMLVs per cell and was used in future experiments. (C) Confocal microscopy of CAR.NK cells
conjugated to DiD-loaded cMLVs (cMLV(DiD)). CAR.NK cells were labeled with 1 µM CFSE and
washed with PBS prior to conjugation to cMLV(DiD). Confocal microscopy was used to visualize the
48

cMLVs on the CAR.NK cell surface. (D) Internalization assay of conjugated cMLVs. CAR.NK cells were
conjugated with carboxyfluorescein-tagged maleimide-labeled cMLVs. The extracellular conjugation was
quenched by trypan blue to differentiate surface-bound and internalized cMLVs 2 hours after
conjugation. Attachment of cMLVs to CAR.NK-cells did not trigger the internalization of particles by the
cells.

As previously reported
141, 171
, the major advantages of extended surface retention of nanoparticles on the surface
of carrier cells are as follows: (1) prevention from immediate particle degradation due to internalization into
degradative intracellular compartments and (2) sustained drug release from the particle-conjugated cells which
allows for effective targeting of the drug to tumor cells. Several studies have shown that nanoparticles can be
endocytosed by a variety of cells
183
, including endothelial cells
184, 185
and macrophages
186
. However, for our
study, it is crucial that the cMLVs remain on the NK cell surface. To address this concern, we performed an
experiment to determine the internalization of these particles after conjugation. To determine whether these NK
cells could also trigger liposome endocytosis, we conjugated NK cells with cMLVs tagged with a PE CF
fluorescein dye, then warmed the cells to 37°C and assessed cell-associated fluorescence over time. Unlike with
other phagocytic cells, attachment of cMLVs to NK cells did not trigger cell uptake of these particles and particles
bound to NK cells remained at the cell surface as shown in Figure 13D.

CAR.NK cells have greater cytotoxic effects against antigen-expressing target cells in vitro and
are less sensitive to PTX

We assessed the ability of CAR.NK cells to trigger cytotoxic effects against the appropriate antigen-expressing
target cells by coincubating nontransduced NK or CAR.NK cells with various target cell lines and reading the
results with flow cytometry. We used lentivirus to transduce SKOV3 cells to express CD19 (SKOV.CD19) to
serve as target cells for our anti-CD19 CAR.NK cells. Both CD19 and Her2-targeting CAR.NK cells
demonstrated significantly greater cytotoxicity against the antigen-expressing target cells (SKOV.CD19 and
SKOV3, respectively) compared with either nontransduced NK cells or CAR.NK cells coincubated with target
cells that did not express the cognate antigen (SKOV3 and MDA.MB.468, respectively, Figure 14A, B). These
49

trends were observed at all effector-to-target ratios (p < 0.01 for 1:1 and 10:1, p < 0.001 for 5:1) and indicated
CAR-mediated cell killing.

Figure 14. Cytotoxicity of CAR.NK cells against CD19
+
or Her2
+
target cells. (A) Cytotoxicity of anti-
CD19 CAR.NK cells. Anti-CD19 CAR.NK cells were cocultured with CD19
-
SKOV3 cells or CD19
+

SKOV.CD19 cells for 24 hours at 1:1, 5:1, or 10:1 effector-to-target ratios and cytotoxicity was measured.
(B) Cytotoxicity of anti-Her2 CAR.NK cells. Anti-Her2 CAR.NK cells were cocultured with Her2
-

MDA.MB.468 cells or Her2
+
SKOV3 cells for 24 hours at 1:1, 5:1, or 10:1 effector-to-target ratios and
cytotoxicity was measured. Summarized statistics are displayed in the graphs (n = 3, mean ± SEM; NS,
not significant; *p < 0.05; **p < 0.01; ***p < 0.001)

Since NK92 cells originate from a patient with NK cell lymphoma, these allogenic cells are irradiated prior to
clinical use to prevent them from proliferating in vivo
149, 187
. In accordance with previous reports
158, 187-189
,
irradiation did not affect the cytotoxic capabilities of our CAR.NK cells (Figure 15). We also performed a cell
viability assay to demonstrate that SKOV3 cells were more sensitive to PTX than NK cells were (Figure 16).
This ensures that the NK cells can carry enough PTX to kill target cells without succumbing to PTX-induced
toxicity themselves. cMLVs conjugated to NK cells also release the majority of their PTX payload by Day 3
(Figure 17).
50

Figure 15. Cytotoxicity comparison between irradiated (5 Gy) and nonirradiated CAR.NK cells. NK or
CAR.NK cells were cocultured with SKOV.CD19 cells for 24 hours at 1:1, 5:1, or 10:1 effector-to-target
ratios and cytotoxicity was measured.

CAR.NK function is unaffected by cMLV conjugation and enhanced with cMLV(PTX)
conjugation in vitro

We ensured that the conjugation of cMLVs to the CAR.NK cell surface does not affect the functionality of the
CAR.NK cell itself. To detect NK cell activation upon antigen binding, we performed an IFN-γ release assay,
coculturing various target cell lines with NK cells with or without cMLV conjugation. None of the CAR.NK
cells reacted when incubated alone or when cocultured with target cells without the cognate antigen, but
coincubation with the correct antigen-expressing target cells resulted in significantly greater percentages of IFN-
γ
+
cells from both anti-CD19 and anti-Her2 CAR.NK cell lines (p < 0.05) demonstrating specificity towards the
appropriate tumor-associated antigen (TAA). When the CAR.NK cells were conjugated to either empty cMLVs
containing no drug (CAR.NK.cMLV(EMPTY)) or PTX-loaded cMLVs (CAR.NK.cMLV(PTX)), IFN-γ release
was not significantly different from that of unconjugated CAR.NK cells (Figure 18A, B).
51

Figure 16. Cell viability assay with NK and SKOV3 cells exposed to PTX. Cells were incubated with various
concentrations of cMLV(PTX). Cell viability percentage was determined by subtracting absorbance values
obtained from media-only wells from the treated wells and then normalized by the control wells containing
cells without drugs.

We repeated the cytotoxicity assays using an effector-to-target ratio of 1:1 with CAR.NK cells that were
unconjugated, conjugated to empty cMLVs (CAR.NK.cMLV(EMPTY)), or conjugated to PTX-loaded cMLVs
(CAR.NK.cMLV(PTX)). CAR.NK.cMLV(EMPTY) did not have significantly affected cell killing, but
cytotoxicity against target cells was significantly increased with CAR.NK.cMLV(PTX) (Figure 18 C, D).
These data indicate that while empty cMLVs do not affect CAR.NK function, the release of PTX from cMLVs
in proximity to the target cells further boosted cytotoxic effects.

Figure 17. PTX release kinetics from free cMLVs and CAR.NK.cMLVs. To obtain the release kinetics of
PTX from cMLVs before and after cell conjugation, cMLV(PTX) and CAR.NK.cMLV(PTX) were
incubated in 10% FBScontaining media at 37 °C and were spun down and resuspended with fresh media
daily. The PTX was quantified from the removed media by HPLC every day.
52

Finally, we monitored NK migration with or without cMLV conjugation. In order to affect an antitumor
response, NK cells must extravasate into and migrate within the tumor site in response to chemoattractants
190
.
To ensure that cMLV conjugation to the NK surface did not impact cell migration, we performed NK cell
transmigration assays. The chemoattractant CXCL9 was used to promote NK cell migration to the lower
chamber of the wells. There were significantly more migrated NK cells in the lower chamber when CXCL9 was
used as an attractant compared to the plain media control (p < 0.05), but there was no significant difference
between conjugated and unconjugated groups, indicating that conjugation of cMLVs to the cell surface did not
impact NK migratory abilities (Figure 18E).

Figure 18. CAR.NK cytokine release and migration when conjugated to cMLVs. (A, B) IFNγ staining
assays. Anti-CD19 (A) or anti-Her2 (B) CAR.NK cells were cocultured with various target cells with
Brefeldin A protein transport inhibitor for 6 hours to detect IFNγ release. Unstimulated CAR.NK cells
served as a negative control. CAR.NK cells were either unconjugated or conjugated with empty cMLVs
53

(CAR.NK.cMLV(EMPTY)) or PTX-loaded cMLVs (CAR.NK.cMLV(PTX)). IFNγ was measured with
intracellular staining. (C, D) Cytotoxicity assays. Anti-CD19 (C) or anti-Her2 (D) CAR.NK cells were
cocultured with various target cells for 24 hours and cytotoxicity was measured. (E) Migration assay.
Unconjugated NK or NK conjugated to cMLV(EMPTY) were plated in the upper chambers of a Transwell
plate. Negative controls had plain media in the lower wells, and CXCL9 was used as a chemoattractant
in the lower wells of non-control groups. After 6 hours of incubation, media from the lower chambers
was collected and NK cells were counted. Summarized statistics are displayed in the graphs (n = 3, mean
± SEM; NS, not significant; *p < 0.05; **p < 0.01; ***p < 0.001)

Figure 19. Biodistribution of free cMLV(DiD) and conjugated CAR.NK.cMLV(DiD). Biodistribution
data 24 hours (A), 48 hours (B), or 72 hours (C) after intravenous injections. NOD/scid/IL2rγ-/- (NSG)
mice bearing subcutaneous SKOV3.CD19 tumors were intravenously injected with 2 × 10
7
CAR.NK cells
conjugated with DiD-labeled cMLVs or an equivalent number of DiD-labeled cMLVs alone (n=3 per group
per time point). After 24 hours, 48 hours, and 72 hours, indicated tissues were removed, weighed, and
macerated with scissors. Specific DiD tissue fluorescence for each organ was quantified using the IVIS
Spectrum imaging system and the mean percentage of injected dose per gram of tissue (% ID/g) was
calculated as the final readout.

CAR.NK.cMLV enhances delivery of cMLVs to the tumor site

After confirming the functionality of our cMLV(PTX)-conjugated CAR.NK cells in vitro, we performed a
biodistribution study to determine if CAR.NK cells enhanced cMLV homing to the tumor site. The fluorescent
54

dye DiD was used to tag cMLVs (cMLV(DiD)) and track their presence in various organs. NSG mice were
subcutaneously injected with SKOV.CD19 cells. Two weeks after tumor inoculation, mice were intravenously
injected with cMLV(DiD) or conjugated CAR.NK.cMLV(DiD). Mice were sacrificed and organs were
analyzed for fluorescence signal at various time points. At 24 hours (Figure 19A and B), most of the cMLVs
from both groups were still circulating in the blood. The CAR.NK.cMLV(DiD) group had significantly more
cMLVs in the blood (p < 0.001), lymph node (p < 0.05), and tumor (p < 0.01), while the cMLV(DiD) group
had significantly more accumulation in the liver (p < 0.001). At 48 hours (Figure 19C and D), most of the
cMLV(DiD) group had accumulated in the liver, but the CAR.NK.cMLV(DiD) group had significantly more
cMLVs in the blood, lymph node, spleen, and tumor (p < 0.001). By 72 hours (Figure 19E and F), most of the
cMLV(DiD) signal was gone, with only small amounts detectable in the liver, blood, and tumor. In contrast, the
CAR.NK.cMLV(DiD) group had significantly more cMLVs in the blood, lymph node, spleen, and tumor (p <
0.001). Overall, cMLV(DiD) without a cell acting as a chaperone were likely cleared by the liver, as hepatic
clearance serves as the main clearance route for particles too large to be cleared by the kidneys
191, 192
. In contrast,
cMLV(DiD) that were conjugated to CAR.NK cells were able to home to the tumor site.

