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Generation and characterization of fully human anti-CD19 chimeric antigen receptor T (CAR-T) cells for the treatment of hematologic malignancies
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Generation and characterization of fully human anti-CD19 chimeric antigen receptor T (CAR-T) cells for the treatment of hematologic malignancies
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Content
Generation and Characterization of Fully Human Anti-CD19
Chimeric Antigen Receptor T (CAR-T) cells for the Treatment
of Hematologic Malignancies
By
Aliya Anvery
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In partial Fulfillment of the
Requirement for the Degree
MASTER OF SCIENCE
(Biochemistry and Molecular Biology)
December 2017
Copyright 2017 Aliya Anvery
ACKNOWLEDGEMENTS
I would like to present my heartfelt gratitude to my adviser Dr. Preet
Chaudhary for allowing me to be part of his laboratory and giving me to
opportunity to learn and complete my thesis. His continuous support,
encouragement and guidance towards my research project and thesis writing
is much appreciated.
I would also like to extend my thanks to all other members of Dr.
Chaudhary’s Lab for their generous help during my research. In particular, I
would like to thank Dr. Sunju Choi for being an excellent mentor and
teaching me molecular cloning techniques, Dr. Venkatesh Natarajan for his
careful review of my thesis and answering my questions, and Drs. Hittu
Matta and Ramakrishnan Gopalakrishnan for their guidance during the tissue
culture phase of my project. It was a pleasure to work with all of them.
Also, I would like to thank the members of my committee, Dr. Zoltan
Tokes and Dr. Judd Rice, for their time and kind support. I would like to
especially thank Dr. Zoltan Tokes for his ever care and involvement
through-out my two-year Master’s program.
Lastly, I would like to thank my family, especially my mother for
always believing in me and being there through the ups and downs of my
graduate studies.
ABSTRACT
Chimeric antigen receptor expression on T cells (CAR-T) is a cell mediated adoptive
immune therapy to treat cancer. It involves in vitro engineering of T cell receptors against
specific kinds of cancer antigens including carbohydrate and glycolipid tumor antigens.
CAR-T cells recognize cell surface tumor antigens in a MHC independent manner. This
makes CAR based therapy more effective than MHC dependent cancer antigen
recognition. A large number of CARs have been developed for targeting and killing both
hematological and non-hematological malignancies. CD19 is a cell surface cancer
antigen on B cells. Recently, clinical trial studies have shown efficacious targeting and
killing of CD19 expressing B cell lymphoma and B cell leukemia with anti CD19 CAR-T
cells. However, severe immunogenic reactions leading to clearance of CAR modified T-
cells have been observed in patients that were infused with anti- CD19 CARs because
these CARs have scFv fragments derived from murine monoclonal antibodies. Murine
antibody fragments not only limits persistence of CAR-T cells, but also lead to allergic
reactions in patients, such as anaphylactic reactions. Moreover, humanized CARs also
have some immunogenic reactions in patients. To counter these limitations, we have
generated an anti-CD19 CAR containing fully human scFv fragment from EUK5-13 fully
human anti-CD19 antibody. We demonstrated that this CAR can be expressed on the
surface of Jurkat T-cells, binds to CD19 antigen on both RAJI (B cell lymphoma) and
Bv173 (B cell leukemia) cancer cell lines and induce cytoplasmic signaling in Jurkart-
NFAT-GFP cells.
Abbreviations
Tumor-associated antigens (TAA)
Major histocompatibility complex (MHC)
Antigen presenting cells (APCs)
Chimeric antigen receptor (CAR)
T-cell receptor (TCR)
Human leukocyte antigen (HLA)
Hodgkin lymphomas (HL)
Non-Hodgkin lymphomas (NHL)
B-cell acute lymphocytic leukemia (B-ALL/ALL)
B-cell chronic lymphocytic leukemia (CLL)
Nuclear factor of activated T-cells (NFAT)
Interleukin-2 (IL-2)
Single chain variable fragment (scFv)
Variable region of heavy chain (vH)
Variable region of light chain (vL)
Transmembrane (TM) domain
Antibody (Ab)
Peripheral blood (PB)
Umbilical cord blood (UBC)
Bone marrow (BM)
Human embryonic stem cells (hESCs)
Natural killer (NK) cells
Human embryonic kidney (HEK) cells
Cytokine release syndrome (CRS)
Macrophage activation syndrome (MAS)
Tumor lysis syndrome (TLS)
Green fluorescent protein (GFP)
Coelenterazine (CTZ)
NanoLuciferase (NLuc)
Table of Contents
Introduction…………………………………………………………………………..1
Lymphoma…………………………………………………………….…..…..2
B-Cell Acute and Chronic Lymphocytic Leukemia………………………...…3
Components of Successful Immunotherapy with CARs……………………....3
Different Generations and Structural Design of CARs………………………..5
Fully Human CAR.…………………………………..………………………..7
Other Cells used to Express CAR.…………………………………...……......8
Delivery of CAR Constructs to Immune Cells with Lentiviruses………...….10
CAR-T Cell Therapy………………………………………………………....11
Limitations of CARs………………………………………………………....14
Objectives of Project……………………………………………………………….16
Materials and Methods………………………………………………….………….17
Generation of Lentiviral Anti-CD19 CAR Construct…………………….….17
Transfection/Virus Generation………………………………………….……18
Production of anti-CD19 scFv N-Luc Fusion Protein…...……………...……19
Viral Transduction……………………………………………………..….….19
In Vitro Binding Assay……………………………………………….……...20
Flow Cytometry for GFP Expression……………………………….…….….20
Results………………………………………………………………………..……...21
Construction of Fully Human Anti-CD19 CAR Plasmids…………..…….…21
scFv Nluc Fusion Protein ……………………...………….…………21
CAR with Cytoplasmic Signaling Domains……………...………….21
Binding Affinity of scFv Nluc Fusion Protein to CD19 Tumor Antigen…....23
RAJI Lymphoma Cell Line……………………………………….….24
BV173 Leukemia Cell Line…………………………………….……24
L428 Cell Line from Hodgkin's Lymphoma…………………………25
Detection of GFP Expression in CAR infected Jurkat Cells……………...….25
RAJI Lymphoma Cell Line………………………………………..…26
BV173 Leukemia Cell Line…………………………………….……27
Discussion…………………………………………………………………………..30
References……………………………………………………….…………..……..32
1
Introduction
The immune system is the body’s defense mechanism from both invasion of pathogens and
intrinsic bodily damage. Innate immune system is non-specific and immediately responses and
reacts to pathogens. It provides defense through skin, mucosal membranes and chemicals in the
blood. Innate immune cells present antigens and activate adaptive immune system. This latter
immune system leads to the production of B-cells and T-cells, special kind of white blood cells.
