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Enhancing the specificity and cytotoxicity of chimeric antigen receptor Natural Killer cells

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Enhancing the specificity and cytotoxicity of chimeric antigen receptor Natural Killer cells

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Content ENHANCING THE SPECIFICITY AND CYTOTOXICITY OF CHIMERIC
ANTIGEN RECEPTOR NATURAL KILLER CELLS
by
DEEMA AYA SHARIF
A Thesis Presented to the
FACULTY OF THE USC MANN SCHOOL OF PHARMACY
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(PHARMACEUTICAL SCIENCES)
August 2023
Copyright 2023 Deema Aya Sharif
Acknowledgements
First, I would like to thank my advisor, Dr. Jianming Xie for his continued guidance and
support through my master’s studies. By accepting me into his lab he provided me the
opportunity to study a research topic that is aligned with my interests and values.
I would also like to express my appreciation for Dr. Liang Rong, the postdoctoral scholar
in our lab, for spending everyday patiently teaching me everything I know about bench work. I
could not have completed this paper without your help.
I also want to thank Dr. Culty and Dr. Haworth for reviewing my thesis as members of
my committee.
Last but not least, I would like to thank my partner for their unwavering support through
my masters research.
ii
Table of Contents
Acknowledgements ................................................................................................................................. ii
List of Figures .......................................................................................................................................... iv
Abbreviations ........................................................................................................................................... v
Abstract .................................................................................................................................................... vii
Chapter 1 - Introduction .......................................................................................................................... 1
1.1 Natural Killer Cells ................................................................................................................... 4
1.2 Chimeric Antigen Receptor Structure ....................................................................................... 5
1.3 Chimeric Antigen Receptor T Cell Therapy Review ................................................................ 7
1.4 Limitations of CAR-T Therapy ................................................................................................. 8
1.5 Advantages of CAR-NK Therapy ............................................................................................. 10
Chapter 2 - A Universal CAR-NK Cell Targeting HIV-1 GP-160 ........................................................ 12
2.1 Abstract ..................................................................................................................................... 12
2.2 Introduction ............................................................................................................................... 13
2.2.1 Human Immunodeficiency Virus and current treatment ................................................. 13
2.2.2 Anti-DNP Chimeric Antigen Receptor Natural Killer Cells ........................................... 15
2.2.3 Generation of Target Cells ............................................................................................... 16
2.3 Results ....................................................................................................................................... 25
2.3.1 Genetic Engineering of NK Cells to Express anti-DNP CAR ........................................ 25
2.3.2 Redirection of CAR-NK Cells Toward HEK293-gp160pos Cells .................................. 28
2.4 Discussion ................................................................................................................................. 32
Chapter 3 - Enhancing Specificity of anti-CD19 CAR-NK Cells By Targeting HLA-DR Loss .......... 34
3.1 Abstract ..................................................................................................................................... 34
3.2 Introduction ............................................................................................................................... 35
3.2.1 CD19 Expression in B Cell Malignancies ....................................................................... 35
3.2.2 CD33 Expression in Myeloid Cell Malignancies ............................................................ 36
3.2.3 Targeting HLA-DR Loss to Enhance Specificity of CAR Therapy ................................ 36
3.3 Results for CD19+ cancer cells ................................................................................................. 38
3.3.1 Engineering aCD19 aHLA-DR iCAR-NK Cells ............................................................. 38
3.3.2 aCD19 aHLA-DR iCAR-NK Inhibition .......................................................................... 43
3.4 Results for CD33+ cancer cells ................................................................................................. 50
3.4.1 Engineering aCD33 aHLA-DR iCAR-NK Cells ............................................................. 50
3.4.2 aCD33 aHLA-DR iCAR-NK Inhibition .......................................................................... 53
3.5 Discussion ................................................................................................................................. 58
Chapter 4 - Conclusion .......................................................................................................................... 63
Chapter 5 - Methods .............................................................................................................................. 67
References ............................................................................................................................................. 71
iii
List of Figures
Figure 1 - Schematic overview of chimeric antigen receptor ……………………………………. 6
Figure 2 - New generations of CAR therapy …………………………………………………….. 7
Figure 3 - A universal CAR-NK cell that targets DNP-conjugated antibodies ………………….. 16
Figure 4 - Design and production of 4 αDNP CAR-NK cells …………………………………… 19
Figure 5 - αDNP CAR-NK Cell ELISA Assay ………………………………………………….. 21
Figure 6 - Cytotoxicity Assay 4 αDNP CAR-NK Cells using 4 DNP-conjugated bNAbs ……… 22
Figure 7 - Cytotoxicity Assay 3 αDNP CAR-NK Cells using 4 different E:T Ratios …………... 25
Figure 8 - Cytotoxicity Assay EF1-NKG2D-3𝜁 to determine bNAbs EC50 value ……………… 30
Figure 9 - Design of Inhibitory CAR Downregulating Activating CAR’s Signal ………………. 38
Figure 10 - Design and Production of 9 αCD19 αHLA-DR iCAR-NK cells ……………………. 41
Figure 11 - αCD19 αHLA-DR iCAR-NK ELISA Assay ………………………………………... 44
Figure 12 - αCD19 αHLA-DR iCAR-NK Cytotoxicity Assay Using 3 Target Cells ……………. 47
Figure 13 - Design and Production of 2 αCD33 αHLA-DR iCAR-NK Cells …………………… 52
Figure 14 - αCD33 αHLA-DR iCAR-NK ELISA Assay ………………………………………... 54
Figure 15 - αCD33 αHLA-DR iCAR-NK Cytotoxicity Assay Using 2 Target Cells ……………. 56
iv
Abbreviations
AIDS - acquired immunodeficiency syndrome
ALL - acute lymphoblastic leukemia
AML - acute myeloid leukemia
APC - antigen presenting cells
APC stain - Allophycocyanin
CAR - chimeric antigen receptor
CLP - common lymphoid progenitor
CML - chronic myeloid leukemia
CRS - cytokine release syndrome
DLBCL - diffuse large B cell lymphoma
DNP - 2,4-dinitrophenyl
ELISA - enzyme linked immunosorbent assay
GvHD - graft versus host disease
HA - human influenza hemagglutinin
HIV - human immunodeficiency virus
HLA-DR - human leukocyte antigen - DR
iCAR - inhibitory chimeric antigen receptor
IFN-γ - interferon gamma
IL-2 - interleukin 2
MHC - major histocompatibility complex
NK - natural killer
v
NKG2D/A - natural killer group 2D/2A
PD1 - programmed cell death protein -1
PE - phycoerythrin
scFv - small chain variable fragment
TAA - tumor associated antigen
TIGIT - T cell immunoreceptor with Ig and ITIM domains
TM - transmembrane domain
TPA - TIGIT-PD1-NKG2A
vi
Abstract
In spite of the potency of a healthy immune system, cancer and HIV infection remain
difficult diseases to treat, and can progress before a diagnosis is made, potentially shortening
patient survival outcomes.This is because immune cells do not have the tools to recognize these
conditions. Here, we demonstrate how we created chimeric antigen receptors (CARs) that
recognize the surface antigens of HIV or B cell leukemias and lymphomas, and engineered them
into Natural Killer cells, effectively arming these cells to recognize and eliminate their target[1].
The first project is to engineer an enhanced anti-HIV CAR-NK cell. HIV-infected cells
express a glycoprotein, gp160, on their surface. Previously, our lab created a universal anti-HIV
CAR-NK cell that can kill HIV-infected cells through the use of adaptor molecules that connect
gp160 to the CAR-NK cells’ receptor[2]. In this thesis, in order to enhance the potency and
cytotoxicity of CAR-NK cells, we utilized NKG2D, 2b4 signal domains, as well as CD28 and
CD3𝜁 in the CAR designs. We found the anti-DNP-NKG2D-2b4-CD3z is the best design for
anti-DNP universal CAR-NK system.
The second project is to engineer dual CAR-NK cells that can specifically target
hematologic malignancies that overexpress certain surface proteins, known as Tumor Associated
Antigens (TAA)[3], and under-express HLA-DR. Previous researchers have created synthetic
receptors, CARs, that recognize these TAAs and engineered them into autologous cytotoxic T
cells (CAR-T). While this treatment has been effective, it causes life-threatening side effects that
could be avoided by engineering CARs into Natural Killer cells (CAR-NK) instead of T cells as
NK cells are shown not to elicit these conditions. Our goal was to create an inhibitory CAR that
vii
can downregulate the CAR-NK cell’s cytotoxicity upon binding HLA-DR, thus limiting the
CAR-NK cell’s activity to only cancer cells. We found the combination of TIGIT, PD-1 and
NKG2A signal domains had the strongest inhibitory effect. We tested our new iCAR designs in
both anti-CD19 and anti-CD33 CAR-NK cells.
viii
Chapter 1 - Introduction
The immune system is a diverse and efficient force that diligently protects its host from
invading pathogens and cancers. And yet, there are still several disease states that are able to
coexist within an organism without eliciting an immune response.
A major step in the development of the adaptive immune system is T-cell priming -
ensuring that these cells can differentiate between the self and the non-self. This step is integral
in preventing autoimmunity. However, at the genomic level, tumor cells share at least 99.9% of
the genomic information of their cells of origin[4]. This similarity causes the tumor cells to be
relatively indistinguishable from healthy cells. Not many tumor cell lines have mutations that are
genetically distinct enough to elicit an immune response[5]. And immune cells do not possess
receptors that recognize tumor antigens or HIV-associated glycoproteins [6].
However, some cancer cell lines begin to overexpress certain cell surface proteins. These