Figure 20. Antitumor efficacy of CAR.NK.cMLV(PTX) in solid tumor xenograft model. Tumor growth
curve. SKOV.CD19 cells were injected subcutaneously into the right flank of NSG mice on Day 0. Mice
were randomized into six groups (n = 5 per group) and treated according to their group description four
times total, 3-4 days apart via tail vein injection. Tumor size was measured with a fine caliper (n = 5,
mean ± SEM; NS, not significant; *p < 0.05; **p < 0.01; ***p < 0.001).

55

CAR.NK.cMLV(PTX) enhances antitumor efficacy in vivo

We established a mouse xenograft model to observe the effects of the anti-CD19 CAR.NK cells in vivo. NSG
mice were subcutaneously injected with SKOV.CD19 cells. Two weeks after tumor inoculation, mice were
randomly divided into six groups and injected with (1) PBS as a control, (2) cMLV(PTX) only, without any
cellular component, (3) nontransduced NK cells only, (4) CAR.NK cells only, (5) mixed cMLV(PTX) + CAR.NK
which were coinjected but not conjugated, and (6) conjugated CAR.NK.cMLV(PTX) cells. Mice treated with
CAR.NK.cMLV(PTX) had significantly slowed tumor growth compared to PBS, cMLV(PTX), and NK groups
(p < 0.001), and significantly slowed tumor growth compared to CAR.NK and CAR.NK + cMLV(PTX) groups
as well (p < 0.01, Figure 20). These data support the hypothesis that both immunotherapeutic effects from the
NK cells and chemotherapeutic effects from the PTX play a role in the killing of tumor cells, as either component
alone was not as effective as when the two were combined. Furthermore, even the mice treated with CAR.NK
+ cMLV(PTX) did not have as great an antitumor response as did the mice treated with CAR.NK.cMLV(PTX).
This demonstrates that the physical conjugation between the drug and the NK cell is crucial to receiving the full
benefits of the treatment system, and that the CAR.NK cells are facilitating the delivery of PTX to the tumor site
for enhanced anticancer effects.

Figure 21. Ex vivo analysis of CAR.NK.cMLV(PTX) treatment. (A) Intratumoral PTX concentration.
Thawed tumor samples were homogenized and PTX concentrations analyzed using HPLC (n = 3, mean ±
SEM; NS, not significant; *p < 0.05; **p < 0.01; ***p < 0.001). (B) TUNEL assay of fixed frozen tumor
sections. Tumor sections were stained with a TUNEL kit according to the manufacturer’s instructions
and imaged with confocal microscopy. Representative images are shown herein. (C) Histology slides
56

for cardiac toxicity. Cardiac tissue was fixed and frozen, and sections were mounted on glass slides. The
frozen sections were stained with hematoxylin and eosin. Histopathologic specimens were examined by
light microscopy. Representative images are shown herein.

CAR.NK.cMLV(PTX) enhances PTX delivery into tumor site

We performed ex vivo analysis of our mouse xenograft tumor model to support our hypothesis that CAR.NK cells
facilitate PTX delivery into the tumor site. Using high performance liquid chromatography (HPLC), we
quantified the intratumoral PTX concentrations in mice treated with PTX, including the groups cMLV(PTX),
CAR.NK + cMLV(PTX), and CAR.NK.cMLV(PTX). The conjugated group, CAR.NK.cMLV(PTX), had
significantly higher PTX concentrations within the tumor tissue compared to the cMLV(PTX) or CAR.NK +
cMLV(PTX) (p < 0.01 and p < 0.001, respectively, Figure 21A).
We also used confocal imaging to visualize apoptotic cells in tumor tissues fixed on glass slides. There were
more terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)
+
cells in groups treated with
CAR.NK cells than in control or NK cell groups, indicating greater cell killing from CAR.NK cells, as shown in
Figure 6B. A higher level of cell apoptosis was observed in the group treated with CAR.NK + cMLV(PTX)
when compared to cMLV(PTX) treatment only, but the degree of cell apoptosis was similar when compared to
CAR.NK treatment. Notably, CAR.NK.cMLV(PTX)-treated tumors had the greatest degree of cell apoptosis,
indicating synergistic efficacy induced by the co-localized delivery of the drug and CAR.NK cells.
Finally, as the therapeutic effect of PTX is limited by its cardiotoxicity, slices of fixed heart tissue stained with
hematoxylin and eosin were imaged with light microscopy. Cardiotoxicity was defined as myofibrillary loss
and disarray, as well as cytoplasmic vacuolization
193
. We observed no damage to the cardiac tissues in any of
the treatment groups (Figure 21C). Since our delivery was targeted, we were able to use a very low dose of
PTX (0.5 mg/kg) compared to those used in conventional PTX-based treatments
193-195
, thus resulting in minimal
cardiotoxicity.

57

DISCUSSION

Our system combines nanoparticle-based drug delivery with immunotherapy to produce a cell-mediated, active
targeting strategy. In vitro, we demonstrate that cMLV conjugation to NK cells does not trigger endocytosis,
even though NK cells have phagocytotic capabilities.
196
The particles remain on the NK cell surface, perhaps
in part due to the size of the cMLVs—previous studies have shown that surface-conjugated particles larger than
50 nm in diameter are not efficiently internalized.
197
Furthermore, the covalent linkage of maleimide-
functionalized cMLVs to free thiols on immune cell surfaces has been shown to be stable for days after initial
conjugation and even after cell division.
171
While the exact mechanisms of this prolonged surface retention
remain to be discovered, the maleimide-thiol conjugation strategy has been shown to be a promising method of
immune cell surface engineering.
We also have demonstrated in vitro that CAR.NK cells can specifically kill antigen-expressing cancer cells, that
cMLV conjugation does not adversely affect NK cell function, and that conjugation of cMLV(PTX) to CAR.NK
cells further augments cytotoxicity. While many studies of CAR.NK cells include results from cytotoxicity
assays but not from cytokine release assays,
157, 158, 189, 198-200
we show that CAR.NK cells release IFN-γ in response
to TAA
+
target cells. Neither CAR.NK cells coincubated with TAA
-
target cells nor nontransduced NK cells
coincubated with any target cells release IFN-γ. These results indicate that the enhanced cytotoxicity of
CAR.NK cells was accompanied by an increase in IFN-γ release. In addition to sensitizing tumor cells to NK
cytotoxicity,
201
IFN-γ release by both primary NK cells and NK cell lines
202
signals to surrounding immune cells,
including T cells, dendritic cells, monocytes, and macrophages, initiating broader adaptive and innate immune
responses.
203, 204

Our in vivo biodistribution study further supports that CAR.NK cells enhance nanoparticle accumulation within
the tumor site. Mice treated with cMLV(DiD) without a cell chaperone had significantly greater cMLV
accumulation in the liver, likely indicating hepatic clearance as commonly observed with larger liposomes.
191

However, the CAR.NK.cMLV(DiD)-treated mice had significantly greater cMLV accumulation at the tumor site.
58

Additionally, significantly higher signal was observed in organs to which NK cells naturally home, such as the
spleen and lymph nodes.
205
Our in vivo and ex vivo data provide evidence that CAR.NK cells facilitate the
delivery of the chemotherapeutic drug PTX to the tumor site, slowing tumor growth and increasing intratumoral
PTX concentrations more effectively than any other treatment group, including coadministered but not conjugated
CAR.NK and cMLV(PTX). Finally, we were able to use a low dose of PTX and did not observe any
cardiotoxicity.
We found that certain doses of PTX can kill tumor cells but not NK92 cells, creating a therapeutic window in
which we can use NK92 cells to deliver this chemotherapeutic drug to kill tumor cells but not the carrier cells.
However, we do not believe that this system is limited to PTX delivery. For example, murine T cells have been
shown to deliver the anticancer drug SN-38 to lymphoma sites in vivo using drug-loaded nanocapsules conjugated
to the cell surface. SN-38 effectively killed lymphoma cells but was not toxic to the T cell carriers.
141
Another
study demonstrated that primary human T cells can enhance antitumor immune responses using surface-
conjugated liposomes carrying the proinflammatory cytokines IL-15 and IL-21.
171
Surface engineering of
immune cells has allowed a number of drugs or adjuvants to be delivered to the tumor site. To our knowledge,
we present the first study of surface-engineered NK cells as well as the first study using CAR.NK cells for tumor-
targeted drug delivery. We believe that our CAR.NK-mediated drug delivery system can be expanded to
include not only the delivery of traditional chemotherapeutic agents, but other anticancer treatments such as
immunomodulators and small molecules that affect the tumor microenvironment.
Cancer immunotherapy has attracted much attention as an alternative or addition to chemotherapy, and currently
a few clinical trials are using CAR-engineered T (CAR-T) cells to target patients with relapsed solid cancers, such
as pancreatic, ovarian, prostate, and lung cancers.
152
However, CAR-T therapy relies on the ex vivo expansion
of the patient’s autologous T cells, which presents logistical issues and delays the start of the treatment while cells
are in preparation (typically 2-3 weeks for the expansion of CAR-engineered immune cells for clinical use).
These issues could be ameliorated in part by using an allogenic cell line instead of autologous cells; while there
59

are few functional cytotoxic T cell lines available, there are several functional, immortal NK cell lines. Of these
NK cell lines, NK92 is the most promising and the only NK cell line used in clinical trials.
206

There are a number of potential benefits to using CAR-engineered NK92 cells over CAR-T cells. CAR-
engineered NK92 cells may provide an alternative “off-the-shelf” vehicle for CAR-based therapy as well as
provide more targeted drug delivery to the tumor site through surface engineering. NK92 cells double every 2-
4 days, allowing for easy expansion, modification, and storage under good manufacturing practice (GMP)
conditions. NK92 cells are identical to the parental cell line, eliminating problems with donor variability.
There would be no lag time required for the ex vivo expansion and modification of autologous immune cells,
which is especially crucial in patients with aggressive cancers, where a treatment delay of days to weeks could
impact outcome.
187
NK92 cells are safe to use clinically if irradiated, which prevents proliferation. This
decreases the risk of off-target effects compared to CAR-T cells. Short-lived CAR-engineered NK92 cells can
be treated as a “living drug”, redosing as necessary. Finally, allogenic NK92 cell-based therapies are less
expensive than autologous CAR-T cell therapies
206
—one group estimated that each CAR-T protocol costs
upwards of $250,000 per patient, but NK92 cells used in the clinic cost around $20,000 per patient.
187

In conclusion, we have demonstrated that CAR.NK cells conjugated to PTX-loaded cMLVs offer targeted drug
delivery and improved antitumor efficacy. We believe that targeted drug delivery using surface-engineered
CAR.NK cells is widely applicable, as both the CAR target and the drug payload potentially can be altered to
treat a variety of cancer types. Overall, this study shows a promising combination of immunotherapy and drug
delivery for enhanced antitumor treatment.

