University of Southern California Dissertations and Theses

Comparative analysis of scFv and non-scFv based chimeric antigen receptors (CARs) against B cell maturation antigen (BCMA)

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Comparative analysis of scFv and non-scFv based chimeric antigen receptors (CARs) against B cell maturation antigen (BCMA)

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Comparative Analysis of scFv and non-scFv based
Chimeric Antigen Receptors (CARs) against B cell
maturation antigen (BCMA)

A thesis submitted by
Pooja Smruthi Keerthipati

In partial fulfillment of the requirements for the degree of
MASTER OF SCIENCE
in
Molecular Microbiology and Immunology
Keck School of Medicine – University of Southern California
May 2019

Adviser: Dr. Preet Chaudhary

ii
AKNOWLEDGEMENTS
First and foremost, I would like to thank Dr. Preet Chaudhary for giving me the opportunity to
work in his lab. I am very grateful for his continuous guidance and support towards my work, and
for helping me apply the knowledge I’ve gained.
I am very thankful to my thesis committee Dr. Keigo Machida and Dr. Ebrahim Zandi for their
constant encouragement and for the intellectual thoughts they shared on many occasions which
stirred my interest further in the field.
I thank Dr. Hittu Matta, Dr. Venkatesh Natarajan, Dr. Ramakrishnan Gopalakrishnan, Dr. Sunju
Choi and Songjie Gong for their continuous guidance and support throughout my time in the lab.
They were always very patient and have given me a lot of freedom allowing me to learn at my own
pace. I would like to convey my heartfelt regards and acknowledge the time and effort spent by
my lab mates Alberto Jeronimo, Dr. Wei-Ying Kuo, Hannalei Mae Zamora, Allen Membreno and
Anthony Morales for their valuable help during the course of my project. I will always remember
the lighter moments spent with all of them. I am thankful to Dr. Venkatesh Natarajan for helping
me edit my thesis.
No one understood the hectic schedule and the state of affairs during my masters better than my
batchmates who were in the same boat as me. I thank them for all the good times we’ve spent in
the last two years.
Lastly, I would like to thank my parents. Without their guidance and support I would not have had
the courage to pursue a career in the field of science.

iii
ABSTRACT

Over the past several years, immunotherapy has reached the forefront of cancer treatment. In
particular, immunotherapy approaches using Chimeric antigen receptor T cells (CAR-T) are
rapidly emerging. Indeed, successful targeting and killing of hematological malignancies with
CAR-T cells is underscored by the two FDA approvals in 2017, one for the treatment of acute
lymphoblastic leukemia (ALL) in patients up to 25 years of age and the other for adults with
advanced B cell lymphomas. Most of the current CAR constructs have a scFv based antigen
binding domain. However, these scFv-based CARs have high affinity for the target antigens and
suffer from non-specific aggregation due to heavy and light chain swapping which leads to
multimer formation. Both of the above lead to tonic signaling of CAR which results in T cell
exhaustion and lack of persistence of T cells and this ultimately results in disease relapse. High
affinity for the target antigen also leads to an increased cytokine production resulting in cytokine
release syndrome and neurotoxicity. Further, murine scFv’s are known to elicit an immune
response and this could limit the persistence of CAR T cells derived from them. To overcome these
limitations, alternative antigen binding domains are sought to improve the efficacy of CAR-T cells.
Centyrins are a type of alternate scaffold proteins from human fibronectin type III repeat with an
immunoglobulin fold. They have affinity and specificity to target molecules. Additionally,
Centyrins are very stable, small in size and engineered to have decreased immunogenicity. There
is also a low chance of domain swapping and multimer formation, hence limiting tonic signaling.
Centyrins can be isolated against any antigen by screening an extensive library of centyrins. We
generated CARs using BCMA centyrin as the antigen binding domain and demonstrate that they
can bind to specific target cells and are functionally active. The results show that centyrins are a
promising alternative for scFv in engineering functional CARs.
iv
TABLE OF CONTENTS
INTRODUCTION……………………………………………………………………………….1
Chimeric Antigen Receptor T cells…………………………………………………………….2
Structural construction of CARs……………………………………………………………….3
Evolution of CAR-T cells………………………………………………………………….......4
Components for successful immunotherapy of CARs……………………………………........6
CARs in the clinic or CARs from bench to bedside…………………………………………...7
Growing CAR-T cells clinical trials…………………………………………………………...8
Multiple Myeloma (MM).…………….…………………………………………………….....9
B cells maturation antigen (BCMA).……………….…………………………………………11
Toxicities of CAR T cells therapy…………………………………………………………….12
Centyrins……………………………………………………………………………..………..14
Gene therapy with Lentivirus vector………………………………………………………..…15
Topanga Assay……………………………………………………………………….………..17
OBJECTIVES OF THE PROJECT…………………………………………………………..19
MATERIALS AND METHODS………………………………………………………………20
Generation of lentiviral BCMA CAR and CD19 CAR constructs (with scFv)……………….20
Generation of lentiviral BCMA centyrin CAR………………………………………………..21
Virus Generation………………………………………………………………………………21
Viral Transduction…………………………………………………………………………….22
Topanga assay for detection of CAR………………………………………………………….22
Co-culture Assay to check engagement of Jurkat cells expressing the CAR…………………23
with target cells
v
ELISA…………………………………………………………………………………………24
RESULTS………………………………………………………………………………….……25
Construction of CAR plasmids……………………………………………………………..…25
Infection of Jurkat cells with CAR constructs……………………………………...…………28
Detection of CAR expression on infected Jurkat cells and its ability to bind to
target antigen……………..………………………………………………………..…………..28
CAR engagement with target cells……………………………………………………………30
Interferon-g Enzyme-linked Immunosorbent Assay (ELISA) ……………………..…………35
DISCUSSION…………………………………………………………………………...………38
REFERENCES……………………………………………………………………….…………40

1

INTRODUCTION

Our immune system is essential for our survival. It has evolved to protect our bodies from attacking
pathogens like bacteria, viruses, parasites and also help fight the intrinsic growth of tumor cells.
Adaptive immunity which is subdivided into humoral immunity (B cells) and cell mediated
immunity (T cells) collectively work to eradicate infections and tumor cells by specific immune
responses. The core step of this immune response is the recognition of antigen by antibodies
(produced by B cells) or T cell receptors, both of which differ in their mode of interaction. B cells
produce antibodies which recognize native antigens and fights it and T cells use their T cell
receptors which interacts with a fragment of the antigen bound to antigen presenting cells.
Adaptive immunity follows a general rule that - it is difficult to activate naïve antigen specific
lymphocytes (B cells and T cells) by antigen alone. Naïve B cells require additional signals from
helper T cells for the proper production of antibodies and naïve T cells require a co-stimulatory
signal from antigen presenting cells (Janeway et al. 2001).
T cells undergo a rigorous process in the thymus before maturity (thymic selection) for efficient
and appropriate expression of T cell receptor. The T cell receptor binds to the antigen presented
by the major histocompatibility complex (MHC) in the antigen presenting cells and this triggers
the T cells to start an immune response against a variety of diseases. By this process, T cells can
recognize tumor associated antigens (TAA) followed by activation of signaling mechanisms which
leads to the killing of tumor cells. But tumors have evolved ways to evade both innate and adaptive
immunity by numerous mechanisms. This includes downregulation of antigen presentation
machinery, lack of costimulatory ligands, upregulation of co-inhibitory receptors, production of
inhibitory factors and recruitment of immunosuppressive cells such a T regulatory cells (Manzo,
2
Heslop and Rooney 2015). All this makes it hard for the T cells to recognize the tumor cells. Hence
the T cells which can recognize the tumor associated antigens are rare and for effective killing of
tumor cells, the tumor specific T cells have to be enriched for effective killing leaving no residual
tumor cells to prevent recurrence. To achieve this, one approach is to isolate the tumor specific T
cells from the cancer patients, expand them in-vivo and transfer them back by infusing it into the
patients. This process is known as Adoptive Cell Transfer (ACT). The use of T cell growth factor
(IL-2) to expand T cells in-vivo often without the loss of effector functions was discovered in 1976,
which paved a way for the success of ACT (Rosenberg and Restifo 2015). To widen the use of
ACT for different types of cancers, genetic engineering techniques were introduced to modify T
cells. For example, T cells engineered to express antitumor receptor could be used for therapy.
Chimeric Antigen Receptor T cells (CAR T cells) represents one such genetically modified
lymphocytes for use in Adoptive cell transfer (ACT) therapy (Rosenberg and Restifo 2015).