include CD19[3], CD33[7], etc. This overexpression is our first clue into figuring out how to
employ the immune system in the destruction of these cancer cells. Moreover, overexpressed cell
surface proteins that are limited to a small subset of healthy cells make even better targets[3].
Indeed, the first approved CAR-T therapy, Tisagenlecleucel[8], was directly targeted at
the cell surface protein CD19[9]. Since tumor cell lines can overexpress this protein, CD19 can
serve as a reliable target to aid CAR-T cells in the recognition and destruction of cancer cells[3].
While CAR-T therapy has been successful in some cases, it is a physically taxing
treatment that leaves patients with severe side effects[10]. First, the foreign nature of CAR-T
1
cells can cause Graft versus Host Disease (GvHD). Second, some patients experience cytokine
release syndrome - a life threatening condition that requires immunosuppressive drugs to
treat[11]. Third, since CD19 is present on healthy B cells, CAR-T therapy can eliminate a
patient's own B cell immune system, leading to B-cell aplasia, which can then cause
hypogammaglobulinemia – a condition wherein the patient has no circulating antibodies to
protect against infection[12]. Subsequent infections have led to the deaths of some patients
undergoing CAR-T therapy[13]. The intensity of this treatment has therefore created a need for a
safer, more selective form of CAR cells. If a new generation of CAR cells can target tumor cells,
but spare healthy cells, the efficacy of this therapy would increase, as well as patient survival
outcomes. We engineered our CAR constructs in Natural Killer cells, which have not caused the
same harsh side effects and have an increased safety profile compared to CAR-T cells [14].
Some blood cancer cell lines overexpress surface antigens, and downregulate human
leukocyte antigens, e.g., HLA-DR[15, 16]. HLA molecules present antigens to the extracellular
matrix that T cells bind to in order to ensure the cells are healthy. A diseased or infected cell, will
display a non-self antigen on their HLA-DR surface protein[17].
In order to decrease “on-target, off-tumor” side effects, we previously developed an
inhibitory CAR-NK cell which can target HLA-DR loss in hematologic malignancies. The
anti-HLA-DR inhibitory CAR can extinguish the signal of the anti-CD19 or anti-CD33
activating CAR. The dual CAR system activates when binding to a tumor associated antigen and
deactivates when binding a self antigen (HLA-DR).
2
In this study, we created six iCAR-NK cells with the same activating CAR, specific for
CD19, and each with a unique inhibitory CAR (iCAR) with varying signal domains like
NKG2A, PD-1, and TIGIT. Our goal was to discern which iCAR can best downregulate the
activating CAR using NKG2D, 2b4, CD28 and CD3𝜁 . We found the combination of TIGIT,
PD-1 and NKG2A signal domains had the strongest inhibitory effect. Following the anti-CD19
iCAR-NK cells, we engineered the same anti-HLA-DR CARs into anti-CD33 CAR-NK cells.
To target HIV-infected cells with CAR-NK cells, we exploited the expression of the HIV
glycoprotein gp-160[18]. We expanded on our lab’s previous research where we created a
universal anti-HIV CAR-NK cell that can eliminate HIV-infected cells through adaptor
molecules that bridge gp160 to the CAR-NK cells’ receptor[2]. The adaptor molecules are
gp160-specific antibodies conjugated with 2,4-dinitrophenyl molecules. By incubating
HIV-infected cells with these DNP-conjugated antibodies, the anti-DNP CAR-NK cells can be
redirected to and kill the HIV-infected cells. By creating DNP-conjugated antibodies for each
subtype of HIV , we can potentially create a system that utilizes one universal CAR-NK cell that
can overcome the vast diversity of HIV .
To enhance the potency and cytotoxicity of our previously established CAR-NK cells, we
utilized NKG2D, 2b4 signal domains, as well as CD28 and CD3 𝜁 in the CAR designs. However,
the signal domains used in our previous CAR-NK cells are CD28 and CD3 𝜁 , which are not
optimal for NK cell activation. Based on the in vitro ELISA of IFN-γ secretion and FACS-based
cytotoxicity assay, We found the anti-DNP-NKG2D-2b4-CD3z is the best design for anti-DNP
universal CAR-NK system.
3
1.1 Natural Killer Cells
Natural killer (NK) cells, first identified in 1975, are a type of immune cell that originates
from a common lymphoid progenitor. They earned their name due to their status of being
consistently active[19]. Conversely, T and B cells, members of the adaptive immune system,
roam the bloodstream in an inactive state, and wait to be activated in the presence of an invading
pathogen[20].
NK cells, classified as CD56
+
/CD3
neg
immune cells, are a cytotoxic type of immune cell
that participates in both the innate and adaptive immune systems[20]. Like CD8+ cytotoxic T
cells, they are able to remove foreign pathogens, and inhibit tumor proliferation, metastasis, and
migration[21]. Additionally, NK cells are able to secrete cytokines, mainly interferon-ɣ (IFN-ɣ).
IFN-ɣ modulates immune cells, leading to other signaling pathways that result in proliferation
inhibition, pathogen recognition, and apoptosis[22].
Similar to other innate immune cells, NK cells originate from the bone marrow where
they undergo education to avoid autoimmunity. Once fully licensed, NK cells are always
activated, unlike CD8+ T cells which need to be activated upon binding major histocompatibility
complex (MHC) on target cells[20]. Because members of the innate immune system respond
earlier to invading pathogens, viruses, and tumor cells, and NK cells serve as the predominant
cytotoxic cells of the innate immune system, they play a pivotal role in tumor suppression and
pathogen elimination[20].
Due to the cytotoxicity and efficiency of NK cells, they have the potential to be
redirected toward hematologic malignancies and human immunodeficiency virus (HIV). Several
4
NK cell lines have been established for preclinical research. We used NK92 for our CAR-NK
experiments, as this cell line is stable and proliferates in the presence of interleukin-2 (IL-2)[20].
1.2 Chimeric Antigen Receptor Structure
One of the hurdles to overcome in immuno-oncology is the lack of tumor specific
antigens to target with therapy. However, certain cancers overexpress surface antigens, which can
be exploited for the development of cancer therapeutics[3]. To redirect NK cells toward these
malignancies, we can engineer synthetic receptors onto their surface that recognize
overexpressed antigens[2]. The use of chimeric antigen receptors (CARs) allows the cells to
eliminate targets that endogenous NK cells may not have recognized.
To recognize the intended antigen, an antigen recognition site typical of a B cell receptor
is required. The heavy chain and light chain are generated and form a single chain variable
fragment (scFv) - a highly specific antigen recognition site that allows the NK cell to find the
target cells. Next, a hinge and transmembrane domain anchor the receptor into the cellular
membrane. They also increase the flexibility of the receptor, allowing it to pivot to find its ligand
and avoid steric hindrance, thus increasing the efficacy of the NK cell. Hidden below the cell
membrane, is a number of intracellular domains (Figure 1)[23]. First generation CAR-T cells
have one activating domain, typically CD3 𝜁 . In order to amplify the CAR’s signal and maximize
the cell’s cytotoxicity, next generation CAR’s have additional costimulatory domains on the
intracellular side of the receptor. Second generation CARs have two activating domains, bound
in tandem, and third generation CARs have a third intracellular domain (Figure 2)[24].
5
Upon binding its target, the receptor’s costimulatory domains are activated, eliciting a
signal transduction pathway that results in cytokine secretion and target cell apoptosis[25].
6
1.3 Chimeric Antigen Receptor T Cell Therapy Review
CAR-T therapy, first approved by the FDA in 2017, has proven to be an efficacious
method to treat hematologic malignancies and prevent relapse[26]. Currently, six approved
CAR-T products are available[27]. Four of which target CD19 in the treatment of relapsing
hematological malignancies: lisocabtagene maraleucel[28], tisagenlecleucel[29], brexucabtagene
autoleucel[29], and axicabtagene cilolecel[30]. And two CAR-T therapies target BCMA for the
treatment of multiple myeloma: idacabtagene vicleucel[31], and ciltacabtagene autoleucel[32].
After being infused into the patient, the CAR-T cells are able to proliferate and destroy many
tumor cells[25]. CD19 was selected as the tumor associated antigen to be the target of the first
7
CAR-T therapy because of its presence on several B-cell leukemias and lymphomas.
Additionally, CD19 is restricted to B cells, minimizing the risk of collateral damage, or “on
target, off tumor” effect[25].
1.4 Limitations of CAR-T Therapy
While CAR-T therapy has been successful in treating relapsing hematologic
malignancies, it often leaves patients fighting cytokine release syndrome, neurotoxicity, and B
cell aplasia[10].
One of the most common side effects of CAR-T therapy is cytokine release syndrome
(CRS). T cell activation leads to a sudden increase in cytokine secretion, which in turns recruits
and activates other immune cells[33]. Severity of CRS can vary, from mild to life-threatening
and typically occurs within five days of initial CAR-T infusion[34, 35]. Symptoms include
high-grade fever, respiratory distress, hypotension, and neurotoxicity. While this does not occur
in all patients, most patients experience some degree of CRS. Certain cases only require
immunosuppressants and some cases require intubation or mechanical ventilation[36]. Treatment
for CRS includes corticosteroids and the monoclonal antibody tocilizumab[37]. However, steroid
use is correlated with a decrease in CAR-T longevity and proliferation. Conversely, tocilizumab,
which targets interleukin-6R, has proven to be effective in treating CRS without compromising
the longevity of the infused CAR-T cells[38]. Therefore, steroid use to treat CRS is correlated
with early relapse, making tocilizumab the preferred treatment[34].
Another potential side effect of CAR-T therapy is neurotoxicity or encephalopathy. While
the exact mechanism for this remains unknown, symptoms manifest as tremors in the
8
extremities, inability to communicate effectively, somnolence, hallucinations, and delirium[35,
36]. Neurotoxicity occurs in almost half of patients receiving CAR-T therapy, and treatment
includes steroids that can cross the blood-brain barrier[10, 35, 36].
Another common complication associated with current CAR-T therapy is the
development of B-cell aplasia. Approved CAR-T therapy that targets CD19 - a tumor associated
antigen often upregulated in B-cell acute lymphoblastic leukemia - also eliminates healthy B
cells. This is because CD19 is present on all B-cells, healthy or otherwise, and on B cell
precursors. Therefore, successful CAR-T therapy that eliminates all malignant cells, also begins
to eliminate healthy B cells, a phenomenon deemed the on-target, off-tumor effect. Due to the
vital role B cells play in the immune system, affected patients become susceptible to
hypogammaglobulinemia, and are at risk of serious infection. Treatment of B cell aplasia
includes periodic intravenous immunoglobulin repletion[10, 12, 33].