METHODS

Cell lines and reagents
MDA.MB.468 (ATCC HTB-132) and SKOV3 (ATCC HTB-77) tumor cell lines were maintained in a 5% CO2
environment in RPMI 1640 (Gibco) media supplemented with 10% FBS, 1% pen-strep, and 2mM L-glutamine.
60

NK92 cells (Dr. Jihane Khalife, Children’s Hospital Los Angeles, ATCC CRL-2407) were maintained in MEM-
α (Gibco) supplemented with 10% FBS, 10% horse serum, 1% NEAA, 1% pen-strep, 1% sodium pyruvate, 0.1
mM 2-β mercaptoethanol, 0.2 mM myo-inositol, and 2.5 µM folic acid. CD19
+
SKOV3 (SKOV.CD19) cells
were generated by transducing SKOV3 cells with lentivirus containing CD19 cDNA and sorting CD19
+
cells with
fluorescence-activated cell sorting (FACS).
PTX was purchased from Sigma-Aldrich (St. Louis, MO). All lipids were purchased from NOF Corporation
(Japan): 1,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phospho-(10-rac-
glycerol) (DOPG), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)but-yramide
(maleimide-headgroup lipid, MPB-PE).

Synthesis of nanoparticles
Liposomes were prepared based on the conventional dehydration-rehydration method.
182, 207
cMLVs were
prepared from 1.5µmol of lipids DOPC: DOPG: MPE-PE = 40:10:50 mixed in chloroform and evaporated under
argon gas before drying under a vacuum overnight to form dried lipid films. The lipid film was rehydrated in
10mM Bis-Tris propane at pH 7.0. After the lipid was mixed through vigorous vortexing every 10 minutes for 1
hour, they underwent three cycles of 15-second sonication (Misonix Microson XL2000, Farmingdale, NY) and
rested on ice at 1-minute intervals after each cycle. A final concentration of 10mM MgCl2 was added to induce
divalent-triggered vesicle fusion. The crosslinking of multilamellar vesicles (cMLVs) was performed by addition
of Dithiothreitol (DTT, Sigma-Aldrich) at a final concentration of 1.5 mM for 1h at 37°C. The cMLVs were
collected by centrifugation at 14,000 g for 5 minutes and washed twice with PBS. The particles were suspended
in filtered water, vortexed and sonicated prior to analysis. Morphological analysis of the multilamellar structure
of vesicles was performed and confirmed by cryo-electron microscopy in previous studies.
182
As shown
61

previously,
175
the hydrodynamic size of cMLVs was measured by dynamic light scattering (Wyatt Technology,
Santa Barbara, CA).
In vitro drug encapsulation and release
As previously reported,
193
the amount of incorporated paclitaxel in the cMLV(PTX) was determined by C-18
reverse-phase high-performance liquid chromatography (RPHPLC) (Beckman Coulter, Brea, CA). The
cMLV(PTX) suspension was diluted by adding water and acetonitrile to a total volume of 0.5 mL. Extraction of
paclitaxel was accomplished by adding 5 mL of tert-butyl methyl ether and vortex-mixing the sample for 1 min.
The mixtures were centrifuged, and the organic layer was transferred into a glass tube and evaporated under argon.
Buffer A (95% water, 5% acetonitrile) was used to rehydrate the glass tube. To test PTX concentration, 1 mL of
the solution was injected into a C18 column, and the paclitaxel was detected at 227 nm (flow rate 1 mL/min). To
obtain the release kinetics of PTX from cMLVs before and after cell conjugation, cMLV(PTX) and
CAR.NK.cMLV(PTX) were incubated in 10% FBS-containing media at 37 °C and were spun down and
resuspended with fresh media daily. The PTX was quantified from the removed media by HPLC every day.
Nanoparticle conjugation with cells and in situ PEGylation
Chemical conjugation of cMLVs to the cells was performed based on a method provided in previous studies.
141,
171
We resuspended 10 x 10
6
cells/mL in serum free MEM-α (Gibco) medium. Equal volumes of nanoparticles
were resuspended in nuclease free water at different cMLV to NK cell conjugation ratios and incubated at 37°C.
The cells and nanoparticles were mixed every 10 minutes for 30 minutes. After a PBS wash to remove unbound
cMLVs from cells, cells were further incubated with 1 mg/ml thiol-terminated 2-kDa PEG at 37°C for 30 minutes
in media to quench residual maleimide groups on cell-bound particles. We performed two PBS washes to remove
unbound PEG. For quantification of cell bound particles, particles were fluorescently labeled with the lipid-
like fluorescent dye DiD (Invitrogen). Particle fluorescence was detected with flow cytometry and a fluorescent
microplate reader. cMLVs were labeled with the lipid-like dye DiD and CAR.NK cells were stained with
62

carboxyfluorescein diacetate succinimide ester (CFSE) (Invitrogen), which allowed the conjugation of cMLVs to
NK cells to be easily detected using confocal microscopy.
Lentiviral and retroviral production and transduction of NK92 cells
Our anti-Her2 CAR construct
178
was cloned into a lentiviral pCCW vector (a pCCL vector
179-181
with an additional
WRE posttranscriptional regulatory region). The CAR consisted of the anti-Her2 scFv 4D5, a CD8 hinge and
transmembrane region, and CD28, 4-1BB, and CD3ζ cytoplasmic regions. Our anti-CD19 CAR construct was
cloned into a MP-71 retroviral vector backbone
177
and contained an anti-CD19 scFv, a CD8 hinge and
transmembrane region, and CD28 and CD3ζ cytoplasmic regions. These plasmids were used to transfect HEK
293T cells in 30mL plates using CaCl2 precipitation methods. Fresh media (high glucose DMEM supplemented
with 10% FBS and 1% pen-strep) was plated onto the cells 4 hours after initial transfection. Supernatants were
harvested and filtered (0.45µm) 48 hours later. NK92 cells were transduced with fresh retrovirus. Lentiviral
supernatant was concentrated (25,000 rpm for 90 minutes at 4°C), resuspended in HBSS, and frozen at -80°C
until later use. NK92 cells were transduced with concentrated lentivirus at MOI 40; the titer was based on
transduction of 293T cells.

CAR detection on NK cell surface
Three days after transduction, anti-CD19 CAR.NK cells (1×10
5
) were incubated with biotinylated Protein L
(Peprotech) at a volume ratio of 1:50 in PBS + 4% FBS at 4°C for 45 minutes and rinsed with PBS. The cells
were subsequently incubated with streptavidin conjugated to FITC (Biolegend) at a volume ratio of 1:500 in PBS
+ 4% FBS at 4°C for 10 minutes, rinsed twice, and read using flow cytometry. Anti-Her2 CAR.NK cells (1×10
5
)
were incubated with rhHer2-Fc chimera (Peprotech) at a volume ratio of 1:50 (2 µg/mL) in PBS at 4°C for 30
minutes and rinsed with PBS. The cells were subsequently incubated with PE-labeled goat anti-human Fc
(Jackson ImmunoResearch) at a volume ratio of 1:150 in PBS at 4°C for 10 minutes, rinsed, and read using flow
cytometry. Nontransduced NK cells served as a negative control.
63

Internalization assay
Quantification of cell cMLVs internalization was performed based on a method previously described.
141, 171
NK
and CAR.NK cells were conjugated with 5 mole % 18:1 PE CF (1,2-dioleoyl-sn-glycero-3-
phosphoethanolamine-N-(carboxyfluorescein) (ammonium salt) (Avanti, Polar Lipids) -tagged liposomes. After
2 PBS washes, cells were transferred to fibronectin (10ug/ml)-coated 96 well plates. After a 2 hour incubation
time, half of the wells were treated with 100 µl trypan blue in HBSS (0.25 mg/mL), an extracellular fluorescence
quenching dye, for 1 min in order to differentiate between membrane-bound and internalized liposomes. Trypan
blue was removed by gentle vacuum aspiration and the cell uptake of liposomes was quantified by a fluorescence
plate reader.
Cytokine release assay
NK cells (1×10
5
per well) were coincubated with target cells in 96-well plates at a 1:1 ratio for 6 hours at 37°C.
1 µg Brefeldin-A (Sigma) was added to each well to prevent protein transport. At the end of the incubation,
cells were permeabilized using the CytoFix/CytoPerm kit (BD Biosciences) and stained for CD8 and IFN-γ using
Pacific Blue-conjugated anti-human CD8 (Biolegend) and PE-conjugated anti-human IFN-γ (Biolegend).
Unstimulated cells served as a negative control. Results were read using flow cytometry.

Cytotoxicity assay
Target cells (1×10
4
) were labeled with 5 µM carboxyfluorescein succinimidyl ester (CFSE, Life Technologies)
as previously described
208
and coincubated with NK cells at various ratios in 96-well plates for 24 hours at 37°C.
The cells were then incubated in 7-AAD (Life Technologies) in PBS (1:1000 dilution) for 10 minutes at room
temperature and analyzed via flow cytometry. Percentages of killed cells were calculated as [CFSE
+
7-AAD
+
cells / (CFSE
+
7-AAD
-
+ CFSE
+
7-AAD
+
)] cells, with live/dead gates based on control wells of target cells to
account for spontaneous cell death.
64

NK92 and SKOV3 cells were seeded in 96-well plates at 2 × 10
4
cells per well in 10% FBS-containing media and
grown at 37 °C in the presence of 5% CO2 for 6 hours. Cells were incubated with various concentrations of cMLV
(PTX) as previously described
193
and cell viability was assessed using the Cell Proliferation Kit II (XTT assay)
from Roche Applied Science (Indianapolis, IN) according to the manufacturer’s instructions. Cell viability
percentage was determined by subtracting absorbance values obtained from media-only wells from the treated
wells and then normalized by the control wells containing cells without drugs.
Transmigration assay
NK cell transmigration assays were performed in 24 mm diameter 3 µm pore size Transwell plates (Costar). NK
cells either conjugated or unconjugated to cMLVs were plated on the upper wells and media was added to the
lower wells. The chemoattractant CXCL9 (0.1mg/ml, Peprotech) was added to the lower wells. After incubation
at 37°C for 6 hours, NK cells that had migrated into the lower chamber were counted.
In vivo biodistribution study
Female 6–10 weeks-old NOD.Cg-Prkdc
scid
IL2Rγ
tm1Wj1
/SZ (NSG) mice were purchased from Jackson
Laboratories (Bar Harbor, ME). All mice were held under specific pathogen-reduced conditions in the animal
facility of the University of Southern California (Los Angeles, CA, USA). All experiments were performed in
accordance with the guidelines set by the National Institute of Health and the University of Southern California
on the Care and Use of Animals. A total of 3.5 x10
6
SKOV3.CD19

cells were inoculated subcutaneously into
the flanks of NOD/scid/IL2rγ-/- (NSG) mice on Day -14, and tumors were allowed to grow until they reached
100mm
3
. On Day 0, mice were injected intravenously through the tail vein with either cMLV(DiD) or
CAR.NK.cMLV(DiD). 24, 48, and 72 hours after injection, mice were sacrificed and organs were analyzed for
fluorescence intensity. DiD tissue fluorescence for each organ was quantified using the IVIS Spectrum imaging
system and the percentage of injected dose per gram of tissue (%ID/g) was calculated.
Xenograft tumor model
65

