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Chimeric Antigen Receptor targeting Prostate Specific Membrane Antigen (PSMA)

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Chimeric Antigen Receptor targeting Prostate Specific Membrane Antigen (PSMA)

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Content Chimeric Antigen Receptors targeting Prostate Specific Membrane
Antigen
By
Parv Barot
A Thesis Presented to the
FACULTY OF THE USC ALFRED E. MANN SCHOOL OF
PHARMACY AND PHARMACEUTICAL SCIENCES
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(PHARMACEUTICAL SCIENCES)
August 2024

ii
Acknowledgements
I am deeply grateful to Dr. Preet M. Chaudhary for the incredible opportunity to join his lab and
for his unwavering support and encouragement throughout my research journey. Despite being a
physician-scientist with an immense workload and a busy schedule, he always found time to
offer his valuable guidance and support. His belief in my abilities allowed me the freedom to
explore various aspects of the lab without restriction, motivating me to take on new
responsibilities and supporting me in every endeavor. I would also like to express my sincere
appreciation to Dr. Hittu Matta and Dr. Sunju Choi for their invaluable guidance and for
imparting their extensive knowledge of cell culture and molecular biology. Dr. Choi's disciplined
nature and strict words were instrumental in my learning and personal growth. My heartfelt
thanks go to Bryant Bravo for his exceptional patience and for always taking the time to teach
me, no matter how many questions I asked. His consistent smile and willingness to help were
greatly appreciated. Additionally, I would like to thank Jennifer, Zhuoyue, Raymond, and Elton
for their constant support and for generously sharing their expertise with me. I am profoundly
thankful to my parents for their unwavering support and continual encouragement in every
aspect of my life. My sincere gratitude extends to Dr. Martine Culty and Dr. Ian Haworth, who,
as my committee members, provided crucial modifications and guidance that greatly enhanced
my work. Their expertise and insights were invaluable. Lastly, I extend my gratitude to all my
friends, relatives, Dr. Preet Chaudhary, and every member of the lab for their collective support
in shaping my journey. Your contributions have made me who I am today. Thank you all.

iii
Table of Contents
Acknowledgements............................................................................................................. ii
List of Figures......................................................................................................................v
Abbreviations.................................................................................................................... vii
Abstract................................................................................................................................x
Chapter 1: Introduction........................................................................................................1
1.1 What is Prostate Cancer? ...................................................................................3
1.2 Pathogenesis of Prostate Cancer........................................................................4
1.3 Prostate Cancer Diagnosis .................................................................................5
1.4 Epidemiology and demographics.......................................................................6
1.5 What is CAR T-cell therapy?.............................................................................9
1.6 Structure of CAR .............................................................................................10
1.7 Types of CAR T-cell therapy...........................................................................14
1.8 Mechanism of Action of CAR T-cell therapy..................................................17
1.9 Success and approvals of CAR T-cell therapies..............................................20
1.10 CAR T-cell therapy toxicity or adverse effects .............................................22
1.11 CAR T-cell therapy for Prostate cancer.........................................................23
1.12 Why PSMA is a target?..................................................................................24
1.13 J591 antibody ................................................................................................27
1.14 Molecular Biology of Plasmid of target construct.........................................28
1.15 Lenti-viral vector ...........................................................................................29
1.16 Topanga Assay...............................................................................................31
1.17 Matador Assay ...............................................................................................32
1.18 ELISA ............................................................................................................33
Chapter 2: Methods............................................................................................................34
2.1 Molecular Biology ...........................................................................................34
2.2 In Vitro Experiments........................................................................................36
2.2.1 Lenti-viral Vector Construction..............................................................36
2.2.2 Lenti-virus Production ............................................................................37
2.2.3 Viral Transduction ..................................................................................38
2.2.4 Co-Culture Assay....................................................................................38
2.2.5 Infection of primary human T-cells with PSMA-CAR virus..................39
2.3 Topanga Assay.................................................................................................39
2.4 Binding Assay and Luminescence Detection ..................................................40
2.5 Matador Assay .................................................................................................40
2.6 ELISA ..............................................................................................................41
2.7 In Vivo experiments.........................................................................................42
2.7.1 In Vivo Testing .......................................................................................42
Chapter 3: Results..............................................................................................................43
3.1 CAR Plasmid Construction..............................................................................43
3.2 Jurkat cell infection..........................................................................................44

iv
3.3 CAR-jurkat cell co-culture with LNCap cell line ............................................44
3.4 T cell infection .................................................................................................45
3.5 Topanga Assay.................................................................................................45
3.6 Matador Assay .................................................................................................46
3.7 ELISA ..............................................................................................................47
3.8 In vivo result ....................................................................................................50
Chapter 4: Discussion ........................................................................................................53
References..........................................................................................................................54