Despite the potentially serious side effects, CAR-T therapy remains an extremely
effective treatment, especially for relapsing malignancies not responding to conventional
treatments[33, 34]. However, to develop CAR-T cells, autologous cells must be taken from the
patient, modified in the lab, and administered back to the patient. This process is long, expensive,
and could lead to some of the side effects listed above[37][33].
Furthermore, CAR-T cells are able to proliferate and survive for an indeterminate number
of days after administration. As a result, patients undergoing CAR-T therapy could achieve
remission from their cancer, but the effector cells remain in their system[12]. This could be
beneficial for tumor surveillance and preventing relapse, but in the absence of which, the CAR-T
9
cells could induce the side effects listed above. This extended lifespan has provoked researchers
to find a mechanism to downregulate or kill the T cells after the patient achieves remission. Such
mechanisms include the use of suicide genes[39], or inhibitory CAR-T cells that can spare
healthy cells[40].
1.5 Advantages of CAR-NK Therapy
Several CAR-T therapies that target CD19 and BCMA have already been approved in the
treatment of hematologic malignancies[41, 42], but due to the limitations listed above, NK cells
may be another lucrative route for immunotherapy, without some of the side effects commonly
associated with CAR-T therapy. Unlike T cells, NK cells do not increase the likelihood of graft
versus host diseases (GvHD)[1]. In fact, the presence of donor NK cells decreases the likelihood
of graft versus host diseases[43]. Moreover, NK cells can prevent GvHD by eliminating the
recipient’s antigen presenting cells (APC)[44].
Although the number of CAR-NK clinical trials are limited, cytokine release syndrome
and neurotoxicity have not yet been observed[14]. Therefore, engineering CARs into NK cells
may be a safer route for immunotherapy.
Moreover, CAR-modified NK cells have also been researched as a therapy to non-cancer
targets, such as human immunodeficiency virus (HIV) and Epstein-Barr virus (EBV).
Another benefit of CAR-NK cells is the potential for an “off-the-shelf” therapy[45].
Unlike CAR-T therapy, which requires autologous cells from the patient, CAR-NK cells can be
10
allogeneic, taken from a healthy donor, modified in the lab, and administered to multiple
patients[27].
One major difference between CAR-T cells and CAR-NK cells is their lifespan. While
CAR-T cells can undergo clonal expansion and persist after activation, CAR-NK cells have
proven to have a shorter lifespan in vivo[46]. This is both a benefit and a drawback. CAR-NK
cells’ short lifespan could limit their cytotoxicity and require more infusions for the patient.
Paradoxically, a shorter lifespan could protect the patient from long term side effects of CAR
treatment, like B cell aplasia and hypogammaglobulinemia[46, 47].
11
Chapter 2 - A Universal CAR-NK Cell Targeting HIV-1 GP-160
2.1 Abstract
Over the last 30 years, researchers have made exciting strides in the fight against the
HIV/AIDS epidemic. Currently, pre exposure prophylactic medications (PrEP) are available to
vulnerable populations to prevent initial infections. HIV functions by attacking a patient's
healthy T cells, incorporating its genetic information into the host cell, and proliferates until the
host cell lyses. This cycle repeats while the patient’s immune system continues to
deteriorate[48]. Therefore HIV patients are further susceptible to diseases[49], fungal
growths[50], and cancers[51] that are rare in healthy patients.
One of the features of HIV that makes it so difficult to treat is the virus’s mutability and
diversity. HIV-1, the more common strain, has 4 groups, M, N, O, and P. Moreover, within the
most common group, M, are 9 more distinct subtypes, A, B, C, D, F, G, H, J, and K. Within
subtype A, are subsubtypes A1-A4, and within F are F1 and F2. Each subtype can vary
genetically up to 35%, each one expressing unique extracellular glycoproteins on the cell
surface[52]. The virus’ diversity can be attributed to its ability to mutate under pressure applied
by the host immune system. This hurdle has prevented a broad spectrum vaccine or cure for HIV .
To overcome the diversity of HIV-1 surface glycoproteins, we have created universal
CAR-NK cells that are specific for the hapten, 2,4-dinitrophenyl (DNP) that is conjugated to
glycoprotein specific antibodies. This creates an adaptor molecule that bridges the infected cell’s
glycoprotein to the cytotoxic CAR-NK cell. Previous research already established an αDNP
12
CAR-NK cell that consists of an EF1 promoter, αDNP small chain variable fragment (scFv),
CD28 transmembrane domain, and CD28 and CD3 𝜁 intracellular signaling domains[2].
To expand on this work and increase the cytotoxicity of our CAR-NK cells, we have
created three more αDNP CARs with varying promoters and intracellular domains. We have
concluded that our novel CAR-NK cell, consisting of an EF1 promoter, αDNP scFV , CD28
transmembrane domain, NKG2D, 2b4, and CD3 𝜁 intracellular domains is a superior
second-generation CAR-NK cell that demonstrates a cytotoxicity rate of nearly 100% when
incubated with gp160
pos
cells and DNP-conjugated antibodies.
2.2 Introduction
2.2.1 Human Immunodeficiency Virus and current treatment
Human Immunodeficiency Virus (HIV) is a retrovirus that affects 38 million patients
worldwide[53]. HIV-1 is a virus responsible for the death of 675,000 Americans since the start of
the HIV/AIDS epidemic in 1981[53]. The virus’ ability to attack, destroy, and evade the immune
system is the reason there is currently no cure or vaccine for HIV .
Despite a 17% decrease, due to the COVID-19 pandemic, an estimated 30,635 new
diagnoses took place in 2021 in the United States alone. While HIV infections in the United
States are largely carried by gay and bisexual men, roughly 80%[54], internationally, the disease
is split evenly between males and females[55]. HIV is also a social injustice issue - in the United
States, the disease disproportionately affects people of color, 42% of patients are African
American, 27% are Hispanic/Latino, and 26% are white. This disparity contributes to the stigma
carried by HIV/AIDS, conflating social issues such as racism, homophobia, transphobia, and
poverty[54].
13
In the last thirty years, researchers have made exciting strides to offer HIV patients a
more hopeful prognosis. Pre-exposure prophylactic medications are offered to high risk
individuals to prevent an initial infection. Anti-retroviral therapies (ART) are able to suppress the
virus’s replication, allowing patients to achieve a viral load so low it cannot be detected in a
diagnostic exam. However ART treatment is not permanent, and if treatment is terminated, the
virus will resume replication[56]. Therefore, one problem persists - there is no absolute cure for
HIV . Once infected with this virus, a patient will remain infected for their entire life. The
following project focuses on creating a CAR-NK cell that is able to detect and eradicate HIV
infected cells, hoping to one day liberate patients from this disease.
One of the hurdles to overcome in the fight against HIV/AIDS is the virus’s mutability
and diversity. Within HIV-1 alone there are four subgroups (M, N, O, P), and within the M
subgroup there are nine subtypes[52]. Each variant of HIV produces distinct glycoprotein160 on
the surface of infected cells. Glycoprotein 160 (gp160) is a dimer consisting of gp120 and
gp41[57]. These proteins are recognized by broadly neutralizing antibodies (bNAbs), but there is
no single antibody that can recognize every variant of gp160[58]. This incentivizes the creation
of a universal therapy that can target all epitopes of HIV-1 without creating an individual therapy
for each and every variant. We have created a CAR-NK cell that recognizes 2,4-dinitrophenyl
(DNP). By conjugating DNP onto pre-existing bNAbs, these antibodies now function as adaptor
molecules, bridging the infected cells and the cytotoxic CAR-NK cells[2].
14
2.2.2 Anti-DNP Chimeric Antigen Receptor Natural Killer Cells
NK92mi cells were selected as the effector cells for CAR modification because they are a
highly cytotoxic natural killer cell line that have been used in early clinical trials of
CAR-modified NK cells[45, 59, 60].
To recognize DNP, an anti-DNP scFv was created and connected to a hinge domain,
transmembrane domain, and several intracellular signaling domains. A number of different
CARs were used and tested throughout this project in order to confirm which CAR was the most
efficient - yielding the most cytotoxic yet specific effect. Previous research established a novel
𝛼 DNP-CD28TM-CD28-CD3 𝜁 universal CAR-NK cell that exhibited promising cytotoxicity and
specificity for mimic HIV infected cells. To expand on these results and amplify the CAR-NK
cell’s cytotoxic signal, we have created three more CARs with an 𝛼 DNP scFv, and the
intracellular domains NKG2D and 2b4. CD28 and CD3 𝜁 are signaling domains that are
endogenous to T cells. Since NKG2D and 2b4 are endogenous to NK cells, they could
hypothetically serve as stronger signaling domains in a CAR-NK cell[61, 62].
The bNAbs used for this project are 10-1074, PG16, and 3BNC117. Of note, each
antibody used targets a different epitope location on the gp160 glycoprotein. In order from
proximal to distal, 3BNC117 targets CD4bs, 10-1074 targets the V3 loop, and PG16 targets the
V1/V2 loops[57, 58]. Hence, another aim of this project is to determine if the binding location of
the antibody relative to the target cell is correlated with the cytotoxicity of the CAR-NK cell.
15
2.2.3 Generation of Target Cells
To test the efficacy of our generated CAR-NK cells, proper target cells need to be
selected. HEK293 cells were chosen as the negative control targets. To mimic infected T cells, a
group of HEK293 cells were transfected with pConBgp160-opt, a vector that contains the full
length of gp160 subtype B[63]. After successful transfection, the HEK293-gp160 cells express
gp160 on their cell surface without actually carrying HIV RNA thus increasing the safety of
these experiments. To select for HEK293-gp160
pos
cells, Geneticin (G418) was added to the cell
16
media, eliminating the HEK293-gp160
neg
cells. To increase the density of the gp160 proteins, the
cells were sorted by fluorescence-activated cell sorting (FACS). We verified the expression of
gp160 on the HEK293-gp160 cells by staining them with VRC01 and a PE-conjugated antibody.
2.3 Results
2.3.1 Genetic Engineering of NK Cells to Express anti-DNP CAR
2,4-dinitrophenyl is a biocompatible chemical compound that can be easily conjugated
onto antibodies, and anti-DNP scFv sequences are readily available for use in CAR designs[64,
65]. Our anti-DNP CARs have an N-terminal HA tag, allowing us to verify CAR expression.
Next, they have the anti-DNP scFv extracellular domain, a CD28 transmembrane domain
anchoring the receptor into the NK cell membrane, next, each CAR being tested has a different
combination of intracellular domains.
Moreover, previous research used the promoter UBC1 to stimulate CAR expression[2].
Therefore, we also used the promoter EF1 to determine if it would lead to a superior CAR-NK
cell. Hence, the four CAR-NK cells used during this project are as follows:
1. UBC1-𝛼 DNP scFv-CD28TM-CD28-CD3 𝜁 referred to as UBC1