Chimeric Antigen Receptor T cells
Chimeric antigen receptors are recombinant receptors which provide antigen binding and T cell
stimulation and activation (Sadelain, Brentjens and Riviere 2013). They redirect the specificity
and function of T cells. In principle, the CAR T cells can target any cell surface molecule,
overcoming tolerance to self-antigens. Physiologic TCRs engage with peptides presented by MHC
complexes, but CARs engage with molecules which don’t require peptide processing or HLA
expression, therefore broadly applicable to HLA diverse patient population (Sadelain et al. 2013).
Downregulated HLA expression, proteasomal antigen processing are few of the many
mechanism’s tumor cells use to escape from TCR-mediated immunity. But HLA independent
recognition and elimination of antigen processing and presentation allows CARs to bypass tumor
escape (Srivastava and Riddell 2015) (Zhou and Levitsky 2012). Once the CARs are expressed in
3
T cells, they act as living drugs which targets the tumor cells. CAR’s not only bind to proteins but
also glycolipids and carbohydrates, hence expanding their target range (Sadelain et al. 2013).

Structural construction of CARs
CARs consist of three parts – an extracellular antigen recognition domain, a transmembrane
domain and an intracellular T cell activation domain (Ramos and Dotti 2011).
The ectodomain is the region of the CAR that is outside the cytoplasm and in the extracellular
space. It consists of signal peptide, antigen recognition region and spacer (Ramos and Dotti 2011)
(Zhang et al. 2017). The antigen recognition region usually falls into three categories: (i) single-
chain variable fragment (scFv) derived from antibodies; (ii) Fabs fragment antigen binding derived
from human libraries or invariant human ligands; (iii) nature ligands that engage their cognate
receptor (Sadelain et al. 2013). scFvs are most commonly used as they are easily derived from
monoclonal antibodies. The light and heavy chains of the scFv’s are fused together by a flexible
linker. The spacer connects the antigen binding domain and the transmembrane domain. The hinge
region of IgG1 is the simplest one used for scFv based antigen binding domain (Zhang et al. 2017).
The transmembrane domain helps to anchor the ectodomain to cell surface and keeps it stable. It
consists of a hydrophobic alpha helix that spans the membrane and the CD28 transmembrane
domain is the most common and stable one at present (Zhang et al. 2017). The endodomain is the
functional end of the CAR structure and generally comprises an activation domain and a co-
stimulatory domain. The most common activation domain used is derived from CD3ζ with
immunoreceptor tyrosine-based activation motifs (ITAMs) and the costimulatory domains derived
from CD28 or 4-1BB are commonly used. After antigen recognition, the signal is transmitted to
the T cell through the co-stimulatory domain (Zhang et al. 2017). Once the CAR interacts with the
4
antigen, it is these signaling domains which mediate the effector functions like various cytokines
and ligands expression, lysis of the target cells etc. An ideal CAR should maximize interaction
with tumor cells and minimize toxicities to normal tissues.

Fig. 1: Structure of Chimeric Antigen Receptor (CAR) (Zhang et al. 2017).
Different parts of CAR including an ectodomain, transmembrane domain and endodomain are
shown.

Evolution of CAR-T cells
CARs were first developed in 1989 (Gross, Waks and Eshhar 1989) and since then, they can be
divided into four generations based on their co-stimulatory domains.
First generation
First generation CARs only use CD3ζ- chain or FcεRIγ (Fc receptor chains) for the activation
signal in the endodomain region (Zhang et al. 2017). The persistence of first-generation CARs is
good, but the expansion ability and anti-tumor efficacy are not satisfactory due to the lack of co-
stimulation (Xu et al. 2018). In order to kill tumor cells, IL-2 had to be administered exogenously
5
(Zhang et al. 2017). Many studies with first generation CAR-T cells didn’t have the desired results
due to low proliferation, short life span in vivo and insufficiently secreted cytokines (Zhang et al.
2017).
Second generation
The second-generation CARs have intracellular signaling domain from different co-stimulatory
protein receptors like CD28, 4-1BB (CD137), OX40 (CD134), DAP10, and ICOS in addition to
CD3ζ- chain at the cytoplasmic end of the CAR (Sadelain et al. 2013). It provides additional
signals to T cells which in turn improves proliferation, promote a sustained response, improve the
life span of CARs in vivo (Zhang et al. 2017). It has been shown that CD28 increases the telomere
length and it increases the persistence and anti-tumor effect of CARs (Xu et al. 2018). CD28
mediated co-stimulation also plays a key role in establishing memory and effector cells (Zhang et
al. 2017). 4-1BB maintains the T cells signal, which helps in survival and memory of T cells and
OX40 sustains proliferation and IL-2 production (Zhang et al. 2017). Comparisons between the
different second-generation CARs is still lacking. Some CD28 and 4-1BB based CARs were
compared in animal models, but each proved superior to the other in different contexts (Sadelain
et al. 2013)
Third generation
Third generation CARs have 2 costimulatory domains added to the activation domain (CD3ζ-
chain) in the endodomain region, for example CD3ζ-CD28-OX40 or CD3ζ-CD28-41BB (Sadelain
et al. 2013) (Zhang et al. 2017). This was designed to increase the cytokine production and the
killing ability, but the outcome was not much different than the second-generation CARs. But there
were only a small number of cases studied and more study is needed to explore the safety and
efficacy of this generation (Zhang et al. 2017).
6
Fourth generation
The fourth generation CARs are different from the first three. It is generated by adding IL-12
(cytokine gene) to the base of the second-generation constructs (Zhang et al. 2017) (Xu et al. 2018).
They are known as T cell redirected for universal cytokine-mediated killing (TRUCKs) (Zhang et
al. 2017). They augment T-cell activation, attract innate immune cells to the target area and also
promote resistance to immunosuppression (Zhang et al. 2017) (Xu et al. 2018).

Fig. 2: T cell receptor (TCR) and the four generations of CARs on surface of T cell
Activation of TCR and the different generation of CARs while in contact with antigen on the
tumor cell. (Hartmann et al. 2017)

Components for successful immunotherapy of CARs
For successful clinical application of CAR T cell therapy, the CAR T cells have to migrate to the
tumor sites after intravenous infusion. It has to recognize the desired antigen, proliferate and
accumulate at the tumor site and execute their effector functions and kill the tumor cells. It has to
evade suppression from tumor microenvironment and persist long enough to eliminate residual
tumor cell growth.
7
The persistence of CAR T cells can be improved by using gamma-retrovirus and lentivirus for
stable integration of CAR expression into the genome. Other therapy techniques like chemotherapy
and radiation prior to treatment with CAR T cell immunotherapy will help with T cell homeostatic
proliferation and persistence (Dudley et al. 2005)

Fig. 3: CAR T cell therapy process (Hartmann et al. 2017).
Blood is collected from the patient and the T cells are isolated, activated and genetically.
engineered to express the CAR construct. After CAR expression, CAR T cells are expanded ex
vivo and are formulated into the final product which can be infused into the patient.