These cells provide a long-lasting immunity, hence, if similar attacks happen again, memory B-
cells and T-cells are stimulated and activated much quicker and provide specific immunity to the
pathogen (Alberts, B., et al. 2002). B-cells are white blood cells that mature in the bone marrow
and fight infections by producing antibodies. On the other hand, T-cells are white blood cells that
mature in the thymus and involve the activation of various cytokines, phagocytes and cytotoxic
T-cells and provide cell mediated immunity.
T-cells have cell surface receptors that can also recognize antigens presented on tumor cells
called tumor-associated antigens (TAA). Upon recognition of TAA, downstream signaling
cascade leads to tumor cell apoptosis. However, these tumor specific T-cells are rare and require
enrichment to effectively kill all tumor cells and prevent recurrence of tumor. Moreover, T-cell
receptors on the surface of T-cells bind to the antigen presented on major histocompatibility
complex (MHC) on antigen presenting cells (APCs) (Jensen, Peter E., 2005). That poses another
limitation of therapy through T-cell receptors and is called tumor escape mechanism. This
involves downregulation of MHC on the tumor cells making them invisible to T-cells.
Consequently, recent approach was developed called chimeric antigen receptor (CAR) cancer
therapy. CAR immunotherapy requires genetically engineering of the TAA-specific TCRs for
2
many different tumor antigens, expressing them in T-cells, and finally infusing them into
patients. Even though application of CAR is limited to cell surface antigens, CARs not only
overcome the limitation of scarcity of tumor specific T-cells, but also introduce HLA-
independent recognition of antigen that enables them to bypass tumor escape mechanism
(Sadelain, Michel, et al. 2013). CARs can work under any HLA backgrounds which overcomes
the barrier matching patient haplotype with TCR.
Lymphoma
Lymphoma is developed from clonal expansion of lymphocytes in the blood. Symptoms of
lymphoma include enlarged lymph nodes, fever, fatigue, weight loss and drenching sweats that
usually occur at night. Lymphoma is classified in two main categories: Hodgkin lymphomas
(HL) and the non-Hodgkin lymphomas (NHL) which are mostly B cell lymphomas and arise
from clonal expansion of B lymphocytes (Ramos, Carlos A., et al. 2014).
Drug-based and cell-based are the two types of immunotherapies for treating lymphoma.
Interferon and monoclonal antibodies are some examples of drug-based immunotherapies.
Monoclonal antibodies against tumor antigens are administered intravenously as drugs. Some of
the monoclonal antibody/drugs for NHL are Rituximab, Obinutuzumab and Ibritumomab
tiuxetan. These drugs are given to patients with chemotherapy or radiation and may also cause
some allergic reactions in patients (Scott, Andrew M., et al. 2012). On the other hand, cell-based
immunotherapies include chimeric antigen receptors (CAR), allogeneic hematopoietic stem cell
transplantation (allo-HCT), in vitro stimulation and reinfusion of autologous T-cells (Karmakar,
S., 2014). CD19 is a tumor antigen on B cell lymphomas and is a good CAR target used in
immunotherapy.
3
B-Cell Acute and Chronic Lymphocytic Leukemia
Both B-cell acute and chronic lymphocytic leukemia (B-ALL/ALL and CLL) are types of
leukemia that start with clonal expansion of CD5
+
CD19
+
B lymphocytes in the bone marrow and
then these cells migrate to the blood and spread to other parts of body (Rozman, Ciril, et al.
1995). Due to presence of CD19 on B lymphocytes, anti-CD19 CAR-T therapy is a good
approach for treating both ALL and CLL.
ALL is an aggressive form of B-cell leukemia. In CLL, B-cells can mature partly but not
completely; thus, their ability of fighting infection is diminished. CLL takes a long time to form
cancer and usually affects people over 50 years old; however, it is generally harder to cure than
acute leukemia (Rozman, Ciril, et al. 1995).
CLL can have either un-mutated or mutated rearranged immunoglobulin (Ig) genes. But
nonetheless, all cases of CLL have a common cellular origin and also a common mechanism of
malignant transformation (Rosenwald, Andreas, et al. 2001).
Components of Successful Immunotherapy with CARs
For successful clinical CAR-T therapy, CAR-T cells need to be able to traffic to the tumor site,
recognize and bind to the desired TAA, proliferate and activate downstream T-cell effector
signals to kill the target tumor cells (Topsent, Jerome, 2015). While performing all the functions
of targeting and killing cancer cells, these CAR-T cells should be able to escape suppression
from tumor microenvironment and persist in patients to eliminate residual tumor cell growth.
CAR-T-cells persistence can be achieved by stable integration of CAR construct with co-
4
stimulatory domains into the T-cell genome using lentiviral transduction techniques (Maude,
Shannon L., et al. 2014).
Before infusing CAR-T cells into patients, chemotherapy and radiation is given to patients for
lymphocyte depletion that would otherwise need cytokines and compete with T-cells for survival
and proliferation (Kochenderfer, J. N., et al. 2013). T-cell infusion occurs soon after conditioning
with chemotherapy and/or radiation.
Efficacious CAR-T-cells migration through the body to the desired antigen expressed sites
depends on chemokine secretion by tumors or TAA and the proper expression of chemokine
receptors on effector T-cells. It was shown that anti-GD2 CAR-T cells migration towards tumor
cells with GD2 antigen was enhanced because these CARs express CCR2b chemokine receptor
that recognize CCL2 chemokine secreted by GD2 presenting tumor cells (Craddock, J. A., et al.
2010).
Fig. 1. Schema of adoptive cellular therapy with CAR-T cells (Kochenderfer, J. N., et al. 2013).