2. EF1-𝛼 DNP scFv-CD28TM-CD28-CD3 𝜁 referred to as EF1
3. EF1-𝛼 DNP scFv-CD28TM-NKG2D-2b4-CD3 𝜁 referred to as 2D-3 𝜁 4. EF1-𝛼 DNP scFv-CD28TM-NKG2D-2b4-CD28-CD3 𝜁 referred to as 2D-28 𝜁 17
Their schematic design is outlined in Figure 4. CAR design 2: EF1- 𝛼 DNP
scFv-CD28TM-CD28-CD3 𝜁 (referred to as EF1) has already been studied as a potential
CAR-NK cell[2]. Unlike other intracellular domains being tested in NK cells, NKG2D is
receptor endogenous to NK cells[62]. Therefore, adding them into a CAR-NK cell may increase
the efficiency of the NK cells, and amplify the cytotoxic signal upon activation of the receptor.
Similarly, 2b4 is another receptor present on certain CD8+ cytotoxic T cells as well as all NK
cells, so adding its domain onto CARs could also increase the efficacy of the CAR-NK cells[62].
Cell line #3, referred to as 2D-3 𝜁 , only possesses CD3𝜁 , while cell line #4, referred to as
2D-28 𝜁 , has CD28 and CD3 𝜁 .
To construct the CARs, the individual domains were purchased and PCR’d in the lab,
then an overlap PCR was used to assemble the domains in the correct order. The gene fragments
were then digested and inserted into a pFUW lentiviral vector. Next, we used that lentiviral
vector to produce lentiviral particles by transfected HEK293T cells.
After extracting the lentivirus, we transduced NK-92mi cells. This cell line secretes and
grows in interleukin-2 (IL-2)[66]. Since the CARs are engineered with an HA tag on the
N-terminal, we are able to verify the success of the transduction protocol by staining the cells
with an anti-HA tag followed by a PE-conjugated antibody.
18
19
2.3.2 Redirection of CAR-NK Cells Toward HEK293-gp160
pos
Cells
To conjugate DNP onto the antibodies, we used N-(2,4-dinitrophenyl)-6-aminocaproic
acid N-succinimidyl ester. Successful conjugation was verified with an SDS-PAGE and a
western blot. The number of molecules per antibody was found by measuring absorbance at 280
nm (antibodies) and 360 nm (DNP). We found that each antibody had an average of two DNP
moieties conjugated to them.
IFN-ɣ is a cytokine secreted by NK cells after their receptors bind their ligand. IFN-ɣ
secretion can be directly correlated to cytotoxicity[22]. To determine which cell line is the most
cytotoxic, we conducted an enzyme linked immunosorbent assay (ELISA), and tabulated the
IFN-ɣ secretion of each (Figure 5).
Each target cell, 293 and 293-gp160
pos
, was stained with each DNP conjugated antibody
(2.5 nM), and then incubated with each effector cell at a 1:1 ratio. 293-gp160
pos
cells, incubated
with 10-1074-DNP, caused the effector cells CAR-NK-2D-3𝜁 to produce 784 pg/mL of IFN-ɣ -
the highest amount of IFN-ɣ, comparatively. When the target cells, 293-gp160
pos
, were incubated
with the antibody PG16-DNP, CAR-NK-2D-3 𝜁 produced 424 pg/mL. The lowest amount of
IFN-ɣ was produced when the target cells, 293-gp160
pos
were incubated with 3BNC117-DNP -
the effector cells, CAR-NK-2D-3𝜁 produced 216 pg/mL (Figure 5).
Following the ELISA assay, we conducted a cytotoxicity assay to discern the most
cytotoxic CAR-NK cell. Using the same DNP conjugated antibodies, each CAR-NK was
incubated with a mix of each target cell. After 8 hours, flow cytometric analysis was used to
identify the surviving cells. We found that 2D-3𝜁 continued to be the most cytotoxic cells. At an
20
antibody concentration of 2.5 nM of 10-1074, PG16, and 3BNC117, 2D-3𝜁 demonstrated a
cytotoxicity of 96%, 95%, and 90%, respectively (Figure 6B). Despite low IFN-ɣ secretion in the
ELISA assay, 2D-28𝜁 was able to kill over 60% of 293-gp160
pos
cells when incubated with
10-1074 and PG16, and roughly 20% of 293-gp160
pos
cells when incubated with 3BNC117.
Conversely, EF1 yielded promising results in the ELISA assay, but when tested in the
cytotoxicity assay, did not exhibit proportional cytotoxicity results. Lastly, UBC1 did not
produce noteworthy IFN-ɣ, and did not exhibit notable cytotoxicity either.
21
22
Due to UBC1’s lackluster performance in the ELISA and cytotoxicity assays, it was
removed from the following cytotoxicity assays. The previous cytotoxicity assay was conducted
at a 5:1 ratio of effector cells to HEK293-gp160
pos
cells. To determine the toxicity of our
effector cells at different ratios with the targets, we conducted more cytotoxicity assays,
at a 1:1, 2.5:1, 5:1, and 10:1 ratio. Since 10-1074 was correlated with the highest
cytotoxicity, this was the only antibody we used for these assays, keeping the
concentration consistent at 2.5 nM each time.
23
We tested EF1, 2D- 3𝜁 , and 2D-28𝜁 . We used no antibody as a negative control, isotype,
and 10-1074. HEK293 and HEK293gp160
pos
were stained with Alexa 647 and CFSE
respectively, then incubated with the aforementioned antibodies. After antibody staining, they
were incubated with a ratio of effector cells, from 1:1, 2.5:1, 5:1, to 10:1 for 8 hours. The
surviving cells were determined using flow cytometric analysis, contour maps and cytotoxicity
percentages are included below (Figure 7).
Evidently 2D-3 𝜁 continues to outperform the other cell lines, and its cytotoxicity
increases with the E:T ratio. EF1 cytotoxicity also increased with the E:T ratio. However 2D-28 𝜁
seemed to plateau around 5:1 to 10:1. While it still demonstrated its cytotoxic effects, it did not
perform as well as 2D-3𝜁 (Figure 7B).
24
25
26
27
28
EF1-NKG2D-2b4-CD3𝜁 EC50 Cytotoxicity Assay
Since previous cytotoxicity assays proved EF1-NKG2D-2b4-CD3 𝜁 to be the most
efficacious 𝛼 DNP CAR-NK cell, we conducted another cytotoxicity assay to deduce the EC50
value using three antibodies at various concentrations. Starting at 5nM of 10-1074, pg16, and
3bnc117, and doing a serial dilution down to 6.4 x 10
-5
nM, we found the EC50 values to be
0.0586 nM, 0.1141 nM, and 0.1857 nM, respectively (Figure 8A).
At antibody concentrations greater than 5 nM, the cytotoxicity started to decrease, likely
due to steric hindrance from an excess of antibodies coagulating the target cells.
29
30
31
2.4 Discussion
The diversity and mutability of HIV is one of the largest obstacles to overcome in the
search for a cure. Patients now have access to pre-exposure prophylactic medication that can
prevent initial infection and anti-retroviral therapy that can suppress HIV replication[52].
However, patients are still weighed down by the constant need for medication, social stigma, and
the lack of an absolute cure. This incentivizes finding a therapy that can eliminate HIV infected
cells, including latent reservoirs that can give rise to further viral proliferation.
Due to this diversity, creating a unique therapy for each subgroup of HIV may not be
plausible. Instead, we have taken pre-existing gp160 specific bNAbs and conjugated DNP
molecules onto them. We then created CAR-NK cells that are specific to DNP and tested out
each one in a series of experiments to determine which is the most cytotoxic and specific.
We started with 4 CAR-NK cells (UBC1, EF1, 2D-3𝜁 , and 2D-28𝜁 ), as well as NK plain
as a negative control. All the CAR-NK cells had relatively similar expressions of their relative
CAR receptors, save for 2D-28𝜁 , which struggled to maintain higher density of its CAR.
Next, we conducted an ELISA assay to determine the IFN-γ production of each cell line.
This is a useful assay to determine the cytotoxicity of each cell line as IFN-γ is a cytokine that
leads to apoptosis of target cells and recruits and activates other immune cells. 2D-3 𝜁
outperformed the other CAR-NK cells in the ELISA assay, producing over 700 pg/mL when
incubated with HEK293-gp160
pos
and 10-1074-DNP. The second highest producer of IFN-γ was
UBC1, the preexisting CAR-NK cell. However, when tested in cytotoxicity assays, EF1 did not
32
perform as strongly as 2D-3𝜁 . This implies that in vivo EF1 could induce cytokine release
syndrome while not being as efficacious as 2D-3𝜁 .
33
Chapter 3 - Enhancing Specificity of anti-CD19 CAR-NK Cells By
Targeting HLA-DR Loss
3.1 Abstract
Despite the efficacy of the immune system, some cancers are able to coexist with the
immune system and are hardly immunogenic. This is because cancer cells share most of their
DNA, an estimated 99.9%, with their genome of origin[4]. Therefore, they are too similar to the
host’s healthy cells for immune cells to recognize them. This similarity is also what obstructs
researchers from finding effective solutions for aggressive malignancies. However, some cancers
overexpress certain antigens on their surface, and this can be exploited to target the malignant
cells more specifically[3]. By creating a synthetic chimeric antigen receptor (CAR) that targets
these overexpressed surface proteins and genetically engineering these receptors into immune
cells, we can eliminate tumor cells using the preexisting cytotoxicity and efficiency of naturally
occurring T cells (CAR-T)[25] and Natural Killer cells (CAR-NK)[1].
CAR-T therapy is a novel individualized immunotherapy that has shown promising
results in the last decade. While the treatment is aggressive, it has shown to be a lucrative option
for patients with relapsing hematologic malignancies[67]. FDA approved CAR-T cells are
specific for the tumor associated antigens CD19[68] and BCMA[69]. CD19 is a surface protein
that is overexpressed on some B cell leukemias and lymphomas. However, since CD19 is also
present on healthy B cells, patients undergoing CAR-T therapy are vulnerable to on-target,
off-tumor effects, leading to dangerous side effects[10]. The side effects are severe enough to
warrant seeking out a solution to increase the treatment’s specificity to only the cancer cells.
34
There are several proposed methods to accomplish this goal, here we have opted to create a
second receptor on our CAR-NK cells that deactivates the cell upon binding HLA-DR. HLA-DR
is a surface protein that is downregulated on some cancer lines.
All the CAR-NK cells have the same activating CAR, and we have created six inhibitory
CARs to determine which option is strong enough to extinguish the activating CARs signal upon
binding HLA-DR. We have successfully produced a CAR-NK cell that exhibits high cytotoxicity
when incubated with CD19
+
HLA-DR
neg
cells, and low cytotoxicity when incubated with
CD19
+
HLA-DR
+
cells.
3.2 Introduction
3.2.1 CD19 Expression in B Cell Malignancies
Despite the diversity and efficiency of the mammalian immune system, cancerous cells
are able to coexist with the immune system without eliciting an immune response. The reason for
this is the lack of tumor specific antigens that are unique to the malignant cells. However, some
cancers, particularly B-cell malignancies, begin to overexpress certain surface antigens, such as
CD19.
CD19 makes a promising target for cancer therapy, as its expression is limited to B cells.
This limits the likelihood of on-target/off-tumor effects. Of the approved CAR-T therapies, both
target CD19 and have proven effective against relapsing cancers that have not responded to other
treatments.
35
3.2.2 CD33 Expression in Myeloid Cell Malignancies
CD33 is a tumor associated antigen that is upregulated on acute myeloid leukemia
cells[70]. Like other cancer types, part of the difficulty in finding adequate treatment is the
discovery of a TAA that is overexpressed on AML cells[71]. The current standard of care is
chemotherapy and stem cell transplants[72]. Despite recent progress, patient prognosis remains