CARs in the clinic or CARs from bench to bedside
The most investigated target for CARs to date is CD19. It is a desirable target as it is present in
most B-cell leukemias and lymphomas but not in any of the normal tissues other than B cell lineage
(Li et al. 1996) (Li, Hayakawa and Hardy 1993). To date, there are two chimeric antigen receptor
T cell therapies approved by the FDA. Kymriah (tisagenlecleucel) developed by Novartis in
collaboration with the University of Pennsylvania became the first CAR-T therapy to receive the
regulatory FDA approval in August 2017. It is used for the treatment of B-cell precursor acute
lymphoblastic leukemia (ALL) that is refractory or in second or later relapse in patients up to 25
years of age. And on May 1
st
, 2018 the FDA approved Kymriah for the treatment of adult patients
with relapsed or refractory (after two or more lines of systemic therapy) large B-cell lymphoma
8
which includes diffuse large B-cell lymphoma (DLBCL), high grade B-cell lymphoma and
DLBCL arising from follicular lymphoma (Novartis website). The second CAR-T therapy with
the FDA approval is Yescarta (axicabtagene ciloleucel) developed by Kite Pharmaceuticals, a
Gilead company. Kite got the approval in October 2017 for the treatment of adult patients with
relapsed or refractory large B-cell lymphoma - diffuse large B-cell lymphoma (DLBCL), an
aggressive type of non-Hodgkin lymphoma. Both Kymriah and Yescarta are genetically modified
autologous T cells expressing CD19 specific CAR, which lyses cells with CD19 antigens (normal
and malignant B cell lineage) (Zheng, Kros and Li 2018).

Growing CAR-T cells clinical trials
As of end of 2016, there are 220 CAR T cell trials of which 188 are ongoing. 128 clinical trials are
in phase I, which primarily evaluates safety and dosing (Hartmann et al. 2017). The first CAR T
cell trials were initiated about 20 years ago, (Kershaw et al. 2006) but the breakthrough was
achieved recently with CD19 specific CAR T cells targeting B cell malignancies (Hartmann et al.
2017). Since then, the number of CAR T cell trials have increased and is still growing. It was
originally introduced in the USA, but then gained importance in the rest of the world, mainly in
China and Europe. Of the current trials, 133 target hematological malignancies and 78 target solid
tumors (Hartmann et al. 2017).

9

Fig. 4: Timeline of cancer CAR T trials
Ongoing trials are indicated by dark blue bars and newly initiated trials are indicated by light
blue bars (Hartmann et al. 2017).

Fig. 5: (A) Hematological tumors vs. Solid tumors. Ongoing trials are indicated by a dark blue
bar and non-active trials are indicated by a light blue bar (Hartmann et al. 2017).
(B) Patient age distribution. Hematological malignancies are indicated by a dark blue
bar and solid tumors are indicated by a light blue bar (Hartmann et al. 2017).
(C) Different generations of CAR constructs used. (Hartmann et al. 2017)

Multiple Myeloma (MM)
Multiple myeloma is a very common hematologic cancer in the United States. It accounts for 10%
of hematologic cancers and 1% of malignancies (Siegel, Miller and Jemal 2018). The main
10
characteristic of multiple myeloma is the expansion and abnormal accumulation of
immunoglobulin-producing plasma cells (PCs) in the bone marrow, and this is associated with
excessive production of monoclonal immunoglobulins in blood and urine (Cho, Anderson and Tai
2018) (Tai and Anderson 2015). Multiple myeloma also causes osteolytic bone lesions and
immunodeficiency which lowers the longevity and quality of life (Cho et al. 2018). In the past two
decades, the clinical outcome for Multiple myeloma patients has improved remarkably especially
due to immunomodulatory drugs, proteasome inhibitors, monoclonal antibodies as
immunotherapies and autologous stem cell transplant. But it still remains incurable for most
patients cause drug resistant clones constantly emerge and persistence of minimal residual disease
is often seen (Cho et al. 2018).
Multiple myeloma develops from a premalignant precursor condition which is still undetermined,
progress to a passive MM and then to an active MM, which finally leads to the end stage Plasma
cell (PC) leukemia. Genetic processes like hyperdiploidy of chromosomes, translocation of
immunoglobulin heavy chain, deregulation of cell cycle genes, alteration of NFkB pathways and
abnormal DNA methylation patterns are seen initially and underlie the progression of Multiple
myeloma (Cho et al. 2018). The MM tumor cells interact with bone marrow accessory cells which
leads to MM cell expansion and also leads to impairing immune inspection and immune effector
functions against the MM cells and enhancing the tumor progression (Cho et al. 2018). Hence
successful therapy for Multiple myeloma should target MM cells and also restore antitumor
activity of the immune effector cells (Hoyos and Borrello 2016). Ideal target for immunotherapy
of MM should be a marker strongly expressed on the surface of MM cells compared to normal
cells. The B cell maturation antigen (BCMA) shows high restricted expression on PCs and not in
11
other tissues, and therefore is a good target for immunotherapy in Multiple myeloma (Carpenter
et al. 2013).
B cell maturation antigen (BCMA)
B cell maturation antigen, also known as tumor necrosis factor receptor superfamily member 17
(TNFRS17) is a type III transmembrane protein. It doesn’t have a signal peptide and contains
cysteine-rich extracellular domains (Madry et al. 1998). BCMA is produced only in late memory
B cells which are committed to differentiate into plasma cells. BCMA is present in all plasma
cells and studies from BCMA-knockdown mice showed that BCMA is important for long life of
plasma cells but is dispensable for overall B cell homeostasis (Cho et al. 2018) (O'Connor et al.
2004). Since BCMA is exclusively expressed on the surface of plasmablasts and differentiated
PCs and not in memory B cells, naïve B cells and CD34+ hematopoietic stem cells and other
normal tissues, it is a good rational to target BCMA for multiple myeloma (Cho et al. 2018).
Also, gene expression and immunohistochemistry studies showed that BCMA mRNA and
protein are more highly expressed in malignant MM cells than in normal plasma cells (Carpenter
et al. 2013).