A total of 3.5 x10
6
SKOV3.CD19

cells were inoculated subcutaneously into the flanks of NSG mice on Day -14,
and tumors were allowed to grow to 70-100mm
3
. Mice were randomly divided into six groups of five mice
each. On Days 0, 4, 7, and 11, the mice were injected intravenously through the tail vein with either PBS,
cMLV(PTX) only, nontransduced NK cells only, CAR.NK cells only, mixed CAR.NK + cMLV(PTX) which
were not conjugated together, or conjugated CAR.NK.cMLV(PTX). 5×10
6
cells per mouse were injected each
time in the groups that were given NK cells. Tumor growth and body weight of the mice were recorded until
sacrifice. The tumor length and width were measured with a fine caliper, and tumor volume was calculated as
½ × (length) × (width)
2
.
Intratumoral PTX concentration measurements ex vivo
Using high performance liquid chromatography (HPLC), the PTX concentration in the frozen tumor tissues was
quantified as previously detailed.
175
Briefly, thawed tumor tissues were chopped and homogenized in ethyl
acetate, with a known concentration of docetaxel added to each sample as an internal standard. The samples
were centrifuged and the organic layer was transferred to a clean tube. The organic layer was evaporated under
a stream of argon and rehydrated in diluted acetonitrile. After running the samples on HPLC, the peak heights
were analyzed to determine intratumoral PTX concentration.
Immunohistochemistry of Tumors, Cardiac Toxicity, and Confocal Imaging
Tumors were excised, fixed, frozen, cryo-sectioned, and mounted onto glass slides. Frozen sections were fixed
and rinsed with cold PBS. After blocking and permeabilization, the slides were washed with PBS and incubated
with a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) reaction mixture (Roche,
Indianapolis, Indiana) for 1 hour and counterstained with 4',6-diamidino-2-phenylindole (DAPI) (Invitrogen,
Carlsbad, CA). Fluorescence images were acquired by a Yokogawa spinning-disk confocal scanner system
(Solamere Technology Group, Salt Lake City, UT) using a Nikon Eclipse Ti-E microscope. Illumination powers
at 405, 491, 561, and 640 nm solid-state laser lines were provided by an AOTF (acousto-optical tunable filter)-
controlled laser-merge system with 50 mW for each laser. All images were analyzed using Nikon NIS-Elements
66

software. For quantifying TUNEL positive cells, four regions of interest (ROI) were randomly chosen per image
at 10x magnification. Within one region, the area of TUNEL-positive nuclei and the area of nuclear staining were
counted by Nikon NIS-Element software, with data expressed as % total nuclear area stained by TUNEL in the
region. For cardiac toxicity, heart tissues were harvested 2 days after the last injection and were fixed in 4%
formaldehyde. The tissues were frozen and then cut into sections and mounted onto glass slides. The frozen
sections were stained with hematoxylin and eosin. Histopathologic specimens were examined by EVOS light
microscopy.
Statistics
The differences between two groups were determined with Student’s t test. The differences among three or more
groups were determined with a one-way analysis of variance (ANOVA).

67

CHAPTER 4: CAR-T CELLS SURFACE-ENGINEERED
WITH DRUG-ENCAPSULATED NANOPARTICLES
CAN AMELIORATE INTRATUMORAL T CELL
HYPOFUNCTION

ABSTRACT

The chimeric antigen receptor T (CAR-T) cell therapy has become a promising cancer immunotherapeutic
method, particularly in treating B cell malignancies; however, this therapy is still in the early stages of
development for the treatment of solid tumors. One limiting factor of CAR-T cell therapy is the suppressive tumor
microenvironment, which inactivates the function of tumor infiltrating lymphocyte (TIL) through the production
of immune-modulating molecules such as adenosine. Adenosine inhibits the function of CD4 and CD8 T cells by
binding to and activating the A2a adenosine receptor (A2aR) expressed on their surface. This immune suppression
pathway can be blocked using the A2aR-specific small molecule antagonist SCH-58261 (SCH), but its
applications have been limited owing to difficulties delivering this drug to immune cells within the tumor
microenvironment (TME). To overcome this limitation, we have used CAR-engineered T cells as active
chaperones to deliver SCH-loaded cross-linked multilamellar liposomal vesicles (cMLVs) to tumor-infiltrating T
cells deep within the immune suppressive TME. Through in vitro and in vivo studies, we have demonstrated that
this system can be used to target immune-modulating drug delivery to the TME. This treatment can prevent or
rescue the emergence of hypofunctional CAR-T cells within the TME.

INTRODUCTION

Chimeric antigen receptor (CAR)-engineered T (CAR-T) cell therapy has demonstrated success in treating
hematological cancers, such as leukemia and B cell lymphoma, in preclinical and clinical trials
209-211
. This success
68

has not been translated to the treatment of solid tumors
212-215
. Unlike hematological cancers, which circulate
throughout the body in the blood stream, solid tumors have their own complex tumor microenvironment (TME),
which provides a unique barrier to immunotherapy
65, 213, 216
. To be effective, immune cells must efficiently
infiltrate the solid tumor mass and have extended persistence of in vivo expanded cells
217-219
. The TME contains
a variety of pro-tumorigenic factors that work to both prevent cancer-killing immune cells from entering the tumor
area and dampen the activation of tumor infiltrating lymphocytes (TILs)
216, 220, 221
. Many of these immune
suppressive mechanisms can also effect adoptively transferred CAR-engineered T cells.
One of the underlying mechanisms responsible for the progressive loss of TIL function in the TME is an inhibitory
pathway involving the A2a adenosine receptor (A2aR) expressed on the surface of activated T cells
222-224
. The
A2aR pathway is triggered by abnormally high concentrations of the extracellular immunosuppressive molecule
adenosine, which has been reported to suppress T cell proliferation and IFN-γ secretion
225-227
. In the TME,
extracellular adenosine triphosphate (ATP) is released in response to tissue damage and cellular stress. ATP in
the extracellular environment is converted into adenosine by ecto-nucleases CD39 and CD73, which are
upregulated in the hypoxic TME
67
. Overexpression of CD73 has been observed in multiple aggressive cancers,
conferring resistance to antitumor agents
228
. Binding of adenosine to A2aR leads to increased intracellular cyclic
AMP (cAMP) production in the TILs. Elevation of intracellular cAMP induces activation of protein kinase A
(PKA) and phosphorylation of the cAMP response element binding protein (CREB), which, in turn, abrogates T
cell receptor (TCR) signaling and IFN-γ production by reducing the activity of the Akt pathway and inhibiting
NF-kB driven gene expression
223, 228
.
Studies have demonstrated that blocking A2aR signaling through either pharmacological inhibition or genetic
deletion can significantly improve antitumor immunity by enhancing the cytotoxic function of T and natural killer
(NK) cells
222, 224, 225, 229
. SCH-58261 (SCH) is one of the most selective and potent antagonists of A2aR
230
. Despite
its therapeutic potential, the clinical application of SCH has been hindered by the drug’s poor solubility and
suboptimal in vivo pharmacokinetic profile. Small-molecule drugs like SCH, which act directly on CAR-T cells,
69

need to be maintained at high concentrations near their site of action in order to be effective. Thus, a carrier
capable of regulating drug circulation time in vivo and efficiently delivering the drug to CAR-T cells in tumors,
while minimizing delivery to other tissue/organ sites, is desired.
Recently, advances in drug delivery nanotechnology have enhanced the therapeutic efficacy of several anticancer
drugs. Compared to free drugs, drug-loaded nanoparticles can improve targeted delivery by prolonging blood
circulation time, controlling and sustaining drug release profiles, altering tissue distribution to reduce systemic
toxicities, and increasing drug concentration in the tumor through the enhanced permeability and retention (EPR)
effect
231
. The EPR effect is highly dependent on adequate vascularization of tumors. Vascularization, however,
may be completely lacking in some tumors that exhibit poor blood supply and hypoxia
232
. Additionally, high
interstitial fluid pressures within the tumor can act to transport therapeutics back into the bloodstream. Most
administered nanoparticles, approximately 95%, are reported to accumulate in organs other than tumor, including,
for example, liver, spleen and lungs
231
. Hence, efficient delivery and distribution of nanoparticles within the tumor
mass remains challenging.
Numerous studies have shown that the addition of targeting moieties on nanoparticles can significantly improve
their tumor specificity and accumulation, but these targeting strategies still rely on passive distribution of the
nanoparticles through the bloodstream
233
. Recently, more advanced strategies have been explored, such as the use
of immune cells as nanoparticle “chaperones” for targeted delivery and active transport into tumors
74, 78, 234
.
Studies have shown that these immune delivery cells can provide effective treatment of tumors by facilitating
drug delivery through active cellular migration and extravasation in response to chemoattractant gradients around
the tumor and inflammatory sites
74, 78, 235
. We believe that this delivery approach involving direct NPs/T cells
conjugation could be used to alter interactions between the TME and endogenous or infused immune cells in vivo.
Therefore, we herein report an approach that enhances the efficacy of CAR-T therapy by chemical conjugation
of adjuvant drug-loaded maleimide-functionalized cross-linked multilamellar liposomes (cMLVs) to the surface
of CAR-T cells ex vivo prior to systemic administration (Figure 22A). We demonstrated that cMLV nanoparticles
70

could be covalently attached to CAR-T cells without affecting the cells’ viability and effector functions. We
further revealed that the therapeutic potential of CAR-T cells could be improved upon surface engineering with
SCH-encapsulated cMLVs.

RESULTS

Nanoparticles stably attached to the surface of CAR-T cells

To improve the efficacy of CAR-engineered T cell therapy, we have used CAR-T cells as chaperones to carry
nanoparticles loaded with SCH, a drug that can inhibit an immune-suppressive mechanism in the TME. To express
CARs on T cells, activated human PBMCs were transduced with a lentiviral vector to deliver anti-CD19 CAR
consisting of CD28 and CD3ζ intracellular signaling domains. FACS analysis of surface CAR expression showed
50% transduction efficiency.
Next, SCH-loaded crosslinked multilamellar liposomal vesicles (cMLVs) were conjugated to the surface of CAR-
T cells. According to previous reports, high levels of free thiols have been detected on the surfaces of T cells, B
cells, and hematopoietic stem cells
78
; therefore, we used thiol-reactive maleimide headgroups present on the lipid
bilayer surface of the cMLVs to stably couple the nanoparticles to the cell surface. The conjugation was performed
in two steps. First, CAR-T cells and cMLVs containing maleimide-functionalized lipids were coincubated to
permit coupling of the liposomes to free thiols on the cell surface. After the initial coupling reaction, the
conjugated cells underwent in situ PEGylation to quench residual reactive groups on the cMLVs. To determine
the maximum number of particles that could be conjugated per T cell, we performed nanoparticle conjugation
reaction at different cMLV-to-T cell ratios (5000:1, 1000:1, 500:1, 250:1 and 100:1). At a ratio of 1000:1, the
conjugation of cMLVs reached a saturation point that resulted in an average of 287±49 surface-bound
nanoparticles per cell (Figure 22B).
71

The average conjugation efficiency of the nanoparticles on the T cell population was 55.9% (Figure 22C and D).
Moreover, single-cell imaging and three-dimensional reconstruction of CART.cMLVs demonstrated that the
nanoparticles were distributed in several clusters on the cell surface (Figure 22E)

Figure 22. Stable conjugation of cross-linked multilamellar liposome (cMLVs). A, Schematic diagram
of cMLV-conjugated CAR-T cells for targeted delivery of small-molecule inhibitors into tumor
microenvironment (TME). Maleimide-functionalized cMLVs loaded with A2AR small-molecule inhibitors
are conjugated to CAR-T cells via cell-surface thiols. B, Quantification graph of conjugated cMLVs per T
cell at different conjugation ratios. cMLVs labeled with the fluorescent dye DiD were coincubated with T
cells over a range of ratios. DiD fluorescence was analyzed to calculate the number of cMLVs on the surface
of each cell. C, Percentage of CD3
+
T cells conjugated with cMLVs at 1000:1 (cMLV:T cell) conjugation
ratio. D, Representative flow cytometry analysis of the percentage cMLV- conjugated CD3
+
T cells. E,
Single-cell confocal microscopy image of DiD-loaded cMLV-conjugated CAR-T cells. These CAR-T cells
were labeled with 1 µM CFSE and washed with PBS prior to conjugation to cMLV(DiD). Confocal
microscopy was used to visualize the cMLVs on the CAR-T cell surface. Scale bar represents 10 µm. (Red:
DiD-labeled cMLVs, Green: CFSE-labeled CAR T cells) (n = 6, mean ± SD; ns, not significant; *p < 0.05;
**p < 0.01; ***p < 0.001). Data represents the mean ± SD of two independent experiments conducted in
triplicate.