poor, with less than 5% of geriatric patients surviving for five years[73].
Up to 90% of AML cells express CD33 and CD123 on their surface[74]. Therefore, we
can target this cell protein when designing a CAR-NK therapy for patients[75]. CD33 is the
target of gemtuzumab ozogamicin - the current mainstream antibody drug conjugate for
treatment of AML. However, due to CD33’s limited expression and slow internalization, this
treatment has demonstrated limited success[76].
Therefore, CD33 could potentially be used as a target for CAR-NK therapy, as its
expression is limited to activated T cells and some natural killer cells as well as healthy B
cells[77].
3.2.3 Targeting HLA-DR Loss to Enhance Specificity of CAR Therapy
CAR-T cells that target CD19 have been effective as a new immunotherapy against B cell
malignancies. However, these potent cytotoxic cells have no ability to distinguish between
healthy B cells and malignant cells, due to healthy B cells also expressing CD19 on their surface.
Multiple routes have been suggested to alleviate or prevent on-target, off-tumor cytotoxicity.
Engineering CAR cells with suicide genes could decrease their lifespan[39], allowing healthy B
cells to replenish their numbers after CAR-T treatment is over. Adding a second activating CAR
is another potential route to increasing specificity - the CAR-T cells only activate after both
36
CARs have bound their ligands. However this would require finding a second tumor associated
antigen that is specific enough to cancer cells so as not to target healthy cells.
Another option to increase specificity of CAR cells is engineering an inhibitory CAR, or
“down-switch” into the immune cells that - upon binding its ligand - shuts down the cells
cytotoxicity[78]. To do this, we need to identify a surface antigen that is present on healthy cells
and absent on cancer cells.
Major histocompatibility complexes (MHC) I and II are surface proteins that present
peptides to the extracellular matrix. MHC I is present on all nucleated cells, and presents
fragments of intracellular proteins[79] to CD8+ T cells. MHC II is present on antigen presenting
cells such as macrophages, dendritic cells, and B cells. They may also be present on atypical
antigen presenting cells like basal cells, mast cells, and eosinophils[17]. There are five different
isotypes of MHC II - HLA-DM, HLA-DO, HLA-DP, HLA-DR, HLA-DQ[80].
However, in certain aggressive cancers, HLA-DR is downregulated or absent[15, 81].
Loss of HLA-DR is associated with lower T cell infiltration and higher patient mortality[82].
This makes HLA-DR an attractive option to target for an inhibitory CAR (iCAR). HLA-DR
deficiency has been documented in up to 34% of AML patients[83] and up to 33% in diffuse
large B-cell lymphoma[84], leading to poorer patient outcomes[85].
37
3.3 Results for CD19+ cancer cells
3.3.1 Engineering aCD19 aHLA-DR iCAR-NK Cells
Previous research established an iCAR-NK cell that consisted of a CD28-CD3 𝜁
activating receptor that recognizes CD19. Its inhibitory CAR consists of a PD1 intracellular
domain that, when bound to HLA-DR, inhibits the activity of the activating CAR. In order to
38
expand on this research, we have created 5 more CAR constructs to test this concept. The
inhibitory signaling domains that we tested are TIGIT, PD1, NKG2A and dimerized NKG2A in
different combinations with each other to determine which one is strong enough to extinguish the
activating CARs signal (Figure 10A).
We chose TIGIT because unlike PD1, it is naturally occurring in endogenous natural
killer cells and could elicit a better inhibitory response in NK cells. Likewise, NKG2A is a
member of the NKG2 family of natural killer cell proteins that functions to inhibit the cell’s
cytotoxicity
5
. Naturally occurring NKG2A dimerizes upon binding its ligand, therefore, we also
tested diNKG2A, to determine if it would elicit more inhibition than its monomer.
We created the iCARs by obtaining their individual genes and completing a PCR protocol
on each one. We then connected them in tandem using an overlap PCR and inserted them into
pFUW vectors. Next, we created lentiviral particles using HEK293T cells. These lentiviruses
were used to transduce NK92-mi cells. The single CAR-NK cell, possessing only an activating
CAR, was transduced once. All the dual iCAR-NK cells were transduced twice, once for each
CAR. To test the success of our transduction protocol, the CARs were designed with an HA tag
on the activating CAR and a Flag tag on the inhibitory CAR on each N-terminus. We verified
expression of the receptors using flow cytometry (Figure 10B). Evidently, all iCAR-NK cells
expressed their activating receptors at the same density as each other. However, while we sorted
the dual CAR-NK cells to achieve the same density several times, they continued to express
slightly different densities of their respective inhibitory receptors.
To confirm the percentage of dual iCAR-NK positive cells, Figure 10E shows the results
of using flow cytometric analysis. NK Plain served as a negative control, not expressing either
39
receptor. CD19 Single expresses only the activating receptor and 99.4% of cells tested positive.
All dual αCD19 αHLA-DR iCAR-NK cells are between 89.0 to 99.5% double positive. To
achieve this, they were enriched by magnetic cell sorting. To determine if the CAR density is
correlated to the CAR-NK cell’s specificity and cytotoxicity, two cell line’s
αCD19+αHLA-DR-PD1 and αCD19+αHLA-DR-TIGIT were sorted at different densities and
given corresponding names (Figure 10 B-E). αCD19+αHLA-DR-PD1 3rd and
αCD19+αHLA-DR-PD1 4th each have the same average number of activating receptors on their
surface. However, PD1-3rd has a medium amount of inhibitory receptors per cell, whereas
PD1-4th has the highest. Conversely, αCD19+αHLA-DR-TIGIT (high) expresses very few
activating CARs and a high amount of inhibitory CARs per cell. αCD19+αHLA-DR-TIGIT
(low) expresses a lower number of activating CARs and a much higher amount of inhibitory
CARs per cell. If these four aforementioned cell lines express varying cytotoxicity based on the
results of the ELISA and cytotoxicity assays, we can determine if the inhibitory CAR density
plays a role in the specificity of the overall dual iCAR-NK cell.
40
41
42
3.3.2 aCD19 aHLA-DR iCAR-NK Inhibition
Previous research already established the iCAR-NK cell αCD19 αHLA-DR PD1 - a dual
CAR-NK cell that can distinguish between CD19
+
HLA-DR
+
and CD19
+
HLA-DR
neg
[86]. Here,
we have constructed five novel cell lines to determine if their specificity is superior to that of
αCD19+αHLA-DR-PD1.
NK92-mi, the cell line used to express the CARs, secretes IFN-γ upon activation.
Therefore, we first conducted an ELISA assay and measured the resulting IFN-γ production. We
used the target cell k562 - which does not express CD19 or HLA-DR - as a negative control.
k562-CD19 served as a positive control that should hypothetically activate all CAR-NK cells due
to its expression of CD19 and lack of HLA-DR. k562-CD19-HLA-DR expresses both CD19 and
HLA-DR, so if the dual iCAR-NK cells are successful, they will limit their activation upon
binding HLA-DR (Figure 11A).
We conducted the ELISA assay at a ratio of 3:1 effector to target cells. The target cells
were plated at 50,000 cells per well and the effector cells were added at 150,000 cells per well.
They were left to incubate for 4 hours before the supernatant was removed off the plate and
tested for IFN-γ.
Evidently, all effector cells expressed low levels of IFN-γ when incubated with k562, less
than 200 pg/mL per well. Next, all the CAR-NK cells activated when incubated with
k562-CD19. aCD19 single released the most IFN-γ, at 859 pg/mL and aCD19-diNKG2A
released the least amount of IFN-γ, at 490 pg/mL. When incubated against
k562-CD19-HLA-DR, all the cells except aCD19-aHLA-DR diNKG2A demonstrated at least
some degree of inhibition. However, aCD19-aHLA-DR-TPA (CD19-TPA) stood out as the best
43
performer amongst the cell lines. CD19-TPA expressed the least amount of non-specific
background killing - only producing 93 pg/mL, whereas CD19 Single produced 200 pg/mL.
Against k562-CD19, CD19-TPA produced 545 pg/mL, which was on the lower end of the
production spectrum compared to the other five cell lines. Against k562-CD19-HLA-DR,
CD19-TPA produced 228 pg/mL, exhibiting an inhibition rate of 58% (Figure 11B).
CD19-PD1-4th and 3rd secreted 625 and 708 pg/mL when incubated with k562-CD19,
respectively. When incubated with k562-CD19-HLA-DR, they secreted 451 and 438 pg/mL - an
inhibition rate of 28% and 38%, respectively (Figure 11B).
44
While the ELISA assay proves that the cells secrete IFN-γ upon binding their activating
ligand, it does not prove that the target cells die upon iCAR-NK cell activation. To determine the
cytotoxicity rates of each iCAR-NK cell, we conducted a cytotoxicity assay (Figure 12). Target
cells were stained with cFSE and were incubated at a 3:1 effector to target ratio for 8 hours. After
incubation, the effector cells’ CD56 surface proteins were stained with APC and analyzed via
45
flow cytometry. Consistent with the ELISA assay, the CAR-NK cells demonstrate the lowest
cytotoxicity when incubated with k562. When incubated with k562-CD19, the iCAR-NK cells
performed similarly, demonstrating between 70 to 85% cytotoxicity. However, when incubated
with k562-CD19-HLA-DR, αCD19+αHLA-DR-TPA stood out as the most specific dual
iCAR-NK cell. It exhibits the least cytotoxicity when incubated with k562 cells, roughly 5%,
high cytotoxicity against k562-CD19, 70%, and the most self-inhibition against
k562-CD19-HLA-DR, 60% cytotoxicity - the lowest cytotoxicity against k562-CD19-HLA-DR
compared to the other iCAR-NK cells. It even outperformed the previously developed
αCD19+αHLA-DR-PD1 iCAR-NK cell.
Figure 12B shows the resulting contour maps analyzed after the 8-hour incubation period.
Each NK was stained with APC that bound to their CD56 surface protein. Endogenous Natural
Killer cells differentiate into CD56
bright
and CD56
dim
cells. CD56
bright
cells readily secrete
cytokines, whereas CD56
dim
cells play a role in antibody mediated cytotoxicity[5].
46
47
48
49
3.4 Results for CD33+ cancer cells
3.4.1 Engineering aCD33 aHLA-DR iCAR-NK Cells
Following the 𝛼 CD19+𝛼 HLA-DR dual iCAR-NK cell experiments, we found that the
most specific and effective inhibitory receptor was 𝛼 HLA-DR-IgG4hinge-TIGIT-PD1-NKG2A
(TPA). Therefore, to determine if this iCAR is also effective with other activating CARs, we
created three new cell lines. The first is a single CAR-NK cell, expressing only an activating
CAR - 𝛼 CD33-CD8hinge-CD28TM-CD28-CD3 𝜁 . The next two cell lines express the same
aforementioned activating CAR with different inhibitory CARs. The first dual iCAR-NK cell,
referred to as 𝛼 CD33-TPA, has the same inhibitory CAR as the corresponding 𝛼 CD19 dual
iCAR-NK cell (𝛼 HLA-DR-IgG4hinge-TIGIT-PD1-NKG2A). The second dual iCAR-NK cell,
referred to as 𝛼 CD33-PD1, has the same inhibitory CAR as the corresponding 𝛼 CD19 dual
iCAR-NK cell (𝛼 HLA-DR-IgG4hinge-PD1).
The development and engineering of our 𝛼 CD33 iCAR-NK cells was largely similar to
that of the 𝛼 CD19 iCAR-NK cells. We acquired the gene fragments of each domain, and
amplified them by PCR. To connect them, we used an overlap PCR and custom primers. We
verified the PCR product length using an agarose gel and inserted the gene fragment in pFUW