Fig. 6: Significance of BCMA in plasma cells (Cho et al. 2018)
BCMA is selectively induced during plasma cell differentiation. It is expressed on late stage B
cells and long-lived plasma cells (PCs) and is required for survival of long-lived PCs. It is
significantly increased on malignant cells compared to normal cells in Multiple Myeloma.
12
Toxicities of CAR T cell therapy
Cytokine release syndrome (CRS)
The most adverse effect following CAR T cell infusion is the cytokine release syndrome (Bonifant
et al. 2016). Second generation CARs improved T cell activation and expansion, cytokine
production and showed good antitumor responses but, it also potentially results in life-threatening
CRS (Bonifant et al. 2016) (Davila et al. 2014). It is an immune activation resulting in elevated
inflammatory cytokines, ranging from mild to severe.
Its symptoms are high fever, nausea, malaise, myalgia, fatigue, tachycardia/hypotension, capillary
leak, cardiac dysfunction, renal impairment, hepatic failure, and disseminated intravascular
coagulation (Bonifant et al. 2016) (Lee et al. 2014). It has been shown that the severity of CRS is
directly related to disease burden: higher the tumor burden, higher the severity of CRS (Bonifant
et al. 2016) (Maude et al. 2014). Systemic corticosteroid has been shown to reverse CRS effects,
but prolonged use dampened the CAR T cell effect. IL-6 receptor blockade mAb, tocilizumab has
demonstrated immediate reversal of CRS (Bonifant et al. 2016) (Davila et al. 2014). It is unknown
if blocking cytokines and receptor partners would maintain antitumor efficacy and further studies
are ongoing (Bonifant et al. 2016) (Davila et al. 2014).
Neurological toxicity
Confusion, delirium, expressive aphasia, obtundation, myoclonus and seizure has been reported in
patients who received CD-19 specific CAR T cells (Bonifant et al. 2016) (Davila et al. 2014) (Lee
et al. 2015). The causative pathophysiology of the side effects is unknown. It is possible that
elevated cytokine levels may be the problem. To date, the neurologic toxicity has been reversible
in majority of cases, but can be fatal. It is also unclear if it is restricted to CD19 specific CAR T
13
cells or if it will be exhibited by targeting other tumor antigens too (Bonifant et al. 2016) (Davila
et al. 2014) (Lee et al. 2015).
On-target/off-tumor recognition
Unfortunately, most CAR T cells target are expressed on normal tissues and hence some degree of
on-target/off-tumor toxicity occurs. The severity ranges from manageable lineage depletion (B-
cell aplasia) to severe toxicity resulting in death. Hence, the correct dosage of CAR T cells is very
important (Bonifant et al. 2016).
Anaphylaxis
Majority of CAR T cells in clinical trials have the antigen recognition domains derived from
murine monoclonal antibodies. Hence, cases of both cellular and humoral rejection of CAR T cells
was seen due to immunogenicity of foreign proteins. There are ongoing efforts to humanize the
expressed protein segments to decrease immunogenicity (Bonifant et al. 2016) (Curran, Pegram
and Brentjens 2012).

Fig. 7: Toxicities of CAR-T cell therapy (Bonifant et al. 2016).
14
Centyrins
In the field of therapeutic proteins, monoclonal antibodies are most widely used due to high affinity
and specificity for target molecules. Recently, engineered protein folds that can bind to specific
targets with high affinity are gaining high interest (Jacobs and O'neil 2015). The advantages of
these alternative scaffold proteins are,
• small in size
• have high affinity
• lack disulphide bonds
• high stability
The immunoglobulin fold is an example of an alternate scaffold molecule (Jacobs and O'neil 2015).
It is found in the variable region of antibodies and also thousands of non-antibody proteins.
Centyrin is one such alternate scaffold protein – the tenth fibronectin type III (FN3) repeat from
human fibronectin (Jacobs and O'neil 2015, Rojas 2014) (Barnett et al. 2016). It occurs in many
animal proteins for ligand binding (Koide et al. 1998). The β-sandwich structure of FN3 closely
resembles that of immunoglobulin domains (Koide et al. 1998). It is interesting to work with as it
can tolerate a number of mutations in the exposed loops and can still maintain the overall
immunoglobulin fold (Jacobs and O'neil 2015). They are also engineered to be of low
immunogenicity (Rojas 2014).
They are very thermostable and are also stable over a wide range of pH. They are small in size
with a molecular weight of only 10KDa (Rojas 2014). Their high affinity to target molecules, small
size and robust biophysical properties makes them a good choice as an alternative for the scFv
region of the antigen binding domain of the CAR T structure. Moreover, centyrins can be isolated
15
against virtually any antigens by designing them from the data present in the extensive centyrin
library (Barnett et al. 2016).

Gene therapy with Lentivirus vector
Gamma retroviruses and lentiviruses are subtypes of retroviruses. They have the RNA genome
that converts to DNA in a transduced cell by reverse transcriptase (Milone and O'Doherty 2018).
In research, gammaretroviral vectors are more common, but recently the use of lentiviral vectors
in clinical trials have been increasing (Milone and O'Doherty 2018). Lentiviral vectors are derived
from the human immunodeficiency virus which is a genus of retroviridae family. They are
extensively used and have been optimized the past two decades. The third generation, self-
inactivating (delivery of desired sequence, but no viral replication in host cells) lentiviral vectors
have been used in multiple clinical trials to introduce genes into mature T cells for CAR T cell
therapy (Carpenter et al. 2013). The regulatory approval of first CAR T cell therapy used lentiviral
vectors.
For lentiviral survival, gag, pol and env are the required basic genes. Gag encodes structural
proteins, env encodes proteins for the viral envelope (glycoprotein) and pol encodes enzymes for
reverse transcription and integration into host cell genome (Milone and O'Doherty 2018) (Escors
and Breckpot 2010).
Lentiviral vectors have three generations, differentiated based on their safety with third generation
being the safest. First generation has a significant amount of HIV genome, with gag and pol genes
and additional viral proteins (Escors and Breckpot 2010) (Milone and O'Doherty 2018). For the
envelope protein, another viral protein, most commonly VSV-G was encoded on a separate
plasmid from other lentiviral genes. The accessory genes - vif, vpr, vpu and nef (survival
16
advantages for lentiviral replication in vivo, but not in vitro) and regulatory genes - tat and rev
(required for viral replication) are included in the first-generation lentiviral vectors (Milone and
O'Doherty 2018). The second-generation lentiviral vectors are safer than the first-generation and
lack the accessory genes – vif, vpr, vpu and nef. Their removal did not affect infection of host cell
(Milone and O'Doherty 2018) (Vannucci et al. 2013). The third-generation further improved
safety. Here, the viral genome is split into separate plasmids and hence virus recombination and
generation are very unlikely (Dull et al. 1998). The gag and pol genes are encoded on a different
plasmid and rev gene is encoded in a different plasmid. Tat gene was removed in third generation
and a constitutively active promoter was engineered upstream of the long terminal repeats (Dull et
al. 1998) (Carpenter et al. 2013). The 3’LTR was modified by introducing deletions to create self-
inactivating lentiviral vectors. It improved the safety by disrupting the promoter and enhancer
activity of LTR (Milone and O'Doherty 2018) (Dull et al. 1998).
Vector Plasmid

Packaging Plasmids

Envelope Plasmid

Fig. 8: Third generation Lentiviral vector
The plasmid with gene of interest has altered LTR sequences for self-inactivation to prevent
recombination. Two packaging plasmids – one encoding gag and pol and the other encoding rev.
Envelope plasmid encoding the envelope protein (env) derived from VSV-G.

Promoter
Promoter
gag-pol
rev
Promoter env (VSV-G)
SIN 5’ LTR Promoter Gene of Interest SIN 3’ LTR
17

Fig. 9: Production of recombinant lentiviral virus (http://www.genetherapynet.com)
Gag and Pol are needed for virus maturation. Vesicular stomatitis virus (VSV-G) is for coding
the envelope glycoprotein. Rev is required for viral replication. All the plasmids are added along
with the vector plasmid containing the gene of interest into HEK 293 T cells for transfection.
The supernatant after 48 – 72 hours will contain the mature virus which can be used for
infection.