CAR-T cells conjugated with nanoparticles maintain T effector functions

We next sought to test whether surface-bound cMLVs could impact key cellular functions of CAR-T cells, such
as cell cytokine secretion, cytotoxicity, and migration. CAR-T cells with and without cMLV conjugation were
co-cultured with either SKOV3.CD19 or K562.CD19 target cells for 6 hours. CART and CART.cMLV stimulated
72

with SKOV3.CD19 target cells induced 17.05±0.07% and 19.15±1.63% IFN-γ
+
T cell populations, respectively,
indicating that both CART and CART.cMLV were able to secrete IFN-γ with similar efficiency (Figure 23A and
B).

Figure 23. Conjugation of cMLV does not alter CAR-T cell function. A, Representative FACS analysis
of CAR-T cells, either unconjugated (CART) or conjugated with empty cMLVs (CART.cMLV), stimulated
with SKOV3.CD19 cells for 6 hours to detect IFN-γ release. Untransduced CAR- T cells served as a
negative control. IFN-γ was measured by intracellular staining. B, The summarized statistics of triplicates
were shown in bar graphs. C, CAR-T cells, either unconjugated (CART) or conjugated to empty cMLVs
(CART.cMLV), were cocultured with SKOV3.CD19 cells for 6 hours, and cytotoxicity was measured.
Untransduced CAR-T cells served as a negative control. (n = 3, mean ± SD; ns, not significant; *p < 0.05;
**p < 0.01; ***p < 0.001). D, Either unconjugated (CART) or cMLVs-conjugated CAR-T cells
(CART.cMLV), were seeded in the upper chambers of a Transwell with or without addition of
chemoattractant CXCL9 to the lower chambers. After 6 hours of incubation, media from the lower
chambers was collected, and CAR-T cells were counted. Summarized statistics are displayed in the graphs
(n = 3, mean ± SD; ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001). All data are representative of
at least three independent experiments.

When cMLVs were labeled with DiD dye, IFN-γ was secreted from both cells with and without surface-
conjugated cMLVs . Moreover, surface conjugation of cMLVs did not reduce CAR-T cell cytotoxicity against
SKOV3.CD19 or K562.CD19 cells (Figure 23C and Figure 24). Lastly, we assessed CAR-T cell transmigration
capabilities in vitro. Comparable percentages of conjugated and unconjugated cells migrated to the lower chamber
of the transwell co-culture system, indicating that CART.cMLV cells maintain their transmigration capabilities
73

(Figure 23D). Thus, the cell surface conjugation of cMLVs does not hinder recognition of target cells, IFN-γ

secretion, cell cytotoxicity, or migration.

Figure 24. In vitro cell cytotoxicity assay of CART and CART.cMLV against K562.CD19 shows no
reduction of T cell cytotoxicity. Anti-CD19 CART cells were co-cultured with K562.CD19 cells for 6
hours and cytotoxicity was measured. The negative control is untransduced T cells. (n = 3, mean ± SD; ns,
not significant; *p < 0.05; **p < 0.01;***p < 0.001)

Conjugation to CAR-T cells increases tumor localization and systemic circulation of cMLVs

To determine whether conjugation of cMLVs to CAR-T cells could improve the accumulation of nanoparticles
to the tumor site, we performed an in vivo biodistribution study. DiD-labeled cMLVs alone (cMLV(DiD)), mixed
with CAR-T cells (CART+cMLV(DiD)), or conjugated to CAR-T cells (CART.cMLV(DiD)) were intravenously
injected into NSG mice bearing SKOV.CD19 tumors, and DiD-tagged cMLV accumulation was monitored in
different organs. At 24 hours, significantly higher cMLV accumulation was detected from CART.cMLV(DiD) in
tumor (p<0.001), spleen (p<0.001), lymph node (p<0.01), and lung (p<0.001). No significant difference in cMLV
accumulation was noted between cMLV(DiD) and CART+cMLV(DiD) groups in any tissues. Additionally, no
significant difference in DiD signal was detected at 24 h in circulating blood in any group (Figure 25A and B).
By 48 hours, the CART.cMLV(DiD) group showed increased cMLV accumulation in the tumor, spleen, lymph
node, and lung when compared to that at 24 hours. Also, at 48 hours, CART.cMLV(DiD) demonstrated higher
74

cMLV accumulation in the blood (p<0.05), tumor (p<0.001), spleen (p<0.01), lymph node (p<0.01) and lung
(p<0.01) compared to both cMLV(DiD) and CART+cMLV(DiD) groups. Notably, CAR-T cell conjugation to
cMLVs resulted in significantly lower cMLV accumulation in the liver compared to both cMLV(DiD) and
CART+cMLV(DiD) groups at 24 (p<0.05) and 48 (p<0.001) hours.

Figure 25. cMLVs bound to CAR-T cells show more efficient infiltration to antigen-expressing tumors
than free cMLVs. Group of 3 NSG mice bearing subcutaneous SKOV3.CD19 tumors were intravenously
injected with 1 × 10
7
CAR-T cells conjugated with DiD-labeled cMLV (CART.cMLV), mixed with DiD-
labeled cMLVs (CART+cMLV), or simply injected with an equivalent number of DiD-labeled cMLVs
(cMLV). After 24 hours A, and 48 hours C, indicated tissues were removed, weighed, and macerated with
scissors. Specific DiD tissue fluorescence for each organ was quantified using the IVIS spectrum imaging
system, and the mean percentage of injected dose per gram of tissue (% ID/g) was calculated as final
readout. B, D Representative images of DiD fluorescence from the tumor tissues. Summarized statistics are
displayed in the graphs. All data are representative of two independent experiments (n = 3, mean ± SD; ns,
not significant; *p < 0.05; **p < 0.01; ***p < 0.001).

Surface-conjugated cMLVs colocalize with CAR-T cells inside the tumor mass

We next evaluated the tumor infiltration properties of carrier CAR-T cells by confocal imaging of histological
SKOV3.CD19 tumor sections that had been treated with cMLV-conjugated, or unconjugated, fluorescently
75

labeled CAR-T cells. Representative confocal images demonstrate that the surface-conjugation of cMLVs does
not impede intratumoral CAR-T cell migration (Figure 26A and B). Both CART.cMLV and CART+cMLV
had comparable infiltration of T cells (Figure 26C). The probability of detecting CAR-T cells in the
CART.cMLV- and CART+cMLV-treated tumor was 25.49±23.4% and 16.10±27.5%, respectively. However, the
colocalization of CAR-T cells and cMLVs was only observed inside tumors treated with CART.cMLV. The
probability of detecting colocalization was 78.5±26.7% in the CART.cMLV-treated tumors compared to no
detection in CART+cMLV-treated tumors, further demonstrating the tumor-targeting property of CAR-T cells
(Figure 26D). These results indicate that cMLV conjugation to the CAR-T cells is able to increase the amount of
cMLVs delivered to the tumor without impeding the migration of T cells.

Figure 26. Surface-conjugated cMLVs colocalize with CAR-T cells inside the tumor mass 48 hours post-
CAR-T cell infusion. A, Representative confocal images of CAR-T cells conjugated with DiD-labeled
cMLVs (red) infiltrating SKOV3.CD19 tumor 48 hours post- injection (CART.cMLVs). B, Representative
confocal image of coadministered DiD-labeled cMLVs (red) and CAR-T cells (green, without conjugation)
infiltrating SKOV3.CD19 tumor 48 hours post-injection (CART+cMLVs). Scale bar, 30 µm. C,
Probabilities of detecting SKOV3.CD19 tumor-infiltrated CAR-T cells from either CART.cMLV or
CART+cMLVs group. D, Probabilities of detecting SKOV3.CD19 tumor-infiltrated CAR-T cells that are
co-localized with cMLVs inside the tumor tissues (n = 6, mean ± SD; NS, not significant; *p < 0.05; **p <
0.01; ***p < 0.001).
76

CAR-T cells conjugated with nanoparticles encapsulated with A2aR antagonist shows improved
antitumor responses in vivo

Figure 27. Anti-CD19 CAR-T cells conjugated with SCH-releasing cMLVs prevented the development of
hypofunction in SKOV3.CD19 tumors. SKOV3.CD19 cells were injected subcutaneously into the right
flank of NSG mice. Mice were randomized into four groups and treated with indicated groups via i.v.
injections. A, Tumor size was measured with a fine caliper. After indicated treatments, flank tumors were
harvested and digested for ex vivo analyses. The quantity and function of tumor-infiltrated CAR-T cells
was evaluated. The percentage of CD45
+
CD3
+
T cells in the tumor at B 2 days and C 14 days. The
percentage of CD45
+
CD3
+
T cells in the spleen at D 2 days and E 14 days. F, Two days post-treatments,
tumor-infiltrated T cells were harvested and stimulated with anti-CD3/CD28, and their IFN-γ secretion
was measured. G, Detection of phosphorylated CREB expression levels in tumor-infiltrated T cells 2 days
post-treatments. (n = 13, mean ± SD; n/s, not significant; *p < 0.05; **p < 0.01; ***p < 0.001).
77