The ex vivo cell processing takes 10 days. The lymphocyte-depleting chemotherapy regimen consists of
fluarabine and cyclophosphamide. All patients receive 25 mg/m
2
of fludarabine daily for 5 dsays. The
5
cyclophosphamide dose depends on the patient’s platelet count. A cyclophosphamide dose of 60 mg/kg daily
for 2 days is administered to patients with a blood platelet count of 100,000/ul or more. A cyclophosphamide
dose of mg/kg daily for 2 days is administered to patients with a blood platelet count between 75,000 and
99,000/ul. Patients with platelet counts less than 75,000/ul are not eligible for the clinical trial. Abbreviations:
CAR, chimeric antigen receptor; PBMC, peripheral blood mononuclear cell.
Different Generations and Structural Design of CARs
CARs have been evolved over time to optimize functionality and their long persistence in
patients. There are four generations of CARs (Smith, Aaron J., et al. 2016). First generation
CARs consist of scFv attached to cytoplasmic signaling domain (e.g., CD3-ζ or Fc receptor
[FCR]-γ chains) via a hinge region and a transmembrane domain. T-cell proliferation is limited
in first generation CARs because this lacks co-stimulatory domain(s). Therefore, second
generation of CARs accounts for the previous limitation of T-cell co-stimulation and
proliferation and modifies first generation CARs by incorporating T-cell costimulatory
molecules (e.g., CD28, OX40, 4-1BB) to CD3-ζ, which not only enhances proliferation of CAR
modified T-cells but also persistence in patients (Bridgeman, J. S., et al. 2014). Third generation
CARs have a second cytoplasmic co-stimulatory domain attached in tandem with the first one.
These co-stimulatory domains are CD28/OX40 or CD28/4-1BB and other domains like CD27
and CD134 can also be used (Fesnak, Andrew D., et al. 2016). Fourth and newest generation of
CARs have one co-stimulatory domain like the second-generation CAR, but has a nuclear factor
of activated T-cells (NFAT) associated with the intracellular region of CAR to signal
transcription of IL-2 genes (Fig. 2). IL-2 cytokine expression cassette to stimulate innate immune
response against cancer cells (Smith, Aaron J., et al. 2016). The benefits of adding additional co-
stimulatory domains in the third generations and having NFAT in the fourth generation over the
6
second-generation CARs are ambiguous at the moment. Due to not knowing clear benefits of
having different numbers of co-stimulatory domains attached to TM domain or having NFAT for
IL-2 expression, ideal design of a CAR is a challenge and needs to be evaluated based on the
specific tumor being treated. However, second and third generation CAR are the most prevalent
structure of CARs.
Antigen specificity of CAR is based on scFv fragments derived from vH and vL regions of a
specific antibody attached together via a linker. These single chain variable regions come either
from mouse monoclonal, humanized or fully human antibodies (Sommermeyer, D., et al. 2017).
For clinical purposes, most effective and safe CAR should be designed to maximize interaction
with tumor antigen and should be less immunogenic and minimize toxicity to patients.
Hinge region connects the extracellular antigen-binding domain of CARs to transmembrane
domain. It also provides flexibility to scFv orientation maximizing interaction with the antigen
(Fesnak, Andrew D., et al. 2016).
Transmembrane (TM) domain is a hydrophobic alpha helix that helps CARs to anchor to T-cell
or other cell surfaces such as Jurkat T-cells and Natural Killer cells. CD3-ζ is signaling domain
for transducing signals from CAR complex and activating the host cells for their downstream
functions such as releasing cytokines and tumor cell lysis.
7
Fig. 2. Chimeric antigen receptor design and evolution (Smith, Aaron J., et al. 2016)
The four generations of CAR T cells are depicted in a manner that emphasizes the differences in the
intracellular domain region of the CAR. It should be noted that OX-40 is also known as CD134 and that 4-
1BB is CD137 for the third generation. Two copies of the same costimulatory molecules are not used in third
generation CAR T cell design. Each generation increases in complexity concerning the addition of
costimulatory molecules or NFAT induced promoter for IL-12 transcription and later translation.
Fully Human CAR
scFv of CARs have been derived from non-human mouse antibodies and some of them are then
humanized by changing some amino acids at the binding region of scFv to mimic human scFv.
Although humanized scFv elicit lower immunogenicity than murine derived single chain variable
fragments, patients treated with CD19-CAR-T-cells with both murine derived and humanized
scFv have developed immune responses and that has caused T-cell clearance. Hence, this has
posed a big problem to the persistence of CAR modified T-cells in patients after infusion
(Bonifant, C. L., et al. 2016 and Sommermeyer, D., et al. 2017).
8
Therefore, there is a need of fully human scFvs to maintain T-cell persistence and subsequent
avoidance of relapses. Anti-human CD19 scFvs have been successfully developed and isolated
from human Ab/DNA-libraries with similar binding characteristics as scFv derived from mouse
(FMC63). Phage display libraries are utilized to study the interactions between antibodies and
tumor antigens and isolate the antibody with the best binding affinity to the antigen (Bazan, J., et
al. 2012). Some of the human scFvs showed improved in vitro functions against tumor cell lines
and CLL as compared to FMC63 and were also more efficient in eliminating lymphoma
xenografts in immunodeficient mice than the FMC63-CAR (Sommermeyer, D., et al. 2017).
EUK5-13 is one of a fully human antibody that can be used to study interactions between its
scFv and CD19 tumor antigen and subsequently can be used to develop a CD19 specific CAR.
The current data suggests that functional fully human CARs against an antigen such as CD19 can
potentially overcome the immunologic barriers that exist with CARs constructed from scFvs that
are not fully human in origin (Sommermeyer, D., et al. 2017). CARs with fully human scFv
suggest promising outcomes in future clinical trials and improve the longevity of CAR-T-cell
persistence and enhance their therapeutic efficacy in patients.
Other Cells used to Express CAR
Natural killer (NK) cells: NK cells are important lymphocytes of the innate immune system and
can be derived from peripheral blood (PB), umbilical cord blood (UBC), bone marrow (BM) and
human embryonic stem cells (hESCs). They are activated by cytokines and react quickly to the
site of infection, tissues from both pathogen-infected and malignant cells.