Topanga Assay
The Topanga assay is a novel luciferase-based assay for the detection of CARs on the surface of
the immune effector cells (Gopalakrishnan et al. 2019). It is economical, fast, sensitive and robust.
All the current methods need flow cytometry for the detection of CARs and many of them require
secondary labelling which is time consuming and labor intensive. Luciferases are very common in
biomedical research due to their high sensitivity and produces low background noise. Firefly
luciferases are the most popular luciferases used in research. But they are quite big, around 61 kDa
and this has hampered their use in fusion protein studies (Gopalakrishnan et al. 2019). Recently,
marine luciferases - Gluc, Nluc, Tluc16 and Mluc7 were discovered which are much smaller in
18
size and much brighter that firefly luciferase. They are approximately 19 kDa and are more stable
than firefly luciferase. The Topanga assay uses recombinant fusion protein called the Topanga
reagent. It is made by joining the extracellular domain of a CAR target in frame with one of the
marine luciferases (Gopalakrishnan et al. 2019).
To develop the Topanga assay for the detection of BCMA CARs, a fusion construct was made by
joining the extra-cellular domain of BCMA containing a signal peptide in frame with Nluc with
the help of a flexible linker, Gly-Gly-Ser-Gly (Gopalakrishnan et al. 2019). The construct has an
N-terminal signal peptide from human CD8 to allow secretion of the fusion protein. It was cloned
using Lentiviral vectors and was transiently transfected into 293 FT cells (Gopalakrishnan et al.
2019). The supernatant was collected after approximately 48 hours and contained the secreted
fusion protein – BCMA-ECD-Nluc fusion protein, i.e., the Topanga reagent (Gopalakrishnan et
al. 2019). This Topanga reagent was used to bind to cells stably expressing the CAR.
Coelenterazine (CTZ) was used as a substrate for NLuc to measure luminescence.

19
OBJECTIVES OF THE PROJECT

To generate Chimeric Antigen Receptors (CARs) with a scFv based antigen binding
domain and a non-scFv based antigen binding domain (centyrin).
• Clone centyrin CAR construct against BCMA using a lentiviral backbone.
• Clone scFv CAR construct against BCMA and CD19 using a lentiviral backbone.
To generate stable cell lines expressing the CARs
• Produce the CAR encoding lentivirus using HEK 293 T cells by transient transfection.
• Infect Jurkat cells with the different cloned CAR constructs and generate stable cell lines
expressing the CARs.
In vitro assays to perform comparative analysis
• Check the CAR expression on infected Jurkat cells and functional affinity to the target
antigen by Topanga assay.
• Co-culture CAR infected Jurkat cells with the target cells to check if they engage.
• Measure the production of IL-2 after the CAR Jurkat cells engage with target cells by
ELISA.

20
MATERIALS AND METHODS
Generation of lentiviral BCMA CAR and CD19 CAR constructs (with scFv binding domain)
pCCL-c-MNDU3-VEGFA-PKG-EGFP-WPRE was the lentiviral vector used and it was a gift
from Fernando Fierro (Addgene plasmid # 89609). The sequence of scFv fragments encoding a
mouse monoclonal antibody against human BCMA (J6MO) and a mouse monoclonal antibody
against human CD19 (FMC63) were codon optimized using GeneArt
TM
software (Thermo Fisher
Scientific). Gene-fragments encoding the optimized scFv sequences were synthesized with an in-
frame and upstream CD8 signal peptide by Integrated DNA Technologies (IDT) and were used as
templates in pfu-PCR reaction using custom primers for amplification. PCR products were
digested with Nhe and Mlu and ligated to the modified pCCL-c-MNDU3-WPRE vector containing
a CAR cassette encoding an in frame CD8 hinge domain, a CD8 transmembrane domain and a
41BB co-stimulatory domain and a CD3ζ activation domain using standard molecular biology
techniques. Ligated products were transformed into Stbl3 competent cells (Invitrogen) and plated
on LB-carbenicillin plates. The resultant colonies were screened using colony-PCR. Plasmids were
purified from positive clones (MIDI prep) using standard procedures.
The final CAR constructs consisted of human CD8 signal peptide, fused in frame to the scFv
fragments (antigen binding domain), the hinge and transmembrane domain of human CD8, the
cytosolic domain of human 41BB (CD137) receptor, the cytosolic domain of human CD3z. We
generated, and sequence confirmed two CAR constructs namely,
pCCLc - MNDU3 – EcoRI – Nhe - mBCMA(J6MO) – Mlu - CD8TM - 4-1BB - CD3z – ter -
sal - WPRE
pCCLc - MNDU3 – EcoRI – Nhe – mCD19(FMC63) – Mlu - CD8TM - 4-1BB - CD3z – ter -
sal – WPRE
21
Generation of lentiviral anti BCMA-centyrin-CAR
The sequence of centyrin which makes it an alternative protein scaffold similar to scFv fragment
against human BCMA (J6MO) was codon optimized using GeneArt
TM
software (Thermo Fisher
Scientific). Gene-fragments encoding the optimized sequences were synthesized by Integrated
DNA Technologies (IDT). The synthesized gene fragments encoding BCMA-centyrin was cloned
into the modified pCCL-c-MNDU3-WPRE vector containing a CAR cassette described in the
previous section. The resulting final CAR construct was sequence confirmed and labelled as,
pCCLc - MNDU3 – EcoRI – Nhe – BCMA centyrin – Mlu - CD8TM - 4-1BB - CD3z – ter -
sal - WPRE

Virus Generation
HEK-293 FT cells and pLP/VSVG envelope plasmid was obtained from Invitrogen. The
packaging plasmid psPAX2 was a gift from Didier Trono (Addgene plasmid # 12260). Lentivirus
was generated in HEK-293 FT cells by transfecting CAR construct along with psPAX2 vector
encoding Gag, Pol, Rev, and Tat genes and pLP/VSVG vector encoding viral envelope
glycoprotein. EGFP vector was used to check transfection efficiency.
A 100mm culture dish with almost fully confluent HEK-293 FT cells was transfected with 10µg
of CAR lentiviral plasmid, 7.5µg of psPAX2 plasmid, 3µg of pLP/VSVG plasmid and 0.25µg of
EGFP (Enhanced Green Fluorescent Protein) encoding plasmid using the standard calcium
phosphate method. Supernatants were collected at 48 h and 72 h post transfection and filtered
through a 0.45µm filter. They were then concentrated by ultracentrifugation at 18,500 rpm at 4°C
for overnight and re-suspended in RPMI medium. Viruses were stored at -80°C until needed.

22
Viral Transduction
Jurkat cell line engineered with a NFAT-dependent EGFP reporter gene (JNG) was kindly
provided by Dr. Arthur Weiss (UCSF) and maintained in RPMI-1640 medium supplemented with
10% FBS. Two million JNG cells were infected with 300µl of concentrated virus supernatant
(BCMA centyrin CAR, BCMA CAR and CD19 CAR) along with 20µl polybrene (8µg/ml). Two
million uninfected JNG cells was used as a control. Cells were centrifuged at 1,800 rpm at 32°C
for 45 min and were left undisturbed overnight at 37°C with 5% CO2. After incubation, the medium
containing viral supernatant and polybrene was replaced by fresh RPMI medium with 10% FBS.
The cells were transferred to a T25 flask for further expansion.