To test whether the pharmacological inhibition of A2aR could prevent the inhibition of adoptively transferred
CAR-T cells by tumor-produced adenosine, thereby improving antitumor reactivity, we monitored tumor growth
and intratumoral CAR-T cell infiltration in vivo. SKOV.CD19 tumor-bearing mice were assigned into four
different groups: (1) PBS (2) CAR-T cells (CART), (3) CAR-T cells conjugated with empty cMLVs
(CART.cMLV), and (4) CAR-T cells conjugated with SCH drug-loaded cMLVs (CART.cMLV(SCH)). The
CART and CART.cMLV groups controlled tumor growth only within the first seven days post-treatment.
Notably, the CART.cMLV(SCH) group showed prolonged tumor growth control, until day 14 post-treatment
(p<0.01). By day 14, the tumor size of CART.cMLV(SCH) was 265±21 mm
3
, while the average size of the PBS,
CART, and CART.cMLV groups were 859 (±122), 759 (±131) and 649 (±24) mm
3
, respectively (Figure 27A).
To explore how SCH affected T cell engraftment, T cells in the tumor and spleen were evaluated on day 2 and 14
post-treatments. On day two, T cells were detected in both the tumor and spleen in all treatment groups.
CART.cMLV(SCH) had higher T cell engraftment in the tumor (5.37±0.5%) than either the CART (2.98±0.5%,
p<0.01) or CART.cMLV (3.26±0.5%, p<0.01) groups (Figure 27B). T cell engraftment in the spleen, on the other
hand, did not show any significant difference among the groups on day 2 (Figure 27D). On day 14, T cells were
still present in both the tumor and spleen in all treatment groups. CART.cMLV(SCH) had a significantly higher
T cell infiltration (8.16±0.6%) than either the CART (5.0±0.5%, p<0.001) or CART.cMLV (5.26±0.4%, p<0.001)
groups (Figure 27C). By day 14, the spleen of mice treated with CART.cMLV(SCH) had significantly higher T

cell infiltration (10.35±1.6%) compared to that of either the CART (5.29±0.8%, p<0.01) or CART.cMLV
(5.27±0.3%, p<0.01) groups (Figure 27E).
Furthermore, we evaluated the functionality of tumor-infiltrating T cells that were exposed to the adenosine-rich
immunosuppressive TME in vivo. On day 2 post-treatments, T cells from various treated xenograft tumors were
isolated for ex vivo analysis. The CART.cMLV(SCH) treatment group showed significantly higher intracellular
78

IFN-γ secretion (MFI = 3733±781) compared to the groups treated with CART (MFI = 612±20, p<0.05) and
CART.cMLV (MFI = 788±138, p<0.05) (Figure 27F).
To determine if the functional preservation of tumor-infiltrating T cells is, at least in part, the result of A2a
receptor blockade, we tested the level of phosphorylated-CREB downstream of A2aR on isolated T cells. Our
data showed that T cells from the CART- and CART.cMLV-treated groups had significantly higher
phosphorylated CREB compared to T cells harvested from the CART.cMLV(SCH)-treated group, indicating that
SCH released from surface-engineered CAR-T cells could block A2a receptor signaling mediated by adenosine
in TME (Figure 27G).

CAR-T cells conjugated with nanoparticles encapsulated with A2aR antagonist is able to rescue
hypofunctional tumor-residing T cells in vivo

79

Figure 28. Anti-CD19 CAR-T cells conjugated with SCH-releasing cMLVs were able to rescue
hypofunctional tumor-infiltrated T cells in SKOV3.CD19 tumors. A, Schematic illustration of targeted in
vivo delivery of CAR-T cells conjugated with cMLVs releasing SCH to inhibit SKOV3.CD19 tumors by
rescuing tumor-infiltrated CART.tEGFR cells. B, Waterfall plot displaying the percent change in
the tumor size from baseline at Day 35 after i.v. injections. C, The percentage of CD45
+
T cells in the tumor
2 days after indicated treatments. D, Representative FACS plots of the percentage of CART.tEGFR cells
in the tumor 2 days after indicated treatment. E, Quantitative graph showing the percentage of
CART.tEGFR cells in the tumor 2 days after indicated treatments. F, Two days post-treatments, tumor-
infiltrated CART.tEGFR cells were harvested and stimulated with anti-CD3/CD28, and their IFN-γ
secretion was measured by intracellular staining. G, Detection of phosphorylated CREB expression levels
in CART.tEGFR cells 2 days after indicated treatments. (n = 10, mean ± SD; ns, not significant; *p < 0.05;
**p < 0.01; ***p < 0.001). All data are representative of at least two independent experiments.

Although tumor-infiltrated CAR-T cells can migrate into the tumor mass, they tend to gradually lose tumor killing
and inflammatory cytokine secretion abilities after entering the adenosine-rich tumor microenvironment
64, 67, 220
.
We hypothesized that hypofunctional tumor-residing T cells could regain their effector functions upon the
blocking of A2aR signaling with SCH. To demonstrate the potential of our conjugated system in this application,
we established an in vivo model with hypofunctional tumor-residing CAR-T cells in the TME by an initial
intravenous infusion of CD19 CAR-T cells that express a truncated epidermal growth factor receptor (tEGFR) to
the tumor bearing mice; these CAR-T cells are designated as CART.tEGFR. The tEGFR surface marker was used
to trace the initial population of hypofunctional tumor-residing CAR-T cells, enabling us to distinguish them from
the subsequent treatment dose of surface-engineered CAR-T cells lacking tEGFR. Ten days after the initial
CART.tEGFR cell transfer, the rescue treatment was infused to mice in five different groups (Figure 28A).
Two days after the treatments, 5 out of 6 tumor-bearing mice that received CART.cMLV(SCH) treatment showed
over 50% reduction in tumor size, with one mouse showing 44% reduction. The combination treatment group of
CART+cMLV(SCH) showed more than 25% reduction in tumor size in 2 out of 6 mice. Tumor-bearing mice that
received either CART or CART.cMLV had no significant reduction in tumor size, and the tumor-bearing mice
that received PBS treatment showed an overall increase in tumor size (Figure 28B). Tumor-infiltrating T cells,
including CAR-positive cells, were isolated from tumors for further ex vivo analysis. As shown in Figure 6C,
CART.cMLV(SCH) treatment resulted in 10.79±0.3% total T cell population, which is significantly higher than
80

all other treatment groups (CART, CART.cMLV, and CART+cMLV(SCH), p<0.001) (Figure 28C). We
further investigated the effect of these treatments on the functionality of the initial hypofunctional CART.tEGFR
cells. Tumors treated with CART, CART.cMLV, and CART+cMLV(SCH) had 25.65±2.8%, 25.35±0.5% and
28.90±3.1% intratumoral CART.tEGFR, respectively, while CART.cMLV(SCH)-treated tumors had 63.08±5.8%
CART.tEGFR cells, significantly higher than all other groups (p<0.001) (Figure 28D and E).
Next, we evaluated the ability of this CART-chaperoned drug to restore inflammatory function of the
hypofunctional CART.tEGFR population. The CART.cMLV(SCH) treatment group showed significantly higher
IFN-γ secretion in CART.tEGFR cells than that of CART, CART.cMLV, or CART+cMLV(SCH) groups
(p<0.001) (Figure 28F). Evaluation of pCREB expression levels in CART.tEGFR cells showed that the
CART.cMLV(SCH) treatment significantly reduced pCREB level in CART.tEGFR cell populations compared to
the CART, CART.cMLV, and CART+cMLV(SCH) treatment groups (p<0.001) (Figure 28G). Taken together,
this collective evidence suggests that the surface-engineered CART system can effectively deliver a small-
molecule inhibitor of A2aR to TME, thereby rescuing hypofunctional tumor-residing T cells in vivo.

DISCUSSION

Our strategy to enhance CAR-T cell efficacy in solid tumors was to conjugate nanoparticles loaded with a small-
molecule inhibitor of the A2a receptor pathway onto the surface of CAR-T cells. In vitro, we demonstrated that
cMLV nanoparticles could be stably conjugated to the CAR-T cell surface while maintaining its ability to release
loaded drug in a sustained manner. Moreover, the surface conjugation did not disrupt CAR-T cell effector
functions, such as cytokine secretion, cytotoxicity, and migration. These findings corroborate previous results
reported by Stephan et al. and Huang et al.
74, 78
, which demonstrated that liposomal nanoparticles with thiol-
reactive maleimide headgroups could be successfully conjugated to the thiol-rich surface of T cells and that this
could be done without altering effector functions and transmigration capabilities of the T cells.
81

Our biodistribution study further shows that CAR-T cells enhance the efficacy of therapeutic drugs by actively
directing drug-loaded nanoparticles to the tumor site in vivo
75, 77, 236
, an event driven by the ability of CAR-T cells
to migrate into the tumor mass through tumor-associated chemokine attraction. The fluorescent signal from
CART.cMLV(DiD) in the tumor was detected as early as 24 hours after infusion. Overall, CART.cMLVS(DiD)
had the highest particle accumulation at the tumor site at both 24 and 48 hours, reemphasizing the importance of
cell-mediated delivery. Moreover, both cMLV(DiD) and CART+cMLV(DiD) resulted in significantly higher
cMLV accumulation in the liver, which is where liposomal nanoparticles are typically cleared from the system
by Kupffer and endothelial cells
237, 238
. However, while CART.cMLV(DiD) showed significantly lower cMLV
accumulation in the liver, increased levels were observed in lymphoid tissues, such as the lymph node, spleen and
lungs. These data provide evidence that CAR-T cell-bound nanoparticles may be retained in circulation for a
longer period of time than free nanoparticles owing to reduced nanoparticle clearance by the liver.
In order to achieve maximal drug action on hypofuctional T cells within the TME, the drug-loaded nanoparticles
must be able to reach the immune cells deep within the tumor mass. In this regard, the CART.cMLV drug delivery
system promotes the colocalization of nanoparticles and CAR-T cells inside the tumor mass due to the innate
mobility of T cells within the tumor to deliver drugs inside the TME
235, 239
. Confocal microscopic images showed
that cMLVs from the CART.cMLV group were able to penetrate deep inside the tumor and colocalize with CAR-
T cells. These results agree with the findings from our in vivo biodistribution study, which shows that a higher
percentage of CART.cMLV nanoparticles accumulated in the tumor compared to unconjugated nanoparticles.
This maximum intratumoral colocalization of the CART.cMLV group could be a major factor contributing to the
higher potency of CART.cMLV(SCH) therapy.
The tumor-targeted CART.cMLV(SCH) therapeutic system was effective at preventing hypofunction of
nanoparticle-conjugated CAR-T cells. Groups treated with CART.cMLV(SCH) demonstrated significant tumor
growth suppression compared to groups without the conjugated drug. Prophylactic CART.cMLV(SCH) treatment
showed high tumor engraftment of T cells with low CREB phosphorylation, indicating the mechanistic
82

importance of SCH A2aR blockade that leads to increased T cell proliferation
225
. This is supported by our
observation that CART.cMLV(SCH) had a higher percentage of tumor-infiltrated T cells and increased IFN-γ
production compared to the other groups
222, 224
.

Figure 29 Tumor infiltrated T cells showed reduced IFN-γ secretion.
Ten days post CAR-T cell therapy, tumor infiltrated T cells were stimulated ex vivo with anti-hCD3 and
anti-hCD28. IFN-γ release was measured with intracellular staining. The positive control was spleenocytes
of non-tumor bearing mice that received CAR-T cell infusion (n = 3, mean ± SD; ns, not significant; *p <
0.05; **p < 0.01; ***p < 0.001).