v
List of Figures
Figure 1: Illustration comparing a normal prostate (left) with a prostate affected by
cancer (right)............................................................................................................4
Figure 2: Graph showing the rate of new prostate cancer cases by age group in the USA
in 2019. ....................................................................................................................7
Figure 3: Age-adjusted prostate cancer mortality and incidence rates by race in the USA
from 2000 to 2019....................................................................................................9
Figure 4: Illustration of the evolution of CAR T-cell generations from 1st to 5th,
highlighting structural and functional enhancements such as co-stimulatory
domains (CD28, 4-1BB) and cytokine inducers (IL-12). ......................................13
Figure 5: Comparison of TCR (left) and SIR (right) developed by Dr. Preet Chaudhary,
highlighting structural components including costimulatory molecules, CD3
complex, and variable regions. ..............................................................................14
Figure 6: Autologous Cell Therapy....................................................................................16
Figure 7: Allogenic Cell Therapy ......................................................................................17
Figure 8: Approved CAR T Cell Therapies. ......................................................................21
Figure 9: Number of Clinical Trials Over Time for CAR and TCR Therapies
(2000-2020)............................................................................................................21
Figure 10 (A): PSMA Expression in Prostate Tissue. (B): Chi-Square Test Result. .........26
Figure 11: Representation of PSMA/GCPII transmembrane protein. ...............................26
Figure 12: lentivector gene engineering transfer system. .................................................30
Figure 13: Matador assay (E:T::3:1)..................................................................................46
Figure 14: Matador assay (E:T::1:1)..................................................................................47
Figure 15: Matador assay (E:T::0.3:1)...............................................................................47
Figure 16: ELISA Results of TNF-alpha Comparison between CAR's and CAR
with 4-1BB.............................................................................................................48
Figure 17: ELISA Results of IFN-Gamma Comparison between CAR's and CAR

vi
with 4-1BB.........................................................................................................................49
Figure 18: ELISA Results of IL2 Comparison between CAR's and CAR with 4-1BB.....49
Figure 19: BLI imaging depicting Tumor burden in mice, Survival days and
any conditions. ...................................................................................................................51
Figure 20: Survival curve of 15 NSG mice included in experiments. ...............................52

vii
Abbreviations
ADT - Androgen Deprivation Therapy
AKT - Protein Kinase B (also known as AKT)
ALL - Acute Lymphoblastic Leukemia
APC - Antigen-Presenting Cell
AR - Androgen Receptor
BBB - Blood-Brain Barrier
BLI - Bioluminescence Imaging
BRCA2 - Breast Cancer 2 Gene
CAR - Chimeric Antigen Receptor
CLL - Chronic Lymphocytic Leukemia
CRS - Cytokine Release Syndrome
CT SCAN - Computed Tomography Scan
DHT - Dihydrotestosterone
DMEM - Dulbecco's Modified Eagle Medium
DNA - Deoxyribonucleic Acid
ECD - Extracellular Domain
EGFRvIII - Epidermal Growth Factor Receptor Variant III
ELAC - Exonuclease, Endo- and Polyphosphatase Family Member 1
ELISA - Enzyme-Linked Immunosorbent Assay
ERK - Extracellular Signal-Regulated Kinase
FBS- Fetal Bovine Serum
FOLH1 - Folate Hydrolase 1
GFP - Green Fluorescent Protein
GSK-3 - Glycogen Synthase Kinase 3
GvHD - Graft-versus-Host Disease
HEK cells - Human Embryonic Kidney Cells
HIV - Human Immunodeficiency Virus

viii
HPC gene - Hereditary Prostate Cancer
ICANS - Immune Effector Cell-Associated Neurotoxicity Syndrome
IFNγ - Interferon Gamma
IL-2 - Interleukin 2
ITAM - Immunoreceptor Tyrosine-based Activation Motif
JAK-STAT - Janus Kinase-Signal Transducer and Activator of Transcription
KLF6 - Krüppel-Like Factor 6
LNCap - Lymph Node Carcinoma of the Prostate
MAPK - Mitogen-Activated Protein Kinase
MHC-1 - Major Histocompatibility Complex Class 1
MRI - Magnetic Resonance Imaging
mTORC2 - Mechanistic Target of Rapamycin Complex 2
NF-κB - Nuclear Factor kappa B
NFAT - Nuclear Factor of Activated T-cells
NHL - Non-Hodgkin Lymphoma
NK cells - Natural Killer cells
NSG - NOD scid gamma
PBMC - Peripheral Blood Mononuclear Cells
PCR- Polymerase Chain Reaction
PDK1 - 3-Phosphoinositide-Dependent Protein Kinase 1
PET SCAN - Positron Emission Tomography Scan
PI3K/AKT - Phosphoinositide 3-Kinase/Protein Kinase B
PLCγ1 - Phospholipase C Gamma 1
PSA - Prostate-Specific Antigen
PSAD - Prostate-Specific Antigen Density
PSCA - Prostate Stem Cell Antigen
PSMA - Prostate-Specific Membrane Antigen
QC - Quality Control

ix
RASSF1a - Ras Association Domain Family Member 1
Rb - Retinoblastoma Protein
RNA - Ribonucleic Acid
RPMI - Roswell Park Memorial Institute Medium
ScFv - Single-Chain Variable Fragment
SER - Serine (Amino Acid)
SOC- Super Optimal broth with Catabolite repression
SRD5a2 - Steroid 5 Alpha-Reductase 2
STAT3 - Signal Transducer and Activator of Transcription 3
SYK - Spleen Tyrosine Kinase
TAA - Tumor-Associated Antigen
TCR - T Cell Receptor
TRUS - Transrectal Ultrasound
VH - Variable Heavy (chain)
VL - Variable Light (chain)
ZAP-70 - Zeta-Chain-Associated Protein Kinase 70
PLCγ1 - Phospholipase C Gamma 1
T-UI - T Uninfected
TITAN - Tumor-Infiltrating T-cell Antigen Discovery
TRAIL - Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand
TRIM - Tripartite Motif
TRP - Transient Receptor Potential
TRPV - Transient Receptor Potential Vanilloid
TSLP - Thymic Stromal Lymphopoietin
TTP - Thrombotic Thrombocytopenic Purpura
TXA - Tranexamic Acid
TXN - Thioredoxin