Topanga Assay for detection of CAR
a. Construction and Production of Marine Luciferase fusion proteins
The nucleotide sequences encoding the extra-cellular domain (ECD) of human BCMA and CD19
were fused in frame to signal peptide-deleted NanoLuc or Nluc (marine luciferase) via a short
intervening Glycine-Serine flexible linker. The construct has an N-terminal signal peptide from
human CD8 to allow secretion of the ECD-Nluc fusion protein. The resulting gene fragment was
cloned into pLenti-EF1α lentiviral expression vector. The fusion protein also carried C-terminal
epitope tags – FLAG and Strep-tagII.
The Topanga fusion proteins were produced in HEK 293FT cells by transient transfection of the
BCMA expression plasmid and CD19 expression plasmid by calcium phosphate method. 293FT
cells were cultured overnight in a 100mm plate and transfected with 10µg of fusion protein
expression plasmid and 0.25µg of EGFP encoding plasmid. Approximately 48 hours after
23
transfection, the supernatants were collected, filtered through a 0.45µm filter and stored at -80°C
in aliquots for further use.
b. Binding Assay and Luminescence detection
Approximately 2x10
5
CAR infected jurkat cells were used per reaction. The cells were spun down
at 1300 rpm for 5 minutes and re-suspended in 100µl of Topanga reagent (filtered supernatant
containing the fusion protein). This was followed by incubation on ice for 45 minutes. Post
incubation, cells were washed 4 times with 1 ml of wash buffer (0.5% FBS in PBS). After the final
wash, the pellet was resuspended in 30µl wash buffer and added to a white 384-well Lumitrac
plate. 20µm Coelenterazine (CTZ) in PBS was used as a substrate to measure luminescence.
Luminescence was read in an endpoint mode using a BioTek synergy H4 hybrid microplate reader
for 10 seconds.

Co-Culture Assay to check engagement of Jurkat cells expressing the CAR with target cells
To check if the Jurkat cells expressing the CAR can engage with the target cells, they were co-
cultured together and left overnight. The Jurkat cell line is engineered with a NFAT-dependent
EGFP reporter gene (JNG), hence when activated, it produces GFP which can be read by
flowcytometric analysis. Approximately 1 million CAR-JNG cells and 1 million target cells were
co-cultured in a 24 well plate and left overnight at 37°C, 5% CO2 incubation conditions. Post
incubation, the cells were analyzed by flow cytometry (BD FACSVerse
TM
).
The target cell lines used were,
L-363 – Plasma cell Leukemia (aggressive form of Multiple Myeloma) derived from peripheral
blood of human.
U-266 – Multiple Myeloma derived from peripheral blood of human.
24
RAJI – Burkitt lymphoma (non-Hodgkins lymphoma) derived from peripheral blood of human.
HL60 – Promyelocytic leukemia (subtype of acute myeloid leukemia) derived from peripheral
blood of human.

ELISA
The IL-2 cytokine secretion was measured using the IL-2 ELISA kit from R&D systems.
ELISA plate (384-well) was coated with capture antibody (50µL per well) diluted to a working
concentration in PBS for overnight at room temperature. Next day, the plate was washed two times
with wash buffer and incubated with blocking buffer (1% BSA in PBS) for 2 hours at room
temperature. Wells were washed three times with a wash buffer and the cell supernatants (25µL
per well) to be assayed for IL-2 was added. After, 2 hours incubation at room temperature, the
plates were washed three times and incubated for additional 2 hours with detection antibody (50µL
per well) diluted in reagent diluent. The plates were washed again and incubated with streptavidin-
HRP conjugate in dark for 20 minutes. Finally, substrate solution (1:1 mixture of color reagent A,
H2O2 and color reagent B Tetramethylbenzidine) was added to each well after washing the plate
three times with wash buffer. The plate was incubated for another 20 minutes and absorbance was
read at 650nm.

25
RESULTS
Construction of CAR plasmids
We generated two scFv based lentiviral CAR constructs targeting BCMA (J6MO) and CD19
(FMC63) and a non scFv based centyrin CAR targeting BCMA.
The final constructs were designated as,
pCCLc - MNDU3 – EcoRI – Nhe - BCMA(J6MO) – Mlu - CD8TM - 4-1BB - CD3z – ter - sal
– WPRE
pCCLc - MNDU3 – EcoRI – Nhe – BCMA centyrin – Mlu - CD8TM - 4-1BB - CD3z – ter -
sal - WPRE
pCCLc - MNDU3 – EcoRI – Nhe – CD19(FMC63) – Mlu - CD8TM - 4-1BB - CD3z – ter - sal
– WPRE
In each case, the cDNA encoding corresponding scFv or centyrin was fused to CD8 signal peptide
and PCR amplified from synthetic gene fragments. It was then cloned in the pCCLc-MNDU3
vector, in frame with the hinge and transmembrane domain of human CD8, the cytosolic domain
(co-stimulatory domain) of human 41BB (CD137) receptor and the signaling domain of human
CD3z. The pCCLc-MNDU3 vector carried the MNDU3 promoter and WPRE as the regulation
element to enhance translation.

26

Fig. 10: A schematic representation of BCMA (centyrin)-CAR construct

Fig. 11: A schematic representation of BCMA (scFv J6MO)-CAR construct
27

Fig. 12: A schematic representation of CD19 (scFv FMC63)-CAR construct.
Following transformation of the recombinant plasmids, positive clones from colony PCR were
purified and their sequences were confirmed.

Fig. 13: Sequence confirmation of BCMA (centyrin)-CAR

28
Infection of Jurkat cells with CAR constructs
For virus generation, the CAR plasmids along with packaging plasmids - pLP/VSVG and psPAX2
and EGFP plasmid (to check transfection efficiency) were transfected into HEK-293 cells through
calcium phosphate transfection method. The viral supernatants were collected 48 and 72 hours
post transfection and concentrated by ultracentrifugation at 18,000 rpm overnight at 4°C.
JNG - Jurkat cell line engineered with a NFAT-dependent EGFP reporter gene was used for the
study. JNG cells were infected with the lentiviruses encoding the different CAR constructs along
with polybrene.

Detection of CAR expression on infected Jurkat cells and its ability to bind to the target
antigen
Topanga assay was done to check the expression of CAR on infected Jurkat cells and its ability to
bind to the target antigen. The Topanga reagent has the extra-cellular domain (ECD) of target
antigen (BCMA and CD19) fused to Nluc (marine luciferase) with a short glycine-serine flexible
linker. The infected jurkat cells were incubated with the respective Topanga reagent and after three
washes were assayed for luminescence using BioTek synergy H4 hybrid microplate reader for 10
seconds.
Figure 15 shows strong binding of Jurkat cells expressing BCMA CAR and significant binding of
Jurkat cells expressing BCMA centyrin CAR with the BCMA-ECD Topanga reagent. No
significant binding was observed with Jurkat cells expressing CD19-CAR (not the target antigen)
or with uninfected parental Jurkat control cells with the BCMA-ECD Topanga reagent as expected.
Figure 16 shows strong binding of Jurkat cells expressing CD19 CAR with CD19-ECD Topanga
reagent and no significant binding of Jurkat cells expressing BCMA CAR or BCMA centyrin
29
CAR (not the target antigen) or with uninfected parental Jurkat cells with the CD19-ECD
Topanga reagent.

Fig. 14: Topanga assay with BCMA - ECD
Strong binding of Jurkat cells expressing BCMA CAR, significant binding of Jurkat cells
expressing BCMA centyrin CAR, and no significant binding of Jurkat cells expressing CD19-
CAR or parental Jurkat cells with the BCMA-ECD Topanga reagent. Statistically significant
differences are shown by (**) at a level of p < 0.001 and (***) at p <0.0001.