We further aimed to restore activity to hypofunctional CAR-T cells (Figure 29). This rescue treatment mirrors a
clinical setting where patients have pre-existing TILs or have previously received CAR-T cell therapy.
CART.cMLV(SCH) treatment resulted in significantly higher IFN-g secretion in the initial hypofunctional
CART.tEGFR cell population upon ex vivo restimulation compared to other treatment groups. In the
CART.cMLV(SCH) treatment group, the CART.tEGFR population also showed lower phosphorylated CREB
and significantly increased tumor infiltration compared to other groups, very likely from terminating adenosine-
induced suppression of T cell proliferation
225
.
We also confirmed that SKOV3.CD19 cells were not directly affected by SCH , indicating that SKOV3.CD19
tumor reduction was solely achieved by the effect of SCH on the tumor-infiltrated CAR-T cells. Moreover, the
immediate reduction of tumor burden after CART.cMLV(SCH) treatment is most likely caused by the recovery
83

of cytotoxicity induced by the CART.tEGFR cell population. This result can be attributed to CART.cMLV(SCH)
treatment, which, at the same dosage, did not reduce, but only suppressed, tumor growth, as observed in our
prophylactic study, and according to previous studies, tumor size reduction occurs 5-6 days after T cell infusion
239
.
While CART.cMLV(SCH) has shown promising results, leading to the regression of solid tumor in our xenograft
model, further improvements and modifications could be made to expand this treatment to different clinical
settings. This delivery platform is highly flexible, and it can be applied to other drugs, cytokines, antibodies, or
any combination thereof. For example, a previous study by Stephan et al. successfully demonstrated that the use
of different therapeutic cells, such as tumor-specific T lymphocytes and hematopoietic stem cells (HSC) as
targeted delivery vehicles, markedly increased the therapeutic efficacy of cytokines and a small-molecule
inhibitor
78
. Moreover, immune regulatory drugs could be delivered in combination with immune checkpoint
blockade, such as anti-PD-1, to further promote antitumor immunity
224, 229
. In a previous review by Iwai et al.,
multiple clinical studies involving αPD1 and αPD-L1, when combined with small-molecule inhibitors of VEGF
and EGFR, for example, have shown potential for the treatment of ovarian and lung cancers
240
. Furthermore, to
overcome the limitations of CAR-T cell therapy, such as the lengthy manufacturing process and toxicities due to
cytokine release syndrome or on target/off tumor recognition
241
, CAR-T cells could also be exchanged for other
“chaperone” cells, such as natural killer (NK) cells, which can be used more universally.
Cell-mediated drug delivery by surface engineering of CAR-T cells with nanoparticles not only enables controlled
drug effect on the carrier cells, but also allows active targeting to the tissue of interest. By using CAR-T cells as
chaperones, we were able to efficiently localize nanoparticles in specific tissues that correspond to T cell
trafficking, including tumor, spleen, lungs and lymph nodes
242
. Overall, this is a promising platform that can
potentially improve the efficacy and specificity of solid tumor therapies.

84

METHODS

Mice
Female NOD.Cg-Prkdc
scid
IL2Rγ
tm1Wj1
/SZ (NSG) mice (6 to 10 weeks old) were purchased from the Jackson
Laboratory (Bar Harbor, ME). All animal studies were performed in accordance with the Animal Care and Use
Committee guidelines of the NIH and were conducted under protocols approved by the Animal Care and Use
Committee of the USC.
Cell culture, reagents and antibodies
The human ovarian cancer cell line SKOV3 (ATCC
®
HTB-77™) and the human chronic myelogenous leukemia
cell line K562 (ATCC
®
CCL-243™) were maintained in RPMI-1640 with 10% FBS, 2 mM L-glutamine, 10 mM
HEPES, 100 U/ml penicillin, 100µg/ml streptomycin and 100µg/ml Normocin™ (InvivoGen, San Diego, CA).
K562.CD19 and SKOV3.CD19 cells were generated by transducing parental K562 and SKOV3 cells with
lentiviral vectors encoding the cDNA of human CD19. All cells were routinely tested for potential mycoplasma
contamination using the MycoSensor qPCR assay kit (Agilent Technologies).
SCH-58261 was purchased from Sigma-Aldrich (St. Louis, MO). All lipids were purchased from NOF
Corporation (Japan): 1,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phospho-
(10-rac-glycerol) (DOPG), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)but-
yramide (maleimide-headgroup lipid, MPB-PE).
Primary antibodies used in this study include 1) anti-HA tag antibody from Abcam (Cambridge, MA); 2)
Alexa488-anti-phospho-CREB antibody (clone 87G3) from Cell Signaling; 3) PE-anti-CD45 (clone HI30), APC-
anti-CD45 (clone HI30), PE-Cy5.5-anti-CD3 (HIT3a), FITC-anti-CD4 (clone RPAT4), Pacific Blue
TM
-anti-CD8
(clone SK1), FITC-anti-CD8 (clone SK1), PE-anti-IFN-γ (clone B27) and APC-anti-EGFR (clone AY13), all
from BioLegend (San Diego, CA); and 4) AlexaFluor-647 goat anti-rabbit antibody from Life Technologies.
Preparation of T cells for adoptive transfer and viral transduction
85

Thawed primary blood mononuclear cells (PBMCs) from healthy donors were cultured in T cell medium (TCM)
containing X-VIVO™ 15 serum-free medium (Lonza, Allendale NJ), 5% (vol/vol) GemCell human serum
antibody AB (Gemini Bio-Products, West Sacramento CA), 1% (vol/vol) Glutamax-100× (Gibco Life
Technologies), 10mM HEPES buffer (Corning), 1% (vol/vol) penicillin/streptomycin (Corning) and 12.25 mM
N-Acetyl-L-cysteine (Sigma). The culture was supplemented with 10 ng/mL human IL-2. The PBMCs were
activated and expanded using Dynabeads
®
human T-expander CD3/CD28 (Invitrogen) at a bead:PBMC ratio of
3:1. Activated PBMCs were transduced with viral vectors 48 hours after activation. During ex vivo expansion,
culture medium was replenished, and the T cell density was maintained between 0.5-1 × 10
6
cells/mL.
Plasmid construction
The lentiviral vector encoding the HA-tagged CD19scFv-28-ζ CAR was constructed based on the CD19 CAR
previously reported
243
. The CD19 single-chain fragment variable (scFv) sequence, followed by the human CD8
hinge region (aa 138-184), was codon optimized and constructed by Integrated DNA Technologies (Coralville,
IA). The CD19/CD8 hinge gene block was combined with the transmembrane and intracellular domains of human
CD28 (aa 153-220) and the intracellular domain of human CD3ζ (aa 52-164) using PCR. The CD8 leader
sequence and HA-tag were inserted upstream of the CD19 scFv to allow for labeling and detection of CAR-
expressing cells. To make the lentiviral vector, this sequence was inserted downstream of the human ubiquitin-C
promoter in the lentiviral plasmid pFUW using Gibson assembly, as previously described
244
.
The retroviral vector encoding the same CD19 CAR (CAR) was constructed based on the MP71 retroviral vector
kindly provided by Prof. Wolfgang Uckert, as described previously
245
. The vector encoding CD19 CAR, along
with truncated EGFR (tEGFR), was then generated by modifying the CD19 CAR plasmid. All primer sequences
for the plasmid construction are available upon request.
Lentivirus and retrovirus vector preparation and transduction
Lentiviral vectors were prepared by transient transfection of 293T cells using a standard calcium phosphate
precipitation protocol as described previously
244
. The viral supernatants were harvested 48h post-transfection and
86

filtered through a 0.45 µg filter (Corning, Corning, NY). Virus supernatants were loaded into a 24-well plate with
5 × 10
5
activated human PBMCs and spun for 90 minutes at 1050g at 25°C.
Retroviral vectors were prepared by transient transfection of 293T cells using a standard calcium phosphate
precipitation protocol. Activated human PBMCs were transduced and expanded as described previously
246
.
Detection of receptor expression on T cell surface
HA-tagged CD19scFv-28-ζ CAR-T cells were washed with PBS and stained with rabbit anti-HA followed by
Alex647-conjugated anti-rabbit antibodies for CAR detection. Retrovirus-transduced cells were stained with
APC-conjugated anti-human EGFR for tEGFR detection. Receptor expression was analyzed using the MACS
Quant flow cytometry analyzer (Miltenyi Biotec, Inc., San Diego, CA).
Synthesis of nanoparticles and drug encapsulation
Liposomes were prepared based on the established dehydration-rehydration method previously reported
247
. To
encapsulate SCH-58261 into cMLVs, 1 mg of SCH in organic solvent was mixed with the lipid mixture to form
dried thin lipid films. To label liposome particles with DiD lipophilic dyes, DiD dyes were added to the lipid
mixture in chloroform at a ratio of 0.01:1 (DiD:lipids). Morphology of multilamellar structure of the vesicles was
analyzed and confirmed by cryo-electron microscopy, as in previous studies
102, 247
. The hydrodynamic size of
cMLVs was measured by dynamic light scattering (DLS) (Wyatt Technology, Santa Barbara, CA). The particles
were suspended in filtered water, vortexed and sonicated prior to analysis.
Nanoparticle conjugation with cells and in situ PEGylation
Chemical conjugation of nanoparticles to the T cells was performed based on a method reported by Stephan et al
78
. For quantification of cell-bound particles, nanoparticles were fluorescently labeled with the lipid-like
fluorescent dye 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine (DiD) before conjugation, and
fluorescence was detected by flow cytometry and fluorescent microplate reader. The surface conjugation of DiD-
labeled cMLVs and CFSE-labeled CAR-T cells was further visualized using confocal microscopy.
In vitro drug encapsulation and release
87

The amount of encapsulated SCH-58261 in the cMLV(SCH) was evaluated by C-18 reverse-phase high-
performance liquid chromatography (RPHPLC) (Beckman Coulter, Brea, CA), as previously reported
53, 102
. To
obtain the release kinetics of SCH from cMLVs before and after cell conjugation, cMLV(SCH) and
CART.cMLV(SCH) were incubated in 10% FBS-containing media at 37 °C and were spun down and resuspended
with fresh media daily. SCH was quantified from the harvested media every day by HPLC.
Cytotoxicity assay
A modified version of a cytotoxicity assay was performed, as previously described in the study by Kochenderfer
et al., to assess the cytotoxicity of CAR-modified T cells
62
. Non-target cells, SKOV3 cells and K562 cells, were
stained with the fluorescent dye 5-(and-6)-(((4-chloromethyl)benzoyl)amino) tetramethylrhodamine (CMTMR)
(Invitrogen). Target cells (SKOV3.CD19 and K562.CD19) were stained with carboxyfluorescein diacetate
succinimide ester (CFSE) (Invitrogen). The cultures were set up in triplicate in a sterile 96-well round bottom
plate (Corning) at effector:target (E:T) cell ratios of 20:1, 10:1, 5:1 and 1:1. Immediately after the incubation, 7-
amino-actinomycin D (7AAD, BD Pharminogen) was added as recommended by the manufacturer. The
fluorescence was analyzed by flow cytometry. Cell cytotoxicity was calculated as [CFSE
+
7AAD
+
cells /
(CFSE
+
7AAD
−
+ CFSE
+
7AAD
+
)] cells.
Transmigration assay
T cell transmigration assays were performed in 24-mm diameter, 3-µm pore size transwell plates (Costar). cMLV-
conjugated and unconjugated CAR-T cells (0.5 × 10
6
/well) were plated on the upper wells, and TCM was added
to the lower wells. The T cell chemoattractant CXCL-9 (0.1mg/ml, Peprotech) was added to the lower wells.
After incubation at 37°C for 6 hours, T cells that migrated into the lower chamber were counted.
In vivo biodistribution study
For the in vivo nanoparticle biodistribution study, a xenograft tumor model was used. To establish the tumor,
SKOV3.CD19