x
Abstract
Chimeric Antigen Receptor (CAR) T-cell therapy has emerged as a promising treatment for
various cancers, including prostate cancer. In this study, we developed CAR T-cells specifically
targeting Prostate-Specific Membrane Antigen (PSMA) to treat prostate cancer. We designed
PSMA-specific next generation CAR comprising a single-chain variable fragment (scFv),
derived from the humanized J591 antibody, into a CAR vector backbone lacking the 4-1BB costimulatory domain, instead incorporating the CD16 co-stimulatory domain. The J591 antibody
was selected for its high specificity and reliability in targeting PSMA, previously utilized in
radio conjugation studies. We hypothesized that the exclusion of the 4-1BB co-stimulatory
domain would mitigate cytokine release syndrome (CRS) and immune effector cell-associated
neurotoxicity syndrome (ICANS) by reducing the excessive release of cytokines such as TNF-α,
IFN-γ, and IL-2. Following restriction enzyme digestion and ligation of the insert DNA
(Deoxyribonucleic Acid) into the vector backbone (4-1BB excluded), we developed lentivirus to
transduce Jurkat cells and performed co-culture experiments to evaluate Nuclear factor of
activated T cells (NFAT) induction against cancer cell lines. Flow cytometry confirmed NFATinduced Green fluorescent protein (GFP) expression, indicating successful transduction.
Peripheral blood mononuclear cells (PBMCs) and T cells were isolated from healthy donors,
activated, and transduced with the CAR construct. Specificity and efficacy assays, including the
Topanga assay, Matador assay, and Enzyme-Linked Immunosorbent Assay (ELISA), were
conducted to assess scFv presence, cytotoxicity, and cytokine release, respectively. Our results
demonstrated a significant reduction in cytokine release for PSMA-PBC2-CAR-T cells
compared to hu-PSMA-scFv-BBz-CAR with 4-1BB costimulatory domain, confirming our
hypothesis. In vivo studies involved engrafting NSG mice with the LNCaP prostate cancer cell
line, followed by administration of the engineered PSMA-PBC2-CAR-T. Tumor regression,

xi
survival curves, and tumor burden analysis indicated enhanced survival in treated subjects,
although some experienced graft-versus-host disease (GvHD), indicating the need for further
optimization. In conclusion, our study provides evidence that CAR T cells targeting PSMA
without the 4-1BB co-stimulatory domain can effectively reduce cytokine release and
demonstrate potential in treating prostate cancer, with in vivo results showing increased survival
rates. Further research is necessary to address GvHD and enhance the therapeutic efficacy of
these engineered CAR T-cells.

1
Chapter 1: Introduction
Prostate cancer is a major health concern for men globally. It ranks among the most frequently
diagnosed cancers. In 2023, worldwide, 1.5 million cases were diagnosed. This translates to 1:8
men facing a prostate cancer diagnosis in their lifetime. Tragically, the disease also claimed an
estimated 246,000 lives in 20211
. In recent trends, prostate cancer is treated using vaccines,
androgen deprivation therapy (ADT), immunotherapy, and surgery. While these options offer
improved success rates, they can also lead to long-term side effects and even the possibility of
relapse. Immunotherapy, with its potential for targeted effects and reduced toxicity, is a highly
researched area2
. One of the immunotherapy approaches, Chimeric Antigen Receptor (CAR) Tcell therapy, has been seen to play a significant role in the treatment of non-solid cancers3
. CAR
T-cell therapy revolutionizes cancer treatment by using genetic engineering to modify a patient's
T cells to express synthetically produced receptors. These receptors can target tumor antigens and
induce cytotoxic or cytolytic effects on cancer cells4
. Activated CAR-T cells bind to the epitopes
on tumor cells, initiating mechanisms to induce cytotoxicity within them. Though this therapy has
shown promising results against hematological malignancies, its effectiveness against solid tumors
remains limited due to factors such as off-target effects, the immunosuppressive tumor
environment, and limited availability of target antigens5
. This thesis focuses on the development
CAR T-cells targeting Prostate-Specific Membrane Antigen (PSMA) receptors in prostate cancer.
PSMA presents a promising target for CAR T-cell therapy due to its overexpression in tumor cells,
potentially minimizing side effects6
. This PSMA-targeted strategy is highly specific and effective.
Our approach involves developing next-generation proprietary CAR T-cells specifically targeted
against PSMA. The development of CAR T-cells begins with constructing a plasmid that expresses
the desired CAR on T cells. This process utilizes molecular cloning techniques to obtain purified