**
***
0
20000
40000
60000
80000
100000
120000
140000
160000
Parental JNG control anti BCMA Centryn
CAR
anti BCMA scFv CAR anti CD19 CAR
Relative luminescense units
BCMA - ECD binding
**
30

Fig. 15: Topanga assay with CD19 - ECD
Strong binding of Jurkat cells expressing CD19 CAR with CD19-ECD Topanga reagent and no
significant binding of Jurkat cells expressing scFv and centyrin BCMA CAR or parental Jurkat
cells with the CD19-ECD Topanga reagent. Statistically significant differences are shown by (**)
at a level of p < 0.001 and (***) at p <0.0001.

CAR engagement with target cells
Jurkat cells expressing BCMA centyrin CAR, BCMA CAR and CD19 CAR were co cultured along
with target cells. Figure 18 shows the target cells used - L-363, U266, RAJI and HL60. One million
of Jurkat cells expressing the CAR was co cultured with one million of target cells for
approximately 24 hours. The Jurkat cell line (JNG) is engineered with a NFAT-dependent EGFP
reporter gene, hence when activated, it produces GFP which can be read by flowcytometric
***
0
20000
40000
60000
80000
100000
120000
Parental JNG control anti BCMA Centryn
CAR
anti BCMA scFv CAR anti CD19 CAR
Relative luminescense units
CD19 - ECD binding
31
analysis. For controls, Jurkat cells expressing the respective CARs were cultured alone with media
and uninfected Jurkat cells were cultured with target cells.
When co-cultured with L-363 (Plasma cell Leukemia), the BCMA centyrin CAR and BCMA CAR
showed expression of GFP, which indicates their engagement with L-363 cells. CD19 CAR
showed no GFP expression as expected (negative control for L-363). With U266 (Multiple
Myeloma), again BCMA centyrin CAR and BCMA CAR showed expression of GFP and CD19
CAR showed no GFP expression (negative control for U266). When cultured with RAJI (Burkitt
lymphoma), CD19 scFv CAR showed the most expression of GFP. Both BCMA centyrin CAR
and BCMA CAR showed GFP expression, but it was lower than the GFP expression when cultured
with L-363 and U266. HL60 (Promyelocytic leukemia) is a negative control for CD19 CAR,
BCMA CAR and BCMA centyrin CAR. As expected, there was no GFP expression when HL60
was co-cultured with all three CARs.
Another important result observed was that, when BCMA CAR and CD19 CAR (both have scFv
antigen binding domain) were co-cultured with media alone (control), it still showed activation at
the basal level. This can lead to tonic signaling which drives the functional exhaustion of CAR-T
cells. And when BCMA centyrin CAR was co-cultured with media alone, it showed very little
basal activation when compared to constructs with scFv antigen binding domain.
BCMA centyrin CAR BCMA scFv CAR CD19 scFv CAR
L-363 (+) L-363 (+) L-363 (-)
U-266 (+) U-266 (+) U-266 (-)
RAJI (+) RAJI (+) RAJI (+)
HL60 (-) HL60 (-) HL60 (-)
Fig. 16. Table showing the target cells used for the co-culture assay with the CAR infected
Jurkat cells. (+) implies the CAR should bind to the target cell, (-) implies the CAR should not
bind to the target cells.
32

Fig. 17: Different target cells used to check the BCMA and CD19 CAR engagement.

A. B. C.
BCMA centyrin CAR alone BCMA CAR alone CD19 CAR alone

D. E. F.
BCMA centyrin CAR with L-363 BCMA CAR with L-363 CD19 CAR with L-363

Fig. 18: Co-culture assay with L-363 (Plasma cell Leukemia)
A, B and C are the controls. (A) is BCMA centyrin CAR co-cultured with media alone overnight,
(B) is BCMA CAR co-cultured with media alone overnight and (C) is CD19 CAR co-cultured
with media alone overnight. (D) is BCMA centyrin CAR co-cultured with L-363 overnight, (E) is

Species Tissue/Organ Tumor
L-363 Human Peripheral Blood Plasma cell Leukemia (aggressive form of Multiple Myeloma)
U-266 Human Peripheral Blood Multiple myeloma
RAJI Human Peripheral Blood Burkitt lymphoma (non-Hodgkins lymphoma)
HL60 Human Peripheral Blood Promyelocytic leukemia (subtype of acute myeloid leukemia)
33
BCMA CAR co-cultured with L-363 overnight and (F) is CD19 scFv CAR co-cultured with L-
363 overnight. The Jurkat cell line (JNG) is engineered with NFAT-dependent EGFP reporter gene
and when activated, produces GFP which is read by flowcytometric analysis. Both the BCMA
CARs showed GFP expression but CD19 CAR did not as L363 lacks CD19 antigen. Both BCMA
CAR and CD19 CAR (with scFv) showed activation at basal level (controls), but BCMA centyrin
CAR showed very low basal activation comparatively.

A. B. C.
BCMA centyrin CAR alone BCMA CAR alone CD19 CAR alone

D. E. F.
BCMA centyrin CAR with U266 BCMA CAR with U266 CD19 CAR with U266

Fig. 19: Co-culture assay with U266 (Multiple Myeloma)A, B and C are the controls - CAR
expressing Jurkat cells cultured with media alone overnight. (D) is BCMA centyrin CAR co-
cultured with U266 overnight, (E) is BCMA CAR co-cultured with U266 overnight and (F) is
34
CD19 CAR co-cultured with U266 overnight. Both the BCMA CARs showed GFP expression
but CD19 CAR did not as U266 lacks CD19 antigen.

A. B. C.
BCMA centyrin CAR alone BCMA CAR alone CD19 CAR alone

D. E. F.
BCMA centyrin CAR with RAJI BCMA CAR with RAJI CD19 CAR with RAJI

Fig. 20: Co-culture assay with RAJI (Burkitt Lymphoma)
A, B and C are the controls - CAR expressing Jurkat cells cultured with media alone overnight.
(D) is BCMA centyrin CAR co-cultured with RAJI overnight, (E) is BCMA CAR co-cultured with
RAJI overnight and (F) is CD19 CAR co-cultured with RAJI overnight. Both the anti BCMA
CARs and anti CD19 CARs showed GFP expression. RAJI cells have high CD19 expression and
lower levels of BCMA expression. Hence CD19 CAR and both the BCMA CARs showed GFP
expression.
35

A. B. C.
BCMA centyrin CAR alone BCMA CAR alone CD19 CAR alone

D. E. F.
BCMA centyrin CAR with HL60 BCMA CAR with HL60 CD19 CAR with HL60

Fig. 21: Co-culture assay with HL60 (Promyelocytic leukemia)
A, B and C are the controls - CAR expressing Jurkat cells cultured with media alone overnight.
(D) is BCMA centyrin CAR co-cultured with HL60 overnight, (E) is BCMA CAR co-cultured
with HL60 overnight and (F) is CD19 CAR co- cultured with HL60 overnight. Both the BCMA
CARs and CD19 CAR showed no GFP expression as HL60 lacks BCMA and CD19 antigen.