NK cells are CD56
+
and CD3
-
and are classified into two subsets, cytotoxic and
immunoregulatory. CD56
dim
CD16
+
NK cells are cytotoxic and account for 90% of natural killer
9
cells in PB. They can lyse pathogen infected or malignant cells in a HLA-independent manner.
Whereas, CD56
bright
CD16
−
cells are immune-regulatory and secrete cytokines such as interferon
to stimulate T-cells for a more efficient killing response (Mandal, Arundhati, et al. 2014). Similar
to CAR T-cells, NK cells are another candidate for adoptive immunotherapy (Guillerey, Camille,
et al. 2016).
Jurkat-NFAT-GFP T-cells: Jurkat cell are an immortalized line of human T-cells. These cells
in culture, compared to primary T-cells, have more rapid growth and stable characteristics.
Unlike primary T-cells, Jurkat cells are not cytotoxic and do not have the ability to kill tumor
cells. Despite being non-cytotoxic, Jurkat cells share other features with primary T cells and
allow expression of CARs on their surface and interaction with TAA. Therefore, these
immortalized cells are a good model for detecting successful expression of CAR on cell surface,
CAR interaction with target tumor cells, and down-regulatory signals produced by CAR
signaling domains by measuring GFP expression in Jurkat cells (Hooijberg, Erik, et al. 2000).
Jurkat-NFAT-GFP T-cells are engineered so that the IL-2 promoter with NFAT binding site is
cloned upstream of GFP gene. The interaction between CAR on Jurkat-NFAT-GFP T-cells and
tumor antigen activates NFAT pathway which activates transcription of GFP reporter gene (Fig.
3).
10
Fig. 3. Example of Jurkat cell activation Stecha, P., et al. 2015).
In this example, upon interaction of CAR on the surface of Jurkat cell with antigen on the desired tumor
cells, cytoplasmic signaling pathway is activated. NFAT promoter is turned on and its downstream GFP
reporter gene is expressed.
Delivery of CAR Constructs to Immune Cells with Lentiviruses
Genus, lentivirus belongs to Retroviridae family. They can integrate their genetic information
into host cell genome; hence are capable of stable and persistent expression of their genetic
information in the host. Lentiviruses are also highly efficient in infecting both dividing and non-
dividing cells with low immunogenicity. Therefore, lentiviral vectors are used for delivery of
CAR constructs into T-cells and other cells such as Jurkat-NFAT-GFP cells and for stable
expression of CAR in these cells. Lentiviruses used for delivering CAR constructs are
replication-defective. That means that these viruses can successfully integrate the gene they are
carrying into the host genome, but viral replication does not occur in host cells.
11
Transfection of lentiviral genes into host cells called packaging cells is achieved by dislocating
viral gene in three parts packaged into three separate vectors (Masoud, Nasri, et al. 2014). One
vector has 5’ LTR to drive expression of package genomic DNA or RNA, and two other
plasmids that code the structural and envelope proteins. Intact viruses are generated after
transfection and are collected from the supernatant. Now, these viruses have the ability of
introducing and integrating desired gene into host cell genome (Fig. 4).
Fig. 4. Lentiviral vectors as gene delivery system.
CAR-T Cell Therapy
CAR-T cell therapy’s efficiency depends on the affinity between CAR and the antigen epitope
and also the location of the epitope being recognized. Further events require activation of T cells
Packaging Cell/HEK
293FT
Gag/Pol + Env + 5’ LTR
CAR Plasmid
Lentivirus
with CAR
Plasmid
Primary
T-cells
Jurkat
T-cells
1
2
3
12
by phosphorylation of tyrosine-based activation motifs (ITAMs) present on the CD3-ζ domain in
the cytoplasmic part of the CAR. (Bridgeman J. S., et al. 2014). Co-stimulatory domains are
required for persistence and proliferation.
CD19 is a glycoprotein transmembrane stable cell-surface marker in B cells and hence B cell
malignancies (Fig. 5); therefore, CD19 is a good target for CAR-T therapy against B cell
lymphomas such as HL and NHL as well as B-cell leukemia such as ALL and CLL (Ramos,
Carlos A., et al. 2014). Anti-CD19 CAR-T cells with CD3 ζ activation domain and co-
stimulatory domains such as 4-1BB, OX40, CD28 and CD134 have shown to eliminate tumor in
clinical trials (Wang, Zhenguang, et al. 2017). Moreover, high remission rates of 70~94% have
been achieved in patients suffering with ALL with the use of CD19-specific CAR T-cells (Wang,
Zhenguang, et al. 2017). In a clinical trial using a second-generation, CD28-containing anti-
CD19 CAR for the treatment of B-cell ALL, of the 5 patients treated, all achieved complete
remission after lymphodepleting doses of cyclophosphamide (Ramos, Carlos A., et al. 2014).
CD19-CAR-T cells have received FDA approval for the treatment of B-ALL (in children) and
Diffuse Large B-cell lymphoma, a type of NHL.
However, currently murine based and humanized CARs are being tested in clinical trials and
cause patients to develop severe immunogenenic reactions leading to T-cell clearance (Bonifant,
C. L., et al. 2016). Persistence of CAR modified T-cell is important to avoid relapses. More
promising results addressing the issue of immunogenicity and lack of CAR-T cell persistence are
hoped to be achieved by CARs containing fully human scFvs.
13
Fig. 5. Consistent expression of CD19 during the development and differentiation of B cells (Mei, Henrik E.,
et al. 2012).
B cells emerge from hematopoietic stem cells (HSC) and acquire maturity in the bone marrow (BM). During
this process, CD19 expression starts at the stage of late pro-B cells and precedes that of CD20, starting in
immature B cells. CD19 and CD20 expression is maintained during B-cell differentiation and activation in the
periphery, while activation and differentiation into memory B cells may fine-tune expression levels. During
activation of B cells in lymphoid organs - such as the spleen and tonsil, resulting in their differentiation into
plasmablasts and finally plasma cells - CD20 expression is downregulated and subsequently lost. At the same
time, CD19 expression is partially downregulated but not extinguished. Being released into circulation, only
few plasmablasts express low amounts of CD20, while most have lost CD20 expression but express CD19.