2
plasmid and sequencing analysis to confirm the desired plasmid of target construct. The confirmed
plasmid is transfected on human embryonic kidney (HEK) 293FT cells to produce lentivirus,
which is subsequently transduced into Jurkat cells expressing GFP under an NFAT promoter (JNG
cells). JNG cells bearing the PSMA-CAR are co-cultured with the Lymph Node Carcinoma of the
Prostate (LNCaP) prostate cancer cell line. Using Flow cytometry, we observed the Green
Fluorescent Protein (GFP) expression upon co-culture with the PSMA-expressing LNCaP cells.
To confirm that PSMA-CAR works when expressed in T cells, we next generated lentiviral vectors
expressing the PSMA CAR construct based on hu-PSMA-1-J591 vL/VH fragments in a vector
comprising the CAR backbone consisting of the CD16 transmembrane and cytosolic domain and
lacking any costimulatory domain. We also generated the corresponding hu-PSMA-scFv-BBzCAR with 4-1BB costimulatory domain. Lentiviral vectors were generated and used to infect CD3
selected human primary T cells isolated from the Peripheral Blood Mononuclear Cell (PBMC)
from a healthy donor. These cells were evaluated by Matador assay for cell death induction by
PSMA-CAR-T cells when co-cultured with PSMA-positive LNCaP cell line. Enzyme-Linked
Immunosorbent Assay (ELISA) for human Interferon-gamma (IFN-γ), Interleukin-2 (IL-2) and
Tumor Necrosis Factor-alpha (TNFα), was done to confirm the cytokine production by PSMACAR-CD16 and PSMA-BBz-CAR T cells. Following successful in vitro results, the study
progresses to an in vivo murine model using 15 NOD scid gamma (NSG) mice. First, we inoculated
LNCaP tumor cells via subcutaneous route (sc) of administration in mice and confirmed the tumor
implantation by bioluminescence imaging (BLI). Subsequently, the mice were divided into three
groups for control group with tumor cells alone, for T-uninfected (T-UI), and PSMA-CAR-T test
group and administered the corresponding treatment. BLI is conducted weekly to measure tumor
growth. The results demonstrate a decrease in tumor burden and prolonged survival in the PSMA-

3
CAR test group. However, the test animals also exhibited adverse effects, including Graft-versusHost Disease (GvHD), a known side effect of this therapy. My thesis describes the development
and preclinical evaluation of immune therapy which targets PSMA in prostate cancer. Our findings
demonstrated the potent anticancer activity of CAR-T cells in both in vitro and in vivo models.
1.1 What is Prostate Cancer?
Prostate cancer is a malignancy arising from the prostate gland, situated in the lower abdomen of
males. The prostate gland's role is to secrete seminal fluid, which helps in the nourishment and
transportation of sperm7
. Prostate cancer arises from the uncontrolled and aberrant proliferation of
cells within the prostate gland. This malignancy predominantly affects males aged 65 and older
and is characterized by a typically indolent progression8
. This gradual progression underscores the
challenges in early detection and treatment, as asymptomatic phases can persist for extended
periods before clinical manifestation. Prostate cancer can remain asymptomatic for up to a decade
while the tumor is localized within the prostate gland9
. By the time clinical symptoms emerge, the
disease is often at an advanced and critical stage, significantly complicating treatment efficacy and
prognosis. While prostate tumors typically exhibit slow growth, the transition from localized to
metastatic disease can profoundly impact clinical management and outcomes. Thus, a thorough
understanding of the molecular and cellular mechanisms involved is crucial10
.

4
Figure 1: Illustration comparing a normal prostate (left) with a prostate affected by cancer
(right)11
.
1.2 Pathogenesis of Prostate Cancer
Prostate cancer is influenced by both genetic and environmental factors, with several pathways contributing
to its initiation and progression. Hereditary risk factors play a significant role, as individuals with close
relatives diagnosed with prostate cancer have a higher risk12. This familial aggregation increases the risk
two-fold if a father or brother is affected, and five to eleven times with two or more first-degree relatives
diagnosed8
. Key genes involved include Hereditary Prostate Cancer (HPC)-1 (Ribonuclease L, RNASEL)
on chromosome 1q24-25, which impairs immune function when mutated; HPC-2 (ElaC ribonuclease Z 2
ELAC2- Elongation Factor, RNA 3) on chromosome 17, where variant alleles increase cancer risk;
Macrophage Scavenger Receptor 1 (MSR1) on chromosome 8p22, where mutations disrupt innate
immunity; and Breast Cancer 2, early onset (BRCA2) and Checkpoint Kinase 2 (CHEK2), which are linked
to DNA repair and cellular response to DNA damage, with mutations increasing cancer risk13,14. Genetic
polymorphisms also contribute, with variations in the Androgen Receptor (AR) gene decreasing
transactivation and binding activity, and polymorphisms in Steroid 5-alpha-reductase 2 (SRD5A2) affecting
enzyme activity and impacting dihydrotestosterone (DHT) levels, thus increasing cancer risk15. Somatic
mutations further contribute to prostate cancer, with loss of heterozygosity in Nuclear Factor X, subunit
3.1. (NFX 3.1) on chromosome 8p21 disrupting cellular processes, and loss of Phosphatase and TENsin
homolog (PTEN) on chromosome 10q leading to uncontrolled cell division. Aberrant DNA methylation