vectors and transformed NEB competent E. coli cells. After collecting the colonies, verifying
their transformation with gel electrophoresis, and sending the positive colonies DNA for
sequencing, we created lentiviral particles by combining the gene product and packaging
plasmids with HEK293T cells. After collecting the viruses and transducing NK92-mi cells, we
stained the transduced cells with an anti-HA tag and PE-conjugated secondary antibody to
identify the activating CAR, and APC to identify the inhibitory CAR (Figure 13). We used flow
50
cytometry to analyze CAR expression and to sort the cells and ensure they have proportional
expression of each receptor. Originally, we had two populations of the 𝛼 CD33-PD1 iCAR-NK
cells. The one with higher expression of both receptor, and overall more varied expression on
each cell (Figure 13B), is named 𝛼 CD33-PD1(high). However, the lower population,
𝛼 CD33-PD1(low) displayed expression that was more proportional to 𝛼 CD33-TPA, allowing for
more accurate comparison between the two cells. Therefore, for the following experiments, only
𝛼 CD33-PD1(low) was used and represented the 𝛼 CD33-PD1 cell line.
𝛼 CD33 single expressed a lower number of activating receptors per cell, 𝛼 CD33-PD1
expressed relatively more activating receptors per cell than 𝛼 CD33 , and 𝛼 CD33-TPA expressed
more than 𝛼 CD33-PD1. Conversely, 𝛼 CD33-PD1 expressed more inhibitory receptors, than
𝛼 CD33-TPA (Figure 13E).
51
52
3.4.2 aCD33 aHLA-DR iCAR-NK Inhibition
To determine if the 𝛼 CD33 iCAR-NK cells exhibit notable specificity and inhibition, we
tested them against the target cells molm13 and molm13-HLA-DR. Molm13 cells are a cell line
established from a patient with relapsing acute monocytic leukemia - a subtype of acute myeloid
leukemia
18
. Molm13 cells are CD33
+
and HLA-DR
neg
. When incubated with the engineered
CAR-NK cells, all three cells should activate upon binding Molm13’s CD33 proteins.
Molm13-HLA-DR is a molm13 cell line that has been transduced to express HLA-DR.
When incubated with our CAR-NK cells, 𝛼 CD33 should still activate, but 𝛼 CD33-PD1 and
𝛼 CD33-TPA should exhibit decreased cytotoxicity as they bind HLA-DR.
We started with an ELISA assay to detect the IFN-γ secretion of each cell line. The
effector cells were incubated at a 3:1 ratio with the target cells, 50,000 target cells to 150,000
effector cells. After a four-hour incubation period, the supernatant was removed and tested for
IFN-γ.
As predicted, 𝛼 CD33 Single activated when incubated with molm13 and
molm13-HLA-DR alike, secreting 299 and 243 pg/mL. 𝛼 CD33 - TPA displayed more activation
with molm13, secreting 487 pg/mL, and secreted 80% less IFN-γ when incubated with
molm13-HLA-DR, roughly 72 pg/mL (p<0.001). 𝛼 CD33 - PD1 exhibited similar IFN-γ
production to 𝛼 CD33 single when incubated with molm13, 251 pg/mL, and inhibited itself
when incubated with molm13-HLA-DR, 91 pg/mL, inhibition of 63% (p<0.001).
53
However, IFN-γ secretion is not necessarily proportional to cytotoxicity rates. Therefore,
we followed up with a cytotoxicity assay (Figure 15). Molm13 and molm13-HLA-DR were
54
stained with CFSE and incubated with the effector cells - 50,000 target cells and 150,000 effector
cells - for eight hours. After which, the effector cells were stained with APC bound to their CD56
surface proteins.
Consistent with the ELISA assay, 𝛼 CD33-TPA exhibits the most cytotoxicity when
incubated with molm13, roughly 93%. When incubated with molm13-HLA-DR, 𝛼 CD33 -TPA
limited its cytotoxicity down to 73% (Figure 15A). 𝛼 CD33 -PD1 demonstrated similar
cytotoxicity to 𝛼 CD33 Single, when incubated against both molm13 and molm13-HLA-DR.
Therefore, like with the 𝛼 CD19 group of dual iCAR-NK cell lines, TPA creates a more specific
and cytotoxic iCAR-NK cell.
55
56
57
3.5 Discussion
While approved CAR therapy has proven to be successful for patients with relapsing
hematologic malignancies, the on-target on-tumor side effects have been a limiting factor in the
overall success of this treatment. Because of the aggressive nature of CAR-T cells, patients are
left with B cell aplasia that causes hypogammaglobulinemia and are subsequently susceptible to
infection. This creates a need to develop CAR therapy that is able to distinguish between healthy
cells and cancerous cells.
Here, we have developed six 𝛼 CD19-𝛼 HLA-DR-CAR-NK cells that activate upon
binding CD19 and deactivate after binding HLA-DR. CD19 is a transmembrane glycoprotein
present on all B cells and some dendritic cells. It is also expressed in certain B cell malignancies
such as non-hodgkin’s lymphoma, and acute lymphoblastic leukemia. Its role as a biomarker of
B cell malignancies makes it an ideal candidate to target for CAR therapy. In fact, 𝛼 CD19
CAR-T therapy have already been approved by the FDA and used in cancer therapy since 2007.
These approved therapies have proven to be successful in treating patients with relapsing ALL.
However, on-target off-tumor effects continue to be an obstacle for clinicians to overcome.
In an attempt to decrease the likelihood of collateral damage, we have created inhibitory
CAR-NK cells that inhibit their own activity after binding HLA-DR. HLA-DR was chosen as the
inhibitory ligand because it is downregulated in some cases of aggressive cancers such as
Hodgkin’s lymphoma, AML, diffuse large B cell lymphoma, and CML. Because of HLA-DR’s
role in activating the adaptive immune system, lack of HLA-DR is associated with lower T cell
infiltration, and higher patient mortality[82, 87].
58
The activating CAR that was used through this project consisted of 𝛼 CD19 scFV , a CD8
hinge and CD28 transmembrane domain, a CD28 costimulatory domain, and a CD3 𝜁 activating
domain. The single CAR-NK cells are able to eliminate the target cells, k562-CD19 at a rate of
90%. All the dual iCAR-NK cells were able to meet similar cytotoxicity levels when incubated
with k562-CD19. However, all CAR-NK cells had high nonspecific background killing when
incubated with k562 alone, except 𝛼 CD19-𝛼 HLA-DR-TPA, a third generation iCAR-NK cell
whose inhibitory CAR receptor consists of an 𝛼 HLA-DR scFV , an IgG4 hinge, and the
intracellular domains of TIGIT, PD1, and NKG2A.
This cell line had the least amount of nonspecific background killing of k562, 70% killing
of k562-CD19, albeit the lowest percentage of the group, and 55% killing of
k562-CD19-HLA-DR. Judging by the ELISA assay, this cell line also demonstrated the most
inhibition compared to the other iCAR-NK cells of the group, producing 500 pg/mL of IFN-γ
when incubated with k562-CD19 and 200 pg/mL of IFN-γ with k562-CD19-HLA-DR. This
equates to an inhibition rate of around 60%.
Another iCAR-NK cell line with notable activation and inhibition is
𝛼 CD19-PD1-NKG2A. Similar to 𝛼 CD19-TPA, this line consists of the same activating CAR,
and an inhibitory CAR consisting of an 𝛼 HLA-DR scFV , an IgG4 hinge, and the intracellular
domains of, PD1, and NKG2A. 𝛼 CD19-PD1-NKG2A demonstrated low nonspecific background
killing when incubated with k562. It also had high cytotoxicity, roughly 80% with k562-CD19,
and 70% with k562-CD19-HLA-DR. Based on the ELISA assay, this cell line produced 800
pg/mL of IFN-γ when incubated with k562-CD19, then 400 pg/mL with k562-CD19-HLA-DR,
showing an inhibition rate of 50%.
59
Similar to 𝛼 CD19-PD1-NKG2A, 𝛼 CD19-diNKG2A-PD1 expressed low nonspecific
killing, high IFN-γ production with k562-CD19, and lower production with
k562-CD19-HLA-DR. In the cytotoxicity assay, it demonstrated the least amount of non-specific
killing, and killed around 80% of the target cells. However, it failed to inhibit its own activation
when incubated with k562-CD19-HLA-DR cells. diNKG2A-PD1 is distinct from PD1-NKG2A
because of its dimerized NKG2A domain. Endogenous NKG2A dimerizes upon binding its
ligand, therefore theoretically, using diNKG2A would more strongly resemble the inhibition of
an endogenous NK cell.
Therefore, of the six cell lines we have created that could prevent on-target off-tumor
effects, three of them were successful. Mainly, 𝛼 CD19-𝛼 HLA-DR-TIGIT-PD1-NKG2A stood
apart as a promising iCAR-NK cell. It did not exhibit nonspecific killing, but high cytotoxicity
with the target cells, and inhibited itself when bound to HLA-DR.
Our next project tests the cytotoxicity and specificity of iCAR-NK cells that are specific
for CD33 positive, HLA-DR negative target cells. CD33 is a sialoadhesin molecule expressed by
myeloid cells. Since 80% of AML patients express higher levels of CD33, it is also used as a
diagnostic marker for AML. Roughly 10-20% of B and T cell malignancies anomalously express
CD33 as well.
Our single 𝛼 CD33 CAR-NK cell was able to kill molm-13 cells at a rate of 88%, and
molm13-HLA-DR at 85%. As predicted, there was insignificant inhibition.
𝛼 CD33-𝛼 HLA-DR-PD1 performed similarly to the single 𝛼 CD33 CAR-NK cells in the
cytotoxicity assay. However, when measuring only IFN-γ, 𝛼 CD33-𝛼 HLA-DR-PD1 demonstrated
self-inhibition at a rate of 60%. Just like the 𝛼 CD19 branch of our project,
60
𝛼 CD33-𝛼 HLA-TIGIT-PD1-NKG2A outperformed the other dual CAR-NK cells, implying the
longer CAR, with the added costimulatory domain created a more precise and specific cytotoxic
cell.
Our initial goal was to create an iCAR-NK cell that maintains its ability to eliminate CD19
or CD33 positive cancer cells, but is also able to downregulate its own cytotoxic activity when it
binds to HLA-DR. CD19 is a biomarker of several malignancies, including B cell lymphoma,
acute lymphoblastic leukemia, and hodgkin’s lymphoma. CD33 is a biomarker of acute myeloid
leukemia. CAR-T therapy has been a successful, albeit aggressive, treatment for patients with
relapsing hematologic malignancies. But it is not without severe side effects that include B cell
aplasia, a serious condition with negative health outcomes for years post-treatment. Aggressive
cancers are able to downregulate HLA-DR expression to evade the patient’s immune system, a
phenomenon associated with lower patient survival. Therefore, lack of HLA-DR can be
exploited to create a CAR-NK cell that is able to kill CD19 or CD33 positive cells, but inhibits
its own cytotoxicity when it binds HLA-DR.
Several methods of decreasing side effects of CAR therapy have been proposed, such as
suicide genes[39], duo CAR cells (with 2 activating receptors), or inhibitory CAR cells. We
propose that the latter option, adding an inhibitory CAR, would be the most successful, as this
will increase the specificity of the cells while maintaining their cytotoxicity.
Here, we have shown that the inhibitory receptor 𝛼 HLA-DR-TIGIT-PD1-NKG2A is able
to downregulate the activating receptor’s signal. Both the 𝛼 CD19 iCAR-NK cells, and the
𝛼 CD33 iCAR-NK cells had minimal nonspecific killing, high cytotoxicity against k562-CD19
and molm13, respectively, and then downregulated the most when incubated with HLA-DR
61
positive cells. Their performance even surpassed that of the preexisting iCAR-NK cell
𝛼 CD19-𝛼 HLA-DR-PD1, with less nonspecific killing, and increased specificity and inhibition.
62
Chapter 4 - Conclusion
Human Immunodeficiency Virus is an extremely mutable and diverse disease that
currently has no universal cure. High risk populations can take pre-exposure prophylactic
medications (PrEP), and infected patients have anti-retroviral therapy (ART) at their disposal, but
there still is no absolute cure or vaccine for HIV .