After successful immigration into deposits in the BM or the lamina propria (LP), all plasma cells lack CD20,
while CD19 expression is maintained by one subset of tissue-resident plasma cells, while another subset
acquires a CD19
-
/CD20
-
phenotype. Other surface molecules such as CD27, CD38 and CD138 do not qualify
as therapeutic targets for global B-cell depletion approaches, as these are not continuously expressed
throughout B-cell differentiation.
14
Limitations of CARs
CAR-T therapy can lead to significant toxicity in patients. Some of the harmful toxic effects in
patients are, cytokine release syndrome (CRS), macrophage activation syndrome (MAS),
neurotoxicity, on-target off-tumor toxicity and tumor lysis syndrome (TLS) (Almasbak, Hilde, et
al. 2016).
CRS is due to immune activation that produces excessive amounts of cytokines such as
interferon-γ, IL-6 and IL-10. Symptoms of CRS include: cardiac dysfunction, high fever, nausea,
fatigue, anorexia, hypotension, renal impairment, hepatic failure, capillary leak, myalgia and
disseminated intravascular coagulation (Bonifant, C. L., et al. 2016). MAS is driven by high
levels of IL-6. Both CRS and MAS can be mitigated by anti-IL-6 antibodies.
When a CAR is developed and CAR-T cells are infused in patients, they target their specific
target without distinguishing whether the same target is on normal cells or the tumor cells. Anti-
CD19 CAR can also lyse normal B cells expressing CD19. This is called on-target off-tumor
toxicity (Fisher, J., et al. 2017). To mitigate this, normal B-cell depleted patients are given
monthly immunoglobulin replacement (Almasbak, Hilde, et al. 2016).
CAR causing allergic reactions are some more limitations of CAR-T therapy (Fig. 6). CARs that
have scFv fragments derived from murine monoclonal antibodies are the most immunogenic and
produce the most allergic responses in patients such as itchy rash, swelling of throat and tongue,
shortness of breath and vomiting. These allergic reactions are called anaphylactic reactions.
Humanized CARs pose lesser allergic responses as compared to murine monoclonal antibodies.
Whereas, fully human scFv is the least immunogenic and stays in the patient for longer amount
of time (Sommermeyer, D., et al. 2017).
15
Fig. 6. Toxicities of chimeric antigen receptor (CAR) T-cell therapy (Bonifant, Challice L., et al. 2016).
Depiction of reported/potential toxicities following the use of CAR T cells: insertional oncogenesis
(theoretical); neurological toxicity; “on-target, off-tumor” toxicity (engagement of target antigen on
nonpathogenic tissues); anaphylaxis/allergy (host reaction to foreign antigen expressed by the CAR T cell);
cytokine release syndrome (systemic inflammatory response following activation of CAR T cells). CRP, C-
reactive protein.
16
Objectives of Project
CAR-T cells immunotherapy is widely being investigated all over the world. Since the concept
of CAR engineered T-cells was initially proposed, several clinical trials were conducted in vivo
including clinical trials targeting CD19. Although they showed remarkable results, a number of
them failed due to immunogenicity and short persistence of CAR-T-cells in patients. The scFv of
CARs is generally derived from murine monoclonal antibodies that elicits anti-mouse responses
in humans leading to clearance of CAR modified T-cells after infusion. To combat this issue of
immunogenecity, humanized scFv fragments and CD19-specific CAR-T cells with co-
stimulatory domains combined to its cytoplasmic region have eased the issue of persistence to
some extent, but there is more that needs to be done to mitigate the issue of immunogenic
responses in patients. It is suggested that fully human antibody will be the best candidate to
diminish the problem of immunogenectiy and short-life of CAR-T cells in vivo. Previously, in
our lab, we have generated humanized scFv against CD19, but we are attempting to generate
anti-CD19 scFv derived from a fully human antibody, EUK5-13 to address the immunogenicity
problems in patients. The objective of this thesis is to generate a fully human version of anti-
CD19-CAR from EUK5-13 CD19 human antibody, express them on Jurkat T-cells and evaluate
its ability to bind to CD19 on both RAJI (lymphoma cells line) and Bv173 (leukemia cell line),
and to also evaluate Jurkat cells activation and downstream cytoplasmic signaling due to the
binding of this CAR to CD19.
17
Materials and Methods
Generation of Lentiviral CD19-CAR Constructs:
The pLENTI-Blast vector was derived from pLenti6v5gw_lacz vector (Invitrogen; ThermoFisher
Scientific) by removal of the LacZ gene. pLenti-MP2 was a gift from Pantelis Tsoulfas
(Addgene plasmid # 36097) and was used to generate the pLENTI-EF1α lentiviral vector by
replacing CMV promoter with human EF1α promoter using standard molecular biology
techniques. psPAX2 was a gift from Didier Trono (Addgene plasmid # 12260). The pLP/VSVG
envelope plasmid and 293FT-cells were obtained from Invitrogen (ThermoFisher Scientific).
The retroviral vector MSCVneo, MSCVhygro, and MSCVpac and the packaging vector pKAT
were obtained from Dr. Robert Illaria’s laboratory. The sequence of the scFV fragment encoding
a fully human monoclonal antibody against human CD19 was codon optimized using GeneArt
TM
software (Thermo Fisher Scientific) and gene-fragments encoding the optimized sequences
synthesized by GeneArt
TM
or Integrated DNA Technologies (IDT).
The gene fragment, T7NCD8-hCD19-EUK5-13-Mlu-xho-xba-pCDNA3R was used as a
template in pfu PCR reaction using custom primers and digested using NheI and MluI restriction
enzymes to get the inset of CD8SP-hCD19-EUK5-13 for fully human anti-CD19 scFv fragment.