5
silences genes like Glutathione S-transferase pi 1 (GSTP1) and Ras association domain family member 1A
(RASSF1A), promoting tumor growth, while overexpression of Krüppel-like Factor 6 (KLF6) and Prostate
Stem Cell Antigen (PSCA) is linked to aggressive prostate cancer forms16. Mutations or loss of expression
in the Retinoblastoma 1 (Rb) gene on chromosome 13q have also been reported, with restoring Rb activity
shown to suppress tumorigenesis. Prostate cancer development involves a complex interplay between
inherited genetic predispositions and somatic mutations, and understanding these factors is crucial for
identifying at-risk individuals and developing targeted therapies7,17
.
1.3 Prostate Cancer Diagnosis1. Digital Rectal Examination: This involves the physical examination of the prostate gland via
the rectal route. Despite its use as an initial clinical assessment for prostate cancer, it still has
some limitations. With a moderate positive prediction of around 50%, it can be indecisive.
However, it is still useful for the preliminary detection18
.
2. Prostate-Specific Antigen (PSA) Testing: Its levels in the blood are used as a biomarker tool to
assess prostate conditions, including cancer. A PSA which is prostate secreted protein level
below 4 ng/ml per milliliter is considered normal, while levels beyond 4 till 10 ng/ml represent
a gray area for diagnosis19. To make this testing more stringent, age-specific cutoff points have
been developed, as PSA levels increase with age due to benign conditions. PSA density
(PSAD), velocity, and the ratio of free PSA versus total PSA are also explored as diagnostic
accuracy tools. However, they have limitations as they are not specific to cancer as there may
be other reasons for elevation as well20
.
3. Transrectal Ultrasonography (TRUS) and Biopsies: TRUS involves imaging of the gland &
vesicles with probe inserted into the rectum, which can also guide biopsy sample collection for
lab testing. TRUS provides information about any structural abnormalities in the prostate

6
gland, such as cyst formation, and biopsy sampling tests demonstrate an important level of
accuracy. Sometimes, repeat and saturation biopsies are performed to confirm diagnosis and
identify specific sites. Saturation biopsy involves taking multiple samples from various regions
of the prostate for tumor detection and more accurate findings21
.
4. Ultrasound-Guided Transperineal and Template-Guided Biopsies: This is an alternative
technique to transrectal biopsies for patients who cannot undergo rectal procedures or require
extensive sampling. In this method, a grid pattern is used for sampling against the perianal
skin. These techniques have a high detection rate and provide more accurate results compared
to other techniques22
.
5. Positron Emission Tomography (PET) scan- It employs specific radiotracers which are
advanced diagnostic tools for prostate cancer due to their ability to detect and visualize
biochemical processes at the molecular level. These scans involve injecting a radioactive
substance that binds selectively to prostate cancer cells, enabling precise detection of malignant
tissue with high sensitivity and specificity. This capability not only aids in accurate staging and
localization of tumors but also provides valuable information for treatment planning and
monitoring response to therapy. PET remains a powerful tool in the armamentarium against
prostate cancer, offering critical insights into disease biology and management23
.
1.4 Epidemiology and demographics
Age-Related Patterns in Prostate Cancer Incidence- Prostate cancer afflicts elderly males, with age
representing a pivotal factor in its onset. This assertion finds robust support in epidemiological
investigations, exemplified by the illustrated data. The graphical representation elucidates a high
rate of incidence in newly diagnosed cases of prostate cancer among individuals above 40 years

7
of age. Subsequently, a gradual uptick in incidence emerges among men in their 40s and 50s,
culminating in a marked escalation within the demographic aged 60 years and above24. The
trajectory persists, depicting a consistent elevation in diagnosis rates with progressive age, peaking
among those surpassing 85 years. This discernible association between advancing age and
heightened susceptibility to prostate cancer underscores the imperative of tailoring screening
protocols to specific age cohorts. Embracing early detection methodologies, inclusive of routine
screenings, holds promise for facilitating prompt interventions and therapeutic interventions,
thereby fostering the prospect of enhanced patient prognosis24,25
.

Figure 2: Graph showing the rate of new prostate cancer cases by age group in the USA in
201926
.

8
Race Disparity in Prostate cancer- Racial disparities in prostate cancer are a critical public health
issue, with African American men disproportionately affected compared to other racial groups.
Epidemiological data indicate that African-American men have a 60% higher incidence rate of
prostate cancer and are more than twice as likely to die from the disease compared to white men27r.
These disparities can be attributed to several factors. Firstly, prostate cancers in African-American
men tend to be more aggressive and are often diagnosed at later stages, contributing to poorer
prognoses and higher mortality rates. Studies have shown that African-American men are 2.5 times
more likely to be diagnosed with advanced-stage prostate cancer compared to their white
counterparts. Secondly, disparities in healthcare access and quality significantly impact
outcomes27,28. African-American men often experience barriers to healthcare, including limited
access to screening and diagnostic services, lower rates of health insurance coverage, and
disparities in the availability of high-quality treatment options. Research has shown that AfricanAmerican men are less likely to participate in routine screenings such as prostate-specific antigen
(PSA) tests, leading to delayed detection and treatment. Addressing these disparities requires a
multifaceted approach, including improving access to healthcare, implementing targeted screening
programs, and providing culturally sensitive health education28
.

9
Figure 3: Age-adjusted prostate cancer mortality and incidence rates by race in the USA from
2000 to 201828
.
1.5 What is CAR-T cell therapy?
It involves the genetic engineering of T cells from individuals to obtain synthetically produced
CAR on their T cells. These receptors are engineered to bind to specific tumor antigens and start
the process of cytotoxicity in tumor cells29. CARs are synthetic receptors engineered onto the
surface of T cells, composed of a fused protein of a single-chain variable fragment (scFv).
Structurally, CARs consist of four parts: scFv, hinge, transmembrane domain, and immunoreceptor
tyrosine-based activation motif (ITAM). The scFv, located at the extracellular domain, serves as
the binding domain for target antigen recognition30
. Upon binding, it activates signaling pathways
that result in T cell activation. The number of co-stimulatory domains plays a major role in this