The reason for this is the virus’ breadth and diversity. Once infected, T cells express a
glycoprotein envelope, gp160, on their cell surface. However, the variety of these glycoproteins
prevents a universal cure from being developed. Patients who are infected with HIV can
sometimes produce broadly neutralizing antibodies that bind to different epitopes of the gp160
protein. The bNAbs used in this study were obtained at no cost from the NIH AIDS Reagent
Program. After acquiring four bNAbs and conjugating two DNP moieties to them, we have
essentially created an adaptor molecule that bridges the diverse gp160 proteins to a single
universal CAR-NK cell that is specific for DNP. Therefore, instead of creating a CAR-NK cell
for each individual variety of gp160, we can create a universal CAR-NK cell, and develop
adaptor antibodies to overcome the diversity of HIV .
Previous research established a universal CAR-NK cell - 𝛼 DNP-CD28TM-CD28-CD3 𝜁
that is able to detect DNP-conjugated antibodies that are bound to gp160 on the target cell’s
surface. Here, we have developed a second-generation 𝛼 DNP CAR-NK cell that consists of
𝛼 DNP-CD28TM-NKG2D-2b4-CD3 𝜁 . This cell line, with added natural killer cell specific
costimulatory domains, expressed strong cytotoxicity and specificity when incubated with
gp160
positive
cells and DNP-conjugated antibodies.
63
CAR-T cells have also been a groundbreaking new option for patients with relapsing
hematologic malignancies. CAR-T therapy combines the specificity of a B cell receptor, the
amplified signal of a synthetic receptor, and the natural cytotoxicity of endogenous immune
cells. 𝛼 CD19 CAR-T cells have allowed patients to achieve complete remission. However,
patients who experience complete remission also develop B cell aplasia - a condition classified
by the absence of naturally occurring B cells. Due to B cell’s role in producing antibodies,
patients with B cell aplasia also experience hypogammaglobulinemia and are susceptible to
infection, thus requiring intravenous immunoglobulin infusions.
B cell aplasia occurs because healthy B cells express CD19, the tumor associated antigen
that CAR-T cells target and kill. To overcome this side effect, CAR-T cells need to downregulate
themselves when they bind a healthy cell. To do this, we have engineered natural killer cells to
express an activating receptor 𝛼 CD19-CD8hinge-CD28TM-CD28-CD3 𝜁 and varying inhibitory
receptors that, upon binding HLA-DR, downregulate the cell’s activating signal.
Previous research established that an 𝛼 HLA-DR-IgG4hinge-PD1 inhibitory receptor can
accomplish this goal. To expand on this, we aimed to create iCAR-NK cells that exhibit less
nonspecific killing, maintain their cytotoxicity against cells that express CD19 or CD33, and
inhibit themselves when they bind HLA-DR. Our experimental iCAR,
𝛼 HLA-DR-IgG4-TIGIT-PD1-NKG2A accomplished this goal. Working in conjecture with either
𝛼 CD19 or 𝛼 CD33 CAR, TPA managed to downregulate the cells, decrease the production of
IFN-γ and overall exhibit lower cytotoxicity against cells that express HLA-DR.
To expand on these results, in vivo experiments need to be carried out to determine if TPA
iCAR-NK cells are able to selectively kill cancer cells and cause fewer overall side effects.
64
Additionally, some iCAR-NK cells did not demonstrate any enhanced specificity, such as
𝛼 HLA-DR-IgG4-diNKG2A. However, 𝛼 HLA-DR-IgG4-diNKG2A-PD1 and
𝛼 HLA-DR-IgG4-PD1-NKG2A demonstrated minor inhibition, implying that PD1 is a superior
inhibitory domain. 𝛼 HLA-DR-IgG4-TIGIT also demonstrated notable inhibition. Therefore, a
receptor that consists of 𝛼 HLA-DR-IgG4-TIGIT-PD1 might exhibit the same inhibition as
𝛼 HLA-DR-IgG4-TIGIT-PD1-NKG2A.
We chose NK cells to carry the CAR receptors because of the potential they hold as a
superior CAR therapy to the established CAR-T cells. While CAR-T therapy has been an
effective and promising treatment option for patients with hematologic malignancies, it is known
to cause serious side effects such neurotoxicity, cytokine release syndrome, and graft versus host
diseases. So far, natural killer cells have not caused any of the aforementioned side effects;
therefore, CAR-NK cells could be a more lucrative therapeutic option. Additionally, CAR-NK
cells have a shorter lifespan after infusion. This could stave off B cell aplasia, as the B cells
could replenish after the CAR-NK cells die off.
Moreover, CAR-T cells are engineered by extracting autologous T cells from the cancer
patient, genetically modifying them in the lab, and infusing them back into the same patient.
Despite this, patients could still begin to target the CAR-T cells[88]. Additionally, the time it
takes to produce a line of CAR-T cells, combined with the fact that each CAR-T batch can only
be given to a single patient extends the time it takes for a patient to receive treatment.
NK cells on the other hand, do not invoke GvHD[89, 90], and can be given to multiple
patients, creating the possibility for an off-the shelf therapy, decreasing the time it takes for each
patient to receive care. Additionally, autologous natural killer cells - taken from patients with
65
AML - are of a lower quality phenotype than natural killer cell lines. They have a decreased
ability to release cytokines or degranulate[91, 92]. Therefore using the NK92mi cell line to
continue CAR-NK studies may lead to shorter treatment times, lower cost, and higher quality of
therapy.
The potential drawbacks to dual iCAR-NK therapy are the possibility of malignant cells
responding to selective pressure by upregulating their HLA-DR to evade the iCAR-NK cells.
66
Chapter 5 - Methods
Cell Culture NK92mi cells were maintained in 1640-RPMI media with L-glutamine and
25 mM HEPES buffer and supplemented with 20% fetal bovine serum (FBS), 1 mM sodium
pyruvate, 1 mM non-essential amino acids, 0.5 mg/mL penicillin-streptomycin-glutamine, and
50 µM ꞵ-mercaptoethanol.
HEK293 and HEK293-gp160 cells were maintained with DMEM media. HEK293-gp160
were also supplemented with G418 (Geneticin) to select for HEK293-gp160
pos
cells.
K562, K562-CD19, K562-CD19-HLA-DR, molm13, molm13-DR cells were cultured in
1640-RPMI media with L-glutamine and 25 mM HEPES buffer and supplemented with 10%
FBS, 1 mM sodium pyruvate, 1 mM non-essential amino acids, 0.5 mg/mL
penicillin-streptomycin-glutamine, and 50 µM ꞵ-mercaptoethanol.
Lentivirus Production Parental vector was pFUW, and packaging plasmids consisted of
pVSV-G, p-RRE, and p-REV
CAR Construction Each fragment of the CAR receptor was acquired and cloned via
PCR. Overlap PCR was used to create a complete fragment, adding E.coli compatible restriction
sites to the ends. Gene fragment inserts were digested and added into E.coli vectors and cloned.
Successful E.coli clones were fermented and DNA was collected and sent for sequencing.
HEK293T cells were used for lentiviral transduction, and successful parental pFUW vectors
were transduced along with the packaging plasmids listed above. After 72 hours, lentiviruses
were collected and NK-92mi cells were transduced. Successful transduction was verified using
67
flow cytometry. Dual CAR-NK cells were transduced twice, this process was repeated for each
CAR.
Reagents Antibodies for flow cytometry were: HA-tag polyclonal rabbit antibody, and
F(ab’)2-donkey anti-rabbit IgG PE conjugated secondary antibody. APC anti-DYKDDDDK Tag
antibody, streptavidin PE-conjugate. IFN-γ ELISA kit purchased from Thermo Fisher Scientific,
Retronectin from Takara Biosciences. RPMI, DMEM, HEPES, PSG, FBS, MEN-NEAA,
Sodium Pyruvate purchased from Thermo Fisher Scientific
Flow Cytometry Staining and sorting performed with BD LSRII flow cytometer (BD
Biosciences). Analysis performed with FlowJo 7.0 software. Cytotoxicity assay analysis
performed on Attune NxT Flow Cytometer (Thermo Fisher)
Cytotoxicity Assay for 𝛼 DNP CAR-NK Cells Each target cell, 293 and 293-gp160
pos
were stained with each DNP conjugated antibody (2.5 nM): isotype control-DNP, 10-1074-DNP,
PG16-DNP, and 3BNC117-DNP. After 20 minutes of incubation, the target cells and antibody
were combined in a 96-well plate, 50,000 of each cell, 100,000 total. Next, 50,000 effector cells:
NK control, CAR-NK-UBC1, CAR-NK-EF1, CAR-NK-2D-3 𝜁 , and CAR-NK-2D-28 𝜁 , were
added to each well. After an 8-hour incubation, the plate was spun down at 1300 rpm for 3
minutes, and the cells were stained with anti-CD56 APC monoclonal antibody at a 1:100 dilution
and Aqua Dead Cell Stain at a 1:500 dilution for 30 minutes. The results were read using the
Attune NxT Flow Cytometer (Thermo Fisher) and interpreted using FlowJo. This experiment
was repeated with different effector:target ratios: 1:1, 2.5:1, 5:1, 10:1.
68
Cytotoxicity Assay for 𝛼 DNP CAR-NK-2D-3 𝜁 EC50 Curve Target cells, 293 and
293-gp160
pos
were stained with each DNP conjugated antibody at varying concentrations from
5nM, to 6.4 x 10
-4
nM. After 20 minutes of incubation, equal parts of the target cells and
antibody were added to a 96-well round-bottom plate, 50,000 of each per well. Next, 50,000
effector cells: CAR-NK-NKG2D-2b4-CD3𝜁 were added to each well. Additionally, a negative
control well, with 50,000 of each target cell, no antibody, and CAR-NK-NKG2D-2b4-CD3 𝜁
were added at a 1:1 ratio with the target cells. 8 hours later, the plate was spun down at 1300 rpm
for 3 minutes and the cells were stained with anti-CD56 APC monoclonal antibody at a 1:100
dilution and Aqua Dead Cell stain at a 1:500 dilution for 30 minutes. The results were read using
the Attune NxT Flow Cytometer (Thermo Fisher) and interpreted using FlowJo.
Cytotoxicity Assay for 𝛼 CD19 and 𝛼 CD33 CAR-NK Cells Each well contained
150,000 target cells, either k562, k562-CD19, k562-CD19-HLA-DR, molm13, or
molm-13-HLA-DR and 50,000 CAR-NK cells. K562-CD19, and k562-CD19-HLA-DR
contained luciferin, while the remaining target cells did not, and therefore required 2 uM cSFE
dye. After staining, target cells and effector cells were incubated at a 1:3 ratio in a round
bottomed 96-well plate for 8 hours. The plate was then spun down at 1300 rpm for 3 minutes,
and the cells were stained with anti-CD56 APC monoclonal antibody at a 1:100 dilution and
Aqua Dead Cell Stain at a 1:500 dilution for 30 minutes. The results were read using the Attune
NxT Flow Cytometer (Thermo Fisher) and interpreted using FlowJo.
ELISA Assay for 𝛼 DNP CAR-NK Cells Each target cell, 293 and 293-gp160
pos
, was
stained with each DNP conjugated antibody (2.5 nM), and then incubated with each effector cell
at a 1:1 ratio. After a 4-hour incubation period, the supernatant was removed from the 96-well
69
round-bottomed plate and an ELISA kit (Thermo Fisher) was used to quantify the amount of
IFN-y secreted. The fluorescence of the resulting plate was read using the BioTek Synergy H1
Machine. Results were interpreted using Microsoft Excel and GraphPad Prism.
ELISA Assay for 𝛼 CD19 and 𝛼 CD33 CAR-NK Cells Each target cell, k562,
k562-CD19, k562-CD19-HLA-DR, molm13, and molm13-DR was incubated at a 1:1 ratio with
their effector CAR-NK cells. After a 4-hour incubation period, the supernatant was collected and
the IFN-y production was measured using an ELISA kit (Thermo Fisher) and the fluorescence
was measured using the BioTek Synergy H1 Machine. Results were interpreted using Microsoft
Excel and GraphPad Prism.
70
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78