Vectors, pLenti-EF1-Nhe-CD8SP-AF2-scFv-Mlu-GGS-Nluc-xho-FlagX4-Streptag-GGS-
8XHis-T2A-Pac and pLenti-EF1ΔXho-Nhe-CD8SP-CD19-mRO05-1-Mlu-MYC-CD8TM-
BBZ-XS-T2A-Pac were digested using NheI and MluI and large fragments from each were
purified. The purified inset was then ligated in each of these purified large fragments to generate
the two desired lentiviral constructs. Ligated products were transformed into Stbl3 competent
cells (Invitorgen) and colonies were screened using colony-PCR selecting CAR-CD19 positive
clones. Purification of plasmids from positive clones was done using plasmid prep (MIDI from
18
50ml O/N culture) following lab protocol. Two plasmids pLenti-EF1-Nhe-CD8SP-hCD19-
EUK5-13-scFv-Mlu-GGS-Nluc-xho-FlagX4-Streptag-GGS-8XHis-T2A-Pac and pLenti-
EF1ΔXho-NheCD8SP-hCD19-EUK5-13-Mlu-MYC-CD8TM-BBZ-XS-T2A-Pac respectively
were the resulting desired constructs confirmed by DNA sequencing.
Both the constructs were designed to have fully human anti-CD19 scFv derived from EUK5-13
fully human anti-CD19 antibody; however, the first one was generated to detect binding of scFv
to CD19 on both RAJI and BV173 cell lines and the second construct was created to evaluate the
expression and signaling of this scFv in Jurkat T-cells. Therefore, for the second construct, insert
was ligated in pLENTI-EF1α vector containing the hinge, transmembrane domain of CD8,
cytosolic domains of human 41BB (CD137) receptor, cytoplasmic domain of human CD3z, a 2A
ribosomal skip sequence and a cDNA encoding a puromycin resistance gene. The first construct,
on the other hand, lacked the transmembrane and cytoplasmic signaling domains attached to
CAR and instead had Nluc to detect binding.
Transfection/Virus Generation
To generate the CAR-encoding lentiviruses, HEK-293 FT-cells were first cultured overnight in
DMEM medium, then transfected with a mixture of 10 µg of the generated CAR lentiviral
plasmid, 7.5µg psPAX2 containing Gag, Pol, Rev, and Tat genes for virus packaging, and 2µg
pLP/VSVG for expression of the virus G glycoprotein using standard calcium chloride and 2x
HBS transfection reagent. Medium was changed at 24 h post transfection. Supernatant containing
anti-CD19 CAR-encoding lentiviruses was collected at 48 h and 72 h post transfection and
filtered through a 0.45µm filter to separate viruses from cell debris. The viruses were then
19
concentrated by ultracentrifugation at 18,500 rpm at 4°C for 2 hours and re-suspended in T-cell
medium. Viruses were stored at -80°C until needed.
Production of Fully Human Anti-CD19 scFv N-Luc Fusion Protein
This above procedure of lentiviral production was repeated for the first construct of fully human
CD19 scFv fused to NLuc except transfection in HEK-293 FT-cells was achieved without viral
packaging plasmids, psPAX2 and pLP/VSVG. Therefore, protein was collected from the
supernatant rather than lentiviruses at 48 h and 72 h. Lentiviruses were not needed as scFv-NLuc
fusion protein was only generated to test the binding of scFv to CD19 on RAJI (lymphoma) and
Bv173 (leukemia) cell lines and not to express in cells.
Viral Transduction
Preparation of Jurkat T-Cells: The Jurkat-NFAT-GFP cell line is a human T lymphocyte-based
cell line for analysis of NFAT pathway activation. This cell line is maintained using RPMI (with
10% FBS) media.
Transduction: Jurkat-NFAT-GFP cells were counted and 1.5 million cells were resuspended in
2ml of un-concentrated viral supernatant collected at 48 h and plated in a well of a 6 well plate. 8
µg/mL polybrene (PB) was added to the well. Cells were centrifuged, spinfection, at 1,800 rpm
at 32°C for 45 min and were left undisturbed at 37°C, 5% CO
2
overnight. After incubating
overnight, Jurkat-NFAT-GFP cells infected with CAR expressing lentivirus were selected with
300ng/mL of puromycin. Jurkat cells infected with CAR had Pac and got selected with
puromycin for 15-20 days in the incubator, whereas the rest of them died.
20
In Vitro Binding Assay with CD19 scFv-Nluc Fusion Protein
The target cell lines (RAJI, Bv173 and L428) were counted and volume was calculated to have 1
million cells. Among these cell lines, RAJI and Bv173 express CD19 while L428 are CD19-
negative. These 1 million cells were incubated overnight with anti-CD19 scFv-Nluc protein
containing supernatant. After extensive washes, NLuc assay buffer containing native
coelenterazine (CTZ) as NLuc substrate (30µl/well of native coelenterazine diluted in PBS) was
added to each well by an automatic dispenser in a well mode using a BioTek synergy H4 plate
reader and light emission as a measure of NLuc activity was measured. Cell lines with medium
alone instead of the scFv were used to monitor background non-specific binding. Negative
control experiment was set-up using the same scFv against a non-B cell line, L428, derived from
Hodgkin’s lymphoma that does not have CD19 on the surface.
Flow Cytometry of GFP Expression
Both target and Jurkat-NFAT-GFP cells were resuspended in RPMI medium. Then, 0.5ml of the
target cell lines and 0.5ml of Jurkat cells expressing anti-CD19 CAR were plated on a 24 well-
plate and incubated at 37°C for 22 hours. Negative controls were prepared the same way by
incubating target cell lines with Jurkat un-infected/parental cells. GFP expression in CAR-
positive cells was then acquired using FACSverse flow machine (BD biosciences).
21
Results
Construction of Fully Human Anti-CD19 CAR Plasmids
Fully human soluble scFv fused with N-Luc and CAR constructs both targeting human CD19
were generated using standard molecular cloning methods. The final constructs were designated
as:
pLenti-EF1-Nhe-CD8SP-hCD19-EUK5-13-scFv-Mlu-GGS-Nluc-xho-FlagX4-Streptag-GGS-
8XHis-T2A-Pac and,
pLenti-EF1ΔXho-NheCD8SP-hCD19-EUK5-13-Mlu-MYC-CD8TM-BBZ-XS-T2A-Pac
In both cases, complementary DNA sequence encoding the same fully human scFv derived from
EUK5-13 human antibody fused to CD8 signal peptide was PCR amplified from synthetic gene
fragments and cloned in frame with a human CD8 signal peptide. The first construct lacked the
hinge, transmembrane and all the cytoplasmic signaling domains and instead had GGS-NLuc,
other tags such as GGS-8XHis and a puromyson resistance gene with the human CD8 signal
peptide. The second construct is a fully human CAR construct where the scFv cloned with a
human CD8 signal peptide, a Myc epitope tag, hinge and transmembrane domain of human CD8,
the cytosolic domain of human 41BB (CD137) receptor, the cytosolic domain of human CD3z, a
2A ribosomal skip sequence and a cDNA encoding a puromycin resistance gene (Fig. 7).