10
activation. Early first-generation CARs had a simplistic design and lacked co-stimulatory domains.
As research advanced, scientists incorporated additional co-stimulatory domains to enhance the
extent of T cell activation and increase cytotoxic effects. CARs differ in their generations based
on their structural composition, with each generation incorporating advancements in design and
functionality29,31
.
1.6 Structure of CAR
The scFv which binds to antigen constitutes the extracellular domain of CAR structure, which
serves a major role in identifying antigen on antigen presenting cells (APC) i.e. tumor cells.
Generated through genetic engineering techniques employing viral or non-viral vectors, the
extracellular scFv domain consists of heavy chain variable (vH) and light chains variable (vL) of
antibodies using linker or hinge region. This ScFv domain exhibits high specificity and affinity
towards its target antigen-binding site32. The linker or hinge region, typically composed of short
peptide chains rich in glycine or serine residues, facilitates the attachment between the heavy and
light chains. Both chains synergistically contribute to antigen recognition. The transmembrane
domain is situated within the lipid bilayer of the T-cell membrane, serving as an anchoring
mechanism that facilitates functional interactions between the scFv and ITAM domains of the CAR
structure32. Comprising hydrophobic or mildly acidic residues such as leucine, isoleucine, valine,
and phenylalanine, the transmembrane domain exhibits a strong affinity for the hydrophobic lipid
core of the cell membrane. Adopting an alpha-helical conformation, variations in the structure,
such as alpha helix tilt, kinks, and bends, may influence the orientation and dynamics of the protein
within the membrane. The transmembrane domain does anchoring, oligomerization, signal
transduction, and intracellular signaling, which are experimentally validated through various

11
biochemical assays33. ITAM are made of amino acids responsible for transducing activating signals
upon antigen recognition in the CAR antigen-binding site. Upon engagement with the target
antigen, ITAMs undergo phosphorylation and activate downstream signaling molecules. Costimulatory domains (4-1BB and CD28) and CD3ζ, enhance T-cell activation. Negative regulatory
mechanisms mediated by phosphatases such as Src homology region 2 domain-containing
phosphatase-1/2 (SHP-1 and SHP-2) prevent excessive T-cell activation, thereby maintaining
immune homeostasis and safeguarding against autoimmune dysfunction34
.
• 1st generation CAR T: These CAR constructs include only the signaling domain of CD3ζ
chain. They lack a co-stimulatory signaling domain, resulting in limited efficiency towards
target antigens35
.
• 2nd generation CAR-T: Developed to improve upon the first generation, these CARs
include a co-stimulatory domain as CD28 or 4-1BB. In addition of CD3ζ signaling chain
which improves activation against cancer33
.
• 3rd generation CAR-T: These CARs are designed by incorporating 2 co-stimulatory
domains, typically CD28 and 4-1BB, along with the CD3ζ signaling domain. The addition
of these three stimulatory domains increases the effectiveness of the treatment36
.
• 4th generation CAR-T: Referred to as T cells redirected for universal cytokine-mediated
killing, these CAR T cells are designed to secrete specific cytokines such as IL-12 upon
antigen recognition. This cytokine secretion aims to create a pro-inflammatory response to
stimulate immunity37
.
• 5th Generation CAR-T: The latest advancement in CAR T cell design, these cells have a
structure like 4th generation CARs but instead of IL-12, they possess IL-2-2R beta. This
enhances cytokine release by following the Signal Transducer and Activator of

12
Transcription 3 (STAT3) and Janus Kinase (JAK) pathways as cellular signaling
pathways38
.
• Synthetic Immune Receptor (SIR-T) - Dr. Preet Chaudhary has developed a novel nextgeneration CAR-T cell therapy that integrates the favorable attributes of both T-cell
receptor (TCR) and CAR technologies. This innovative therapy incorporates multiple costimulatory domains characteristic of TCRs, enhancing signal transduction pathways
critical for robust immune responses. Simultaneously, it leverages CAR's single-chain
variable fragment (scFv) design, integrating both vH and vL chains to optimize antigen
specificity and binding affinity. This approach effectively circumvents the requirement for
Major Histocompatibility Complex class I (MHC-1) binding seen in traditional TCR
therapies, while addressing CAR's challenges related to simplicity, high toxicity, and the
potential for dysregulated cytotoxic release leading to cytokine release syndrome (CRS) or
immune effector cell-associated neurotoxicity syndrome (ICANS). Dr. Chaudhary's
innovative therapy represents a significant advancement in the field, promising improved
safety and efficacy profiles for CAR-T cell therapies.

13
Figure 4: Illustration of the evolution of CAR T-cell generations from 1st to 5th,
highlighting structural and functional enhancements such as co-stimulatory domains
(CD28, 4-1BB) and cytokine inducers (IL-12).

14
Figure 5: Comparison of TCR (left) and SIR (right) developed by Dr. Preet Chaudhary,
highlighting structural components including costimulatory molecules, CD3 complex, and
variable regions.
1.7 Types of CAR T-cell therapy
CAR T-cell therapies are of two types based on the source of T-cells.
1. Autologous CAR T-cell therapy- The term "autologous" originates from "auto," meaning
self, indicating that the patient's own T cells are equipped with CAR before being
reintroduced into their body. The process begins with harvesting the patient's own T cells
which are genetically engineered to express a CAR that target specific antigens found on
cancer cells. This approach minimizes the risk of GvHD since the immune system
recognizes these cells as self. However, this method is time-consuming due to the need for
cell culturing and genetic modification, and it carries the potential for manufacturing errors
and stimulated in a lab setting before reintroduction into the patient's body39
.
It is eight step process through which any patient needs to pass to get autologous CAR T-cell
therapy treatment.