Abstract (if available)

Abstract In spite of the potency of a healthy immune system, cancer and HIV infection remain difficult diseases to treat, and can progress before a diagnosis is made, potentially shortening patient survival outcomes. This is because immune cells do not have the tools to recognize these conditions. Here, we demonstrate how we created chimeric antigen receptors (CARs) that recognize the surface antigens of HIV or B cell leukemias and lymphomas, and engineered them into Natural Killer cells, effectively arming these cells to recognize and eliminate their target.

The first project is to engineer an enhanced anti-HIV CAR-NK cell. HIV-infected cells express a glycoprotein, gp160, on their surface. Previously, our lab created a universal anti-HIV CAR-NK cell that can kill HIV-infected cells through the use of adaptor molecules that connect gp160 to the CAR-NK cells’ receptor. In this thesis, in order to enhance the potency and cytotoxicity of CAR-NK cells, we utilized NKG2D, 2b4 signal domains, as well as CD28 and CD3𝜁 in the CAR designs. We found the anti-DNP-NKG2D-2b4-CD3z is the best design for anti-DNP universal CAR-NK system.

The second project is to engineer dual CAR-NK cells that can specifically target hematologic malignancies that overexpress certain surface proteins, known as Tumor Associated Antigens (TAA), and under-express HLA-DR. Previous researchers have created synthetic receptors, CARs, that recognize these TAAs and engineered them into autologous cytotoxic T cells (CAR-T). While this treatment has been effective, it causes life-threatening side effects that could be avoided by engineering CARs into Natural Killer cells (CAR-NK) instead of T cells as NK cells are shown not to elicit these conditions. Our goal was to create an inhibitory CAR that can downregulate the CAR-NK cell’s cytotoxicity upon binding HLA-DR, thus limiting the CAR-NK cell’s activity to only cancer cells. We found the combination of TIGIT, PD-1 and NKG2A signal domains had the strongest inhibitory effect. We tested our new iCAR designs in both anti-CD19 and anti-CD33 CAR-NK cells.

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Asset Metadata

Creator Sharif, Deema Aya (author)

Core Title Enhancing the specificity and cytotoxicity of chimeric antigen receptor Natural Killer cells

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Degree Conferral Date 2023-08

Publication Date 07/21/2023

Defense Date 07/19/2023

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

Tag B-cell malignancies,CAR-NK,CAR-T,chimeric antigen receptor,HIV,immuno-oncology,immunotherapy,leukemia,lymphoma,natural killer cells,OAI-PMH Harvest

Format theses (aat)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Haworth, Ian (committee chair), Culty, Martine (committee member), Xie, Jianming (committee member)

Creator Email dasharif@usc.edu,dsharif25@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC113282076

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Source 20230721-usctheses-batch-1071 (batch), University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

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