cells in PBS solution were injected subcutaneously into the flanks of NOD/scid/IL2Rγ
-/-
(NSG)
mice. DiD-labeled cMLVs, CD19 CAR-T cells (5 × 10
6
) mixed with DiD-labeled cMLVs, CD19 CAR-T cells (5
× 10
6
) surface-conjugated with DiD-labeled cMLVs, or PBS were intravenously injected into the tumor-bearing
88

mice. After 24 and 48 hours, indicated tissues were removed, weighed, and macerated with scissors. DiD-specific
tissue fluorescence (Abs 644 nm, Em 665 nm) was quantified for each organ using the Xenogen IVIS spectrum
imaging system by the USC Imaging Core scientists blinded to the groups, and the percentage of injected dose
per gram of tissue (%ID/g) was calculated.
Quantification of accumulated nanoparticles at tumor sites
SKOV3.CD19

tumors were implanted into NSG mice, as described above, and CFSE-labeled CAR-T cells and
DiD-labeled cMLVs were injected into tumor-bearing mice. At the indicated times, tumors were excised, fixed,
frozen, cryo-sectioned, and mounted onto glass slides. Fluorescence of CFSE-labeled CAR-T cells and DiD-
labeled cMLVs was visualized using a Zeiss 700 Confocal Laser Scanning Microscope (Inverted) (Carl Zeiss,
Germany). Quantification analysis was performed using Zeiss Zen microscope software
In vivo xenograft experiments for prevention study
SKOV3.CD19

tumors were implanted into NSG mice, as described above. After tumors were established, 10 mice
were randomly assigned to each treatment group. Tumor growth was measured using calipers and calculated using
the formula (width
2
× length)/2. Five mice from each group were sacrificed on day two and day 14 post-treatment.
The tumor and spleen from each mouse were harvested for further ex vivo analysis.
In vivo xenograft experiments for rescue study
SKOV3.CD19

tumors were implanted into NSG mice, as described above. After tumors were established, all the
mice were injected with 3 × 10
6
CART.tEGFR cells. Ten days after initial adoptive CAR-T cell transfer, the mice
were randomly assigned to receive the following treatments: (1) PBS; (2) CAR-T cells; (3) CAR-T cells
conjugated to empty cMLVs (CART.cMLV); (4) a mix of CAR-T cells and cMLV(SCH) (CART+cMLV(SCH));
and (5) CAR-T cells conjugated to SCH-loaded cMLVs (CART.cMLV(SCH)). Each mouse was injected with
2.5 × 10
6
CAR-positive T cells. For mice treated with unconjugated cMLVs, 10
9
drug-loaded cMLVs were co-
infused with CAR-T cells. Forty-eight hours after the second adoptive T cell transfer, the mice were sacrificed.
The spleen and tumor were harvested for ex vivo assays.
89

Ex vivo analysis
Two functional analyses were performed: (1) anti-CD3/anti-CD28-induced intracellular IFN-γ cytokine staining
and (2) phospho-CREB expression in CAR-T cells. For intracellular IFN-γ staining, a total of 0.5 × 10
6
cells were
stimulated with human CD3/CD28 antibodies and 10 ng/mL Brefeldin A. The culture was incubated for 6 hours
at 37°C in 96-well round bottom plates. Fluorophore-conjugated human CD3, CD45, CD4 and CD8 antibodies
were used for immunostaining. Cytofix/Cytoperm solution (BD Bioscience) was used to permeabilize cell
membrane and perform intracellular staining according to the manufacturer’s instruction. For intracellular
phospho-CREB staining, cells were fixed with 4% paraformaldehyde (PFA), followed by permeabilization in
methanol for 30 minutes on ice. Cells were then stained with Alexa488-conjugated anti-human phospho-CREB
for 30 minutes at 4°C. Flow cytometry analysis was carried out using the MACSQuant® Instrument from
Miltenyi Biotec (Auburn, CA).
Statistics
The differences between two groups were determined with Student’s t test. The differences among three or more
groups were determined with a one-way analysis of variance (ANOVA). Prism (GraphPad) was used to calculate
statistical significance of the difference in mean values and P value

90

CHAPTER 5: CONCLUSION AND FUTURE
PERSPECTIVES

In summary, we have successfully developed drug delivery platforms utilizing cross-linked multilamellar
liposomal nanoparticles as a novel strategy of combination therapy. Through extensive in vitro and in vivo studies,
we have demonstrated a variety of targeting strategies to improve overall antitumor efficacy. The first presented
study has investigated the effect of co-delivery of a chemotherapeutics and a compound targeting cancer stem
cells (CSCs) for treatment of breast cancer in mice. Specifically, we have utilized cMLVs to co-encapsulate
doxorubicin (Dox, chemotherapeutics) and salinomycin (Sal, targeting CSCs). The efficient targeting effect of
cMLV(Dox+Sal) on CSCs was validated through in vitro experiments using breast cancer stem cell makers. In
accordance with the in vitro combination treatment, in vivo breast tumor suppression by cMLV(Dox+Sal) was
more effective than single drug cMLV treatment or treatment with combination of cMLV(Dox) and cMLV(Sal).
To further validate the efficacy of the treatments, a long term survival study needs to be performed following the
antitumor efficacy study.
Overall, this study has demonstrated a proof of concept delivery system that can serve as a potential platform for
a combination therapy, allowing co-delivery of an anti-cancer agent and a CSC inhibitor for the elimination of
breast cancer cells together with breast cancer stem cell.
As previously mentioned, passive targeting strategies by nanoparticles are often insufficient in poorly
vascularized tumors. We aimed to create a cell-based therapy which combines immunotherapy with drug delivery
to provide more targeted treatment of solid tumors. Following studies have successfully demonstrated that our
immune cell-mediated delivery system using cMLVs can target various tumor antigens and can carry a variety of
anticancer agents, including but not limited to chemotherapeutics and immunomodulatory drugs. First, we
provided compelling evidence that CAR.NK cells facilitate the delivery of the chemotherapeutic drug PTX to the
91

tumor site, slowing tumor growth and increasing intratumoral PTX concentrations as the CAR can improve
specificity and tumor-targeting ability of carrier cells.
We have further shown that this delivery system can be broadened to include immunomodulatory drugs such as
adenosine A2a receptor antagonists. In the last study, CART.cMLV(SCH) has shown promising results, leading
to the regression of solid tumor in our human ovarian cancer xenograft model through enhancing the efficacy of
tumor infiltrated CAR-T cells.
However, further improvements and modifications could be made to expand this treatment to different clinical
settings. First, we demonstrated a proof of concept design using αhCD19-CAR T cells that target a CD19
transduced ovarian cancer cell line. Further engineering is required to design a CAR that can sensitively and
specifically target endogenous solid tumor antigens. Second, apart from the A2a receptor antagonist, cMLVs
could also deliver drugs that interfere with other immune inhibitory pathways. Diacylglycerol kinase (DGK), for
example, is an enzyme highly expressed in T cells. Elevated levels of DGK has been shown to limit Ras/ERK
activation, which suppresses T cell cytokine secretion and cell cytotoxicity
248
. Riese et al. showed that CAR- T
cells from DGK knockout mice have enhanced antitumor functions in vivo
248
. Likewise, blocking dgkα and dgkζ
with small molecule inhibitors has also been shown to increase CAR- T cell function in vivo and elicit solid tumor
regression (unpublished data). Moreover, immune regulatory drugs could be delivered in combination with
immune checkpoint blockade, such as anti-PD-1, to further promote anti-tumor immunity. Upon activation of T
cells, PD-1 becomes upregulated, which leads to immunosuppression in the TME, and multiple studies have
successfully used PD-1 antibody to enhance CAR- T cell function in vivo
249-251
. As this system has the ability to
conjugate multiple drugs to the T cell surface, either through coencapsulation of multiple drugs in a single cMLV
or by conjugating two nanoparticles loaded with different drugs to the surface of the same T cell, any combination
of these immune suppression blockades could be adapted for use in this immune cell-conjugated nanoparticle
delivery system.
92

Conjugation of drug-loaded nanoparticles with CAR-T/NK cells is an effective strategy to armor these cells
against immunosuppressive agents in the TME. This platform is highly flexible, and can be applied to other drugs,
cytokines, antibodies, or any combination thereof. The major advantage of this strategy is its active targeting to
the tissue of interest. By using CAR-T/NK cells as chaperones, we were able to efficiently localize cMLV
nanoparticles in specific tissues that corresponds to T/NK cell trafficking –such as the tumor, spleen, lungs and
lymph nodes. Overall, this is a promising platform that can greatly improve the efficacy and specificity of solid
tumor therapies.

93

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Abstract (if available)

Abstract A formulation of crosslinked multilamellar liposomes (cMLVs) was utilized as a nanocarrier platform to maximize overall drug delivery and therapeutic efficacy. My research goal was to utilize innovative drug delivery approaches to investigate and treat various types of cancers and diseases. The cMLV drug delivery system was developed and optimized for co-delivery of drugs to simultaneously target two cell populations: cancer cells and breast cancer cells. Having optimized the delivery system to simultaneously target these two populations, this innovative approach can eventually overcome the major challenge of cancer treatment, which is the failure of chemotherapy likely due to the presence of the cancer stem cells (CSCs). In this project, breast cancer stem cells were extensively characterized both in vitro and in vivo. The project was focused on the formulation of cMLVs co-encapsulating doxorubicin (cytotoxic drug) and salinomycin (CSC inhibitor), allowing for simultaneous delivery of the two drugs to the tumor site for enhanced anti-tumor effect. The breast cancer stem cells were identified in murine and human breast cancer cell lines as well as in the in vivo breast tumor-derived cells using several putative breast cancer stem cell markers. Data obtained from the in vitro and in vivo studies revealed that the combinatorial delivery system of Dox and Sal in a single cMLV can be highly effective at inhibiting CSCs and breast cancer cells, suggesting intriguing potential as a promising new method for breast cancer therapy. ❧ Recent research focuses on “immunobioengineering” with the aim of employing engineering tools and principles to quantitatively understand the immune system in health and disease and to develop novel molecular and cellular immunotherapies by precisely modulating disease-specific immune responses. Two latest projects were optimizing chimeric antigen receptor (CAR) engineered immune cells such as T cells and NK cells for treating solid tumors. I have investigated underlying mechanisms to enhance the anti-tumor efficacy of CAR-T cells in the immunosuppressive tumor microenvironment and developed surface engineering of CAR T cells and CAR NK cells with nanoparticles to enhance drug delivery to solid tumors.

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University of Southern California Dissertations and Theses

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Asset Metadata

Creator Kim, Yu Jeong (author)

Core Title Optimization of nanomedicine based drug delivery systems for the treatment of solid tumors

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Pharmaceutical Sciences

Publication Date 03/05/2018

Defense Date 11/10/2017

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

Tag cancer immunotherapy,cancer therapy,drug delivery,nanomedicine,OAI-PMH Harvest

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Wang, Pin (committee chair), Okamoto, Curtis (committee member), Shen, Wei-Chiang (committee member)

Creator Email yujeongk@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-480957

Unique identifier UC11266730

Identifier etd-KimYuJeong-6075.pdf (filename),usctheses-c40-480957 (legacy record id)

Legacy Identifier etd-KimYuJeong-6075.pdf

Dmrecord 480957

Document Type Dissertation

Rights Kim, Yu Jeong

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