15
1) Patient evaluation: Patients undergo a thorough medical examination to assess their disease
status and overall health, ensuring they meet the inclusion criteria for therapy.
2) Leukapheresis is a procedure where blood is drawn from the patient, and PBMC having T
cells are separated out through an intravenous (IV) route. The collected blood is then
separated, with T cells isolated and collected while other components are returned to the
patient.
3) T-cell activation and modification: The isolated T cells are activated and expanded in the
laboratory using specific growth factors and cytokines to stimulate proliferation.
Subsequently, the activated T cells are modified to express CAR by transducing them with
genetically engineered plasmid constructs.
4) CAR-T cell expansion: The altered/modified CAR-T cells are expanded in number within
the laboratory until enough is obtained for infusion back into the patient.
5) Characterization: Quality control (QC), purity, and potency testing are conducted to assess
the modified T cells for any adverse genetic alterations. Various characterization assays are
performed to check the quality and efficacy.
6) Lymphodepletion: Before administration, patients undergo chemotherapy to suppress their
immune system, creating space for the genetically modified immune cells to effectively
expand.
7) CAR-T cell infusion: Once the cells undergo characterization, they are introduced into the
patient's bloodstream via the IV route. Patients usually undergo hospitalization with
medical oversight during this process.

16
8) Follow-up: Patients are closely monitored for CAR T-cell-related toxicities and adverse
effects, including neurotoxicity. Regular follow-up assessments are conducted to ensure
patient safety and efficacy of the treatment40,41
.
2. Allogeneic CAR T-cell therapy involves harvesting T cells from a healthy donor and then
genetically modifying them to express CAR tailored to specific antigens. However, this
promising approach is challenged by the significant risk of GvHD42. GvHD occurs when
the donor T cells recognize the recipient's tissues as foreign, leading to potentially severe
immune reactions. Managing this immunological mismatch is crucial, highlighting the
complexity and careful considerations needed in allogeneic CAR T-cell therapy to ensure
safety and efficacy43
.
Figure 6: Autologous Cell Therapy.

17
Figure 7: Allogenic Cell Therapy44
1.8 Mechanism of Action of CAR T-Cell Therapy
There are 5 step cascade process in the mechanism of action of CAR-T.
1. Antigen Recognition and binding- The scFv component of CAR-T cells plays a pivotal role in
antigen recognition. Upon encountering a tumor-associated antigen (TAA), the scFv,
composed of linked vH and vL chains, facilitates precise binding to the antigen epitope on the
surface of tumor cells. This interaction occurs at the extracellular domain of the CAR T-cell,
initiating a cascade of events that culminates in the formation of an immunological synapse
between the CAR T-cell and the target antigen45
.
2. CAR T-Cell Activation- Following antigen recognition, CAR-T cells undergo activation
characterized by the phosphorylation of ITAM within the CD3ζ signaling domain of the CAR
construct. The engagement of the scFv with the TAA induces a conformational change that
triggers ITAM phosphorylation, essential for the recruitment and activation of downstream

18
signaling molecules such as Zeta-chain-associated protein kinase 70 (ZAP-70) and Phospholipase
C gamma 1 (PLCγ1). This signaling cascade leads to the release of cytotoxic molecules,
cytokines, and chemokines, driving CAR T-cell effector functions including target cell lysis
and immune modulation within the tumor microenvironment46
.
3. Signal Transduction: Phosphorylated ITAMs serve as docking sites for downstream signaling
molecules, including ZAP-70 (Zeta-chain-associated protein kinase 70) and Syk (Spleen
tyrosine kinase), which propagate a complex signaling cascade. This activation induces various
pathways involved in CAR-T cell effector functions, such as the activation of NF-κB (Nuclear
Factor kappa-light-chain-enhancer of activated B cells) and MAPK (Mitogen-Activated
Protein Kinase) pathways, ultimately regulating gene expression, cytokine production, and
cellular proliferation. Additionally, the Phosphoinositide 3-Kinase (PI3K)/Akt pathway is
activated, contributing to CAR T-cell survival, proliferation, and metabolic activities crucial
for sustained anti-tumor responses. These signaling events orchestrate the potent anti-tumor
activity of CAR T-cells and contribute to their therapeutic efficacy in treating malignancies39
.
• Mitogen-Activated Protein Kinase (MAPK) Pathway: The MAPK pathway is important
signaling cascade which is responsible for proliferation, differentiation, and survival. Upon
CAR T-cell activation, phosphorylation events initiated by Src family kinases Various
transcription factors, such as Elk-1 and c-Fos, are phosphorylated by activated Extracellular
Signal-Regulated Kinase (ERK) as it enters the nucleus, initiating the transcriptional regulation
of genes47
.
• PI3K/Akt Pathway: Responsible for regulating cell growth, metabolism, and survival, this
pathway undergoes activation upon CAR T-cell stimulation. Phosphorylated ITAM motifs
recruit and trigger the activation of PI3K, resulting in the generation of phosphatidylinositol