22
Fig.7. A schematic representation of the fully human CD19-CAR construct: pLenti-EF1ΔXho-NheCD8SP-
hCD19-EUK5-13-Mlu-MYC-CD8TM-BBZ-XS-T2A-Pac
Generated plasmids were transformed into Stbl3 competent cells and plated on carbenicillin
plates. Colony PCR followed by agarose gel electrophoresis was used to detect positive
recombinant clones (Fig. 8). One of the positive clones of each construct was used for plasmid
isolation using standard MIDI-Prep method. DNA sequence was then confirmed by automated
sequencing.
scFV
Fully human
Anti-CD19
Nhe
scFV
5’LTR
Mlu
3
’
LTR
EF1α
Prom
Digestion with Nhe& Mlu
scFV
5’LTR
3
’
LTR
EF1α
Prom
Huluc63
SP
Fully human
Anti-CD19
Digestion
with Nhe&
Mlu,
Ligation
Pac
(500bp)
Pac
(500bp)
23
Fig.8. 8 colonies were picked for colony PCR. One primer specific for insert and the second primer specific
for vector was used. After gel electrophoresis, positive clones were detected and used for plasmid isolation.
Binding Affinity of anti-CD19 EUK5-13 scFv NLuc fusion protein to CD19 Tumor Antigen
Before generating a CAR using an scFv derived from the fully human EUK5-13 antibody, we
decided to determine the binding specificity of human EUK5-13 scFv to CD19. For this purpose,
scFv from EUK5-13 was expressed as a fusion protein with C-terminal NLuc (NanoLuc;
Promega). The fusion protein also carried Flag, Streptag and His tag for protein detection and
purification. Two cancer cell lines, RAJI derived from B-cell lymphoma and Bv173 derived
from B-cell leukemia both express CD19. Fully human anti-CD19 scFv fused with Nluc was
tested to bind to CD19 present on these cell lines (Fig. 9 A & B). In contrast, L428, a cell line
derived from Hogkin’s lymphoma, does not express CD19 and was used as a negative control to
confirm binding specificity of scFv to only CD19 (Fig. 9 C). Non-specific binding was
monitored with media alone. As shown in Fig 9A-C, RAJI and Bv173 cells showed significant
binding with EUK5-13-NLuc fusion protein, while L428 cells failed to do so.
24
A
B
GGSG-N-Luc –RAJI parental
GGSG-N-Luc –Bv173 parental
25
C
Fig. 9. Binding assay to detect the binding affinity of fully human anti-CD19 scFv to CD19 using RAJI and
Bv173 cells. (A & B) High luciferase activity showed successful binding of fully human scFv to CD19 on both
RAJI (B-cell lymphoma) and Bv173 (B-cell leukemia) cell lines. (C) Fully human scFv is specific to binding to
CD19 and so did not show luciferase activity/binding to L428 cell line. As predicted, medium alone with each
of the cell lines did not show significant luciferase activity. This further confirmed binding affinity between
fully human anti-CD19 scFv and CD19. RAJI cells showed more luciferase activity than Bv173 because from
previous studies it is shown that RAJI express more CD19 than Bv173 and thus more binding detected to
scFv.
Detection of GFP Expression in CAR Infected Jurkat cells
The fully human anti-CD19 CAR was tested for its ability to express on cell surface and activate
NFAT signaling in Jurkat T cells expressing GFP under an NFAT promoter when cocultured
with CD19-expressing RAJI and Bv173 target cells. Jurkat-NFAT-GFP cells (J-N-G) were stably
transduced with EUK5-13-scFv-BBz-PAC lentiviral construct and selected with puromycin. The
GGSG-N-Luc –L428 parental
26
cells were subsequently co-cultured with CD19-expressing RAJI and Bv173 cell lines.
Activation of Jurkat cells and NFAT pathway was detected by measuring GFP expression using
flow cytometry (Fig. 10 A-H) and (Table 1).
A
B
C
27
D
E
F
28
G
H
Fig. 10. Flow cytometry for testing binding of CAR expressed on Jurkat cells to CD19 and activation of
Jurkat-NFAT-GFP cells with measuring GFP expression. (A) Jurkat T-cells alone with media did not show
activation and GFP expression in P2 gate. (B) Jurkat cells expressing EUK5-13-based fully human anti-CD19
CAR showed very little activation of effector cells and GFP expression. (C) RAJI lymphoma cells alone for
background did not express GFP. (D) RAJI cells with Jurkat parental cells expressed some GFP suggesting
that something else other than CAR on Jurkat parental cells can have some interaction with either CD19 or
some other antigen on RAJI cells. (E) RAJI cells incubated with Jurkat-NFAT-GFP cells transduced with
lentivirus containing fully human anti-CD19 CAR showed CAR expression and its specific binding with
CD19 on RAJI cells and activation of Jurkat cells and a lot of GFP expression. (F) Bv173 leukemia cells alone
in media did not have GFP expression, as predicted. (G) Bv173 cells with Jurkat parental cells did not
activate Jurkat cells and did not express GFP. (H) Bv173 cells with Jurkat-NFAT-GFP cells transduced with
29
the lentiviral CAR construct expressed fully human anti-CD19 CAR on their surface and that CAR activated
downstream signaling in these Jurkat cells and expressed a lot of GFP.
Table 1. Data showing results of flow cytometry experiment
Events mean the number of cells that expressed GFP. It is very clear that a large number of our fully human
anti-CD19 CAR on Jurkat cells bound to CD19 on both RAJI and Bv173 cells and activated expression of
GFP.
Thus, the results from the table and the graphs confirm that the generated fully human CD19-
specific CAR was successfully expressed on Jurkat-NFAT-GFP cells and bound to CD19 on
both RAJI and Bv173 cells and activated downstream signaling in Jurkat cells.