Interferon-γ Enzyme-linked Immunosorbent Assay (ELISA)
To detect if there is an increased cytokine secretion when Jurkat cells expressing the CAR engages
with target cells, we measured the secretion of IL-2 via ELISA. From the co-culture experiment,
the remaining supernatant was collected. IL-2 secretion in uninfected Jurkat cells was checked as
36
a control for background interference and in the CAR Jurkat cells alone to check the basal level of
IL-2 produced by them.
When L-363 cells were co-cultured with Jurkat cells expressing BCMA centyrin CAR and BCMA
CAR, IL-2 levels were significantly higher. In comparison, Jurkat cells expressing CD19 CAR did
not secrete significant level of IL-2 as L363 does not have the target antigen – CD19. The
uninfected Jurkat cells (control) had very low level of IL-2 secretion. With U-266, BCMA CAR
showed significantly high secretion of IL-2 compared to the uninfected Jurkat control. BCMA
centyrin CAR showed IL-2 secretion, but it was not as high as BCMA CAR and CD19 CAR
showed low levels of IL-2 secretion (no target antigen). When co-cultured with RAJI, CD19 CAR
showed the most secretion of IL-2 and it was significantly very high compared to the control. Both
the BCMA CARs showed lower secretion of IL-2 compared to CD19 CAR. With HL60 (negative
control), all three - CD19 CAR, BCMA CAR and BCMA centyrin CAR showed no significant
secretion of IL-2.

Uninfected
Jurkat cells
CD19 scFv
CAR
BCMA scFv
CAR
BCMA centyrin
CAR
Media alone 1:1 1:1 1:1 1:1
L-363 1:1 1:1 1:1 1:1
U-266 1:1 1:1 1:1 1:1
RAJI 1:1 1:1 1:1 1:1
HL60 1:1 1:1 1:1 1:1

Fig. 22: Ratio of uninfected, infected CAR expressing Jurkat cells and target cells used for
co-culture.

37

Fig. 23: IL-2 secretion measured by ELISA
IL-2 secretion by CAR expressing Jurkat cells upon activation when cultured with target cells.
BCMA centyrin CAR and BCMA CAR showed significant secretion of IL-2 when co-cultured
with L363 and U266 and RAJI. BCMA CAR secreted more IL-2 compared to BCMA centyrin
CAR. With HL60 (negative control) they showed no significant IL-2 secretion. CD19 CAR
showed a significant secretion of IL-2 when co-cultured with RAJI but not with L363, U266 and
HL60.

0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Alone L363 U266 RAJI HL60
O.D. at 650 nm
ELISA - IL-2 secretion
UI-JNG CD19 CAR BCMA scFv CAR BCMA centyrn CAR
38
DISCUSSION
Chimeric antigen receptor T cells have risen to the forefront of immunotherapy for the treatment
of cancer. With two FDA approvals for CAR T cells directed against CD19 receptors, this therapy
is believed to be very promising as clinical trials demonstrated expansion and long-term
persistence. Most of the CAR-T cells in current use have an scFv based antigen binding domain.
Though they have been successful in clinical trials, they do come with few disadvantages. They
have high affinity for the target antigens and due to their instability, there is a potential for
multimer formation due to heavy and light chain swapping (Barnett et al. 2016). All the above
factors leads to tonic signaling (T cell exhaustion) and lack of persistence of T cells, ultimately
resulting in disease relapse (Barnett et al. 2016). Further, CAR-T cells engineered using murine
scFv’s elicit immune response in humans leading to limited persistence. Hence alternative
strategies for the binding domain are needed to improve the efficacy of CAR-T cells.
Centyrins are a type of alternate scaffold proteins derived from human fibronectin type III repeat.
They can tolerate a number of mutations, yet maintain the overall Ig fold (Jacobs and O'neil 2015).
They have multiple advantages compared to scFvs, which include high specificity, stablility,
smaller size and lower immunogenicity (Rojas 2014). Further, due to lower possibility for domain
swapping and multimer formation, CARs engineered using centyrins are less likely to exhibit tonic
signaling. Centyrins can be isolated against any antigen by screening centyrin library (Jacobs and
O'neil 2015).
In this project, we generated CAR constructs against BCMA using centyrin as the antigen binding
domain and compared it to conventional scFv derived CAR. CD19 CAR with scFv was used as a
control as it is well established with a number of positive clinical trial results. The Topanga assay
showed expression of BCMA centyrin CAR on Jurkat cells and its ability to bind to the target
39
antigen, thereby demonstrating that the antigen binding domain with centyrin is functionally active
when linked to the rest of the CAR backbone. As anticipated, binding of BCMA centyrin CAR
with target antigen was much lower than that of BCMA CAR, which suggests that centyrins can
serve as a better alternative to minimize tonic signaling. Further, co-culture assay demonstrated
that JNG cells expressing both BCMA CAR and BCMA centyrin CARwas activated by target
myeloma cells (L-363 and U266). BCMA CAR and CD19 CAR (both with scFv antigen binding
domain) showed T cell activation at the base level, which usually leads to T cell exhaustion. It is
noteworthy that in comparison, BCMA centyrin CAR showed very little base level T cell
activation. Further, upon co-culture with the target myeloma cells (L-363 and U266) BCMA
centyrin CAR showed a significant expression of IL-2, but was not as high as BCMA CAR,
showing centyrins may be a better alternative to lower chance of cytokine release syndrome in
CAR-T therapy.
Collectively, these results demonstrate that CAR T cells with centyrin as the antigen binding
domain can specifically bind to target cells are functionally active. The future direction for this
project is to continue the in vitro studies with primary human T cells and follow with in vivo
studies in immune-deficient mice. If the in vivo studies show promising results, then CAR-T
cells using centyrins as an antigen binding domain is a promising alternative platform for clinical
studies.

40
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Abstract (if available)

Abstract Over the past several years, immunotherapy has reached the forefront of cancer treatment. In particular, immunotherapy approaches using Chimeric antigen receptor T cells (CAR-T) are rapidly emerging. Indeed, successful targeting and killing of hematological malignancies with CAR-T cells is underscored by the two FDA approvals in 2017, one for the treatment of acute lymphoblastic leukemia (ALL) in patients up to 25 years of age and the other for adults with advanced B cell lymphomas. Most of the current CAR constructs have a scFv based antigen binding domain. However, these scFv-based CARs have high affinity for the target antigens and suffer from non-specific aggregation due to heavy and light chain swapping which leads to multimer formation. Both of the above lead to tonic signaling of CAR which results in T cell exhaustion and lack of persistence of T cells and this ultimately results in disease relapse. High affinity for the target antigen also leads to an increased cytokine production resulting in cytokine release syndrome and neurotoxicity. Further, murine scFv’s are known to elicit an immune response and this could limit the persistence of CAR T cells derived from them. To overcome these limitations, alternative antigen binding domains are sought to improve the efficacy of CAR-T cells. Centyrins are a type of alternate scaffold proteins from human fibronectin type III repeat with an immunoglobulin fold. They have affinity and specificity to target molecules. Additionally, Centyrins are very stable, small in size and engineered to have decreased immunogenicity. There is also a low chance of domain swapping and multimer formation, hence limiting tonic signaling. Centyrins can be isolated against any antigen by screening an extensive library of centyrins. We generated CARs using BCMA centyrin as the antigen binding domain and demonstrate that they can bind to specific target cells and are functionally active. The results show that centyrins are a promising alternative for scFv in engineering functional CARs.

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Asset Metadata

Creator Keerthipati, Pooja Smruthi (author)

Core Title Comparative analysis of scFv and non-scFv based chimeric antigen receptors (CARs) against B cell maturation antigen (BCMA)

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Publication Date 05/01/2019

Defense Date 03/21/2019

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

Tag BCMA,chimeric antigen receptors,OAI-PMH Harvest

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Machida, Keigo (committee chair), Chaudhary, Preet M (committee member), Zandi, Ebrahim (committee member)

Creator Email keerthip@usc.edu,poojasmruthik@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-164011

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Legacy Identifier etd-Keerthipat-7384.pdf

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Rights Keerthipati, Pooja Smruthi

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