19
(3,4,5)-trisphosphate (PIP3). PIP3 functions as a binding site for Akt, which becomes
phosphorylated and activated by both phosphoinositide-dependent kinase-1 (PDK1) and
mammalian target of rapamycin complex 2 (mTORC2). Once activated, Akt modulates various
downstream effectors, including mTORC1, glycogen synthase kinase 3 (GSK-3), and the FoxO
family of transcription factors, to support cell survival, protein synthesis, and metabolic
adaptation48
.
• Nuclear Factor-Kappa B (NF-κB) Pathway: Vital for regulating inflammation, immune
responses, and cell survival, the NF-κB pathway undergoes activatio1
n upon CAR T-cell
stimulation. This activation involves the phosphorylation and subsequent degradation of IκB
(inhibitor of κB) proteins, which typically sequester NF-κB dimers in the cytoplasm. With IκB
degraded, NF-κB dimers, usually comprised of p50 and RelA (p65) subunits, translocate to the
nucleus. There, they oversee the transcription of genes associated with inflammation, immune
responses, and cell survival. NF-κB activation in CAR-T cells prompts the production of
proinflammatory cytokines such as IL-2 and TNF-α, along with an upsurge in anti-apoptotic
proteins, thus bolstering CAR T-cell activation and viability49
.
4. Cytokine secretion- CAR-T cells execute anti-tumor activities through multifaceted
mechanisms, prominently including direct cytotoxicity achieved by the release of cytolytic
molecules that form pores in the cancer cell membrane, culminating in cellular lysis.
Concurrently, activated CAR-T cells secrete a spectrum of pro-inflammatory cytokines such
as IFN-γ, TNF-α, and IL-2, pivotal in orchestrating immune responses and promoting cancer
cell demise. These cytokines play integral roles in activating downstream signaling pathways
and modulating the tumor microenvironment to potentiate anti-tumor immune responses.
Furthermore, CAR-T cells exhibit memory-like characteristics, enabling heightened

20
responsiveness upon antigen re-encounter, thus bolstering their efficacy in combating
malignancies50
.
1.9 Success and approvals of CAR T-cell therapies
CAR T-cell therapy has shown remarkable efficacy in treating various liquid tumors, as evidenced
by its successful approvals and treatment outcomes in diseases like acute lymphoblastic leukemia
(ALL), chronic lymphocytic leukemia (CLL), and non-Hodgkin lymphoma (NHL). Notably, CAR
T-cells designed to target CD19 have demonstrated exceptional effectiveness in managing these
blood cancers51. However, its application in solid tumors encounters obstacles, including the
immunosuppressive nature of the tumor microenvironment and the variable expression of antigens.
Current research endeavors strive to surmount these challenges by directing attention towards
liquid tumors, identifying potential targets such as epidermal growth factor receptor variant III
(EGFRvIII) and glypican-2 (GD2). In the context of prostate cancer, ongoing investigations are
exploring multiple targets, including PSMA, PSCA, and the androgen receptor (AR)52
.

21
Figure 8: Approved CAR T-Cell Therapies53
.
Figure 9: Number of Clinical Trials Over Time for CAR and TCR Therapies (2000-2020)54
.

22
1.10 CAR T-cell therapy toxicity or adverse effects
This therapy is often associated with significant post-administration adverse effects, notably CRS
and ICANS, both of which can be severe and potentially fatal but manageable with appropriate
interventions. CRS, the most prevalent complication of CAR T-cell therapy, is triggered by CAR
T-cell binding to target antigens, leading to the release of cytotoxic molecules and proinflammatory cytokines, including IL-6, IL-10, and IFN-gamma. Elevated cytokine levels can
affect multiple organ systems, resulting in symptoms such as fever, hypotension, hypoxia, and
organ toxicity55. CRS typically manifests within one to two weeks post-administration, with onset
usually occurring within two to three days. Clinical trials such as CYCARTA and Kymriah have
reported incidence rates of grade 3 CRS at 13% and 49%, respectively. CRS symptoms generally
resolve within approximately three weeks, with management strategies involving tocilizumab, an
anti-IL-6 receptor antagonist, for low-grade CRS, and corticosteroids for fever and hypotension.
Tocilizumab is particularly pivotal in managing CRS as it mitigates symptoms without
compromising therapy efficacy56
.
Neurological events associated with this therapy manifest through symptoms such as confusion,
muscle weakness, convergent and non-convergent seizures, and cerebral edema. These events
typically occur in two distinct phases: an initial phase within the first five days post-therapy and a
subsequent phase thereafter. The initial phase, often milder and shorter, coincides with CRS, during
which elevated levels IL-6 facilitate increased blood-brain barrier (BBB) permeability, allowing
cytokines to infiltrate the central nervous system (CNS) and precipitate ICANS57. Management
strategies for ICANS involve neuroimaging techniques such as MRI (Magnetic Resonance
Imaging) and CT (Computed Tomography) scans to assess neurological status. Intravenous
dexamethasone is commonly administered to mitigate neurotoxicity58. In addition to neurological

23
complications, other notable effects of this therapy include B-cell aplasia,
hypogammaglobulinemia, and cytopenia, all of which heighten susceptibility to infections. GvHD
poses a significant challenge in allogeneic CAR T-cell therapy, where donor T cells recognize
recipient tissues as foreign, triggering an immune response. Acute GvHD typically manifests
within 100 days post-transplantation, affecting the skin, gastrointestinal tract, and liver, with
symptoms such as rash, diarrhea, abdominal pain, and liver dysfunction. Treatment often involves
immunosuppressive agents like corticosteroids. Chronic GvHD, occurring beyond 100 days posttransplantation, can involve multiple organs including the skin, mouth, eyes, lungs, joints, and
liver, leading to long-term complications. Management requires prolonged immunosuppressive
therapy and supportive care to alleviate symptoms and improve patient outcomes. Both acute and
chronic GvHD significantly impact therapy success, necessitating