30
Discussion
CD19 is a known antigen in B cell malignancies such as ALL, CLL and lymphomas. Chimeric
antigen receptors targeting CD19 have shown promising results in clinical trials and lead some
patients to have complete remission. However, thus far, anti-CD19 CARs have been derived
from murine monoclonal antibodies which have also been humanized; and even though there
have been efficacious results using these CARs, both of these CARs elicit immunogenic and
allergic reactions in patients and poses a problem to persistency of CAR-T cells in patients.
To overcome these shortcoming with the use of murine based and humanized CARs, we have
designed a fully human anti-CD19 scFv derived from EUK5-13, a fully human anti-CD19
antibody. A scFv of fully human nature will be less immunogenic than both murine and
humanized anti-CD19 CARs. Due to being less immunogenic, it will take a longer time for it be
cleared from the patients’ bodies and thus will have longer persistence.
We generated a CAR construct of fully human anti-CD19 scFv attached to a CD8
transmembrane domain, 4-1BB co-stimulatory domain and CD3-ζ signaling domain, and also a
soluble scFv-Nluc construct with the same scFv attached to GGS-NLuc and other tags such as a
GGS-8XHis tag. It was demonstrated that this scFv had a robust binding affinity to CD19 on
both RAJI (B-cell lymphoma) and Bv173 (B-cell leukemia) cell lines using scFv-NLuc fusion
protein. CAR construct with the transmembrane domain was then expressed in Jurkat-NFAT-
GFP cells to demonstrate expression of our fully human anti-CD19 CAR on the surface of Jurkat
cells and the activation of downstream signal in these Jurkat cells upon binding of CAR to CD19
on RAJI and Bv173 cell lines by detecting GFP expression. When CAR is expressed on the
surface of Jurkat cells and bound to the specific tumor associated antigen, CD19, only then it can
activate NFAT pathway and GFP expression in Jurkat cells. Jurkat-NFAT-GFP cells infected
31
with our fully human anti-CD19 CAR showed high GFP expression when tested its interaction
with CD19 on both RAJI and Bv173 cells.
The results confirmed a good interaction between the EUK5-13 derived fully human scFv and
CD19 and that this CAR can be expressed in Jurkat T-cells and is capable of activating
downstream functions in these cells. Based on these results, in future, we plan to express our
fully human anti-CD19 CAR in primary T-cells and demonstrate the activity of those CD19-
CAR modified T-cells to kill both RAJI and Bv173 cells in vitro and subsequently in vivo for
both B-cell lymphoma and B-cell leukemia. We also plan to develop next generation CARs
using different signaling domains based on EUK5-13 and comparing the in vitro and in vivo
activities of different fully human EUK5-13 CARs. We will then select the best CAR for human
clinical trials.
In conclusion, we have successfully generated a fully human CAR to target CD19, and have
observed its remarkable binding to CD19 and activation of downstream signaling in Jurkat T-
cells. We are hopeful that fully human CD19-specific CAR developed from EUK5-13 will
present a good treatment approach to B-cell malignancies and will also address the issues of
immunogenencity and T-cell clearance in vivo.
32
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Abstract (if available)
Abstract
Chimeric antigen receptor expression on T cells (CAR-T) is a cell mediated adoptive immune therapy to treat cancer. It involves in vitro engineering of T cell receptors against specific kinds of cancer antigens including carbohydrate and glycolipid tumor antigens. CAR-T cells recognize cell surface tumor antigens in a MHC independent manner. This makes CAR based therapy more effective than MHC dependent cancer antigen recognition. A large number of CARs have been developed for targeting and killing both hematological and non-hematological malignancies. CD19 is a cell surface cancer antigen on B cells. Recently, clinical trial studies have shown efficacious targeting and killing of CD19 expressing B cell lymphoma and B cell leukemia with anti CD19 CAR-T cells. However, severe immunogenic reactions leading to clearance of CAR modified T-cells have been observed in patients that were infused with anti-CD19 CARs because these CARs have scFv fragments derived from murine monoclonal antibodies. Murine antibody fragments not only limits persistence of CAR-T cells, but also lead to allergic reactions in patients, such as anaphylactic reactions. Moreover, humanized CARs also have some immunogenic reactions in patients. To counter these limitations, we have generated an anti-CD19 CAR containing fully human scFv fragment from EUK5-13 fully human anti-CD19 antibody. We demonstrated that this CAR can be expressed on the surface of Jurkat T-cells, binds to CD19 antigen on both RAJI (B cell lymphoma) and Bv173 (B cell leukemia) cancer cell lines and induce cytoplasmic signaling in Jurkart-NFAT-GFP cells.
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Anvery, Aliya
(author)
Core Title
Generation and characterization of fully human anti-CD19 chimeric antigen receptor T (CAR-T) cells for the treatment of hematologic malignancies
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Biochemistry and Molecular Biology
Publication Date
11/15/2017
Defense Date
10/24/2017
Publisher
University of Southern California
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Tag
B-cell acute lymphocytic leukemia (B-ALL/ALL),B-cell chronic lymphocytic leukemia (CLL),chimeric antigen receptor (CAR),coelenterazine (CTZ),green fluorescent protein (GFP),Hodgkin lymphomas (HL),human embryonic kidney (HEK) cells,NanoLuciferase (NLuc),non-Hodgkin lymphomas (NHL),nuclear factor of activated T-cells (NFAT),OAI-PMH Harvest,single chain variable fragment (scFv),tumor-associated antigens (TAA),variable region of heavy chain (vH),variable region of light chain (vL)
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Chaudhary, Preet (
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Tags
B-cell acute lymphocytic leukemia (B-ALL/ALL)
B-cell chronic lymphocytic leukemia (CLL)
chimeric antigen receptor (CAR)
coelenterazine (CTZ)
green fluorescent protein (GFP)
Hodgkin lymphomas (HL)
human embryonic kidney (HEK) cells
NanoLuciferase (NLuc)
non-Hodgkin lymphomas (NHL)
nuclear factor of activated T-cells (NFAT)
single chain variable fragment (scFv)
tumor-associated antigens (TAA)
variable region of heavy chain (vH)
variable region of light chain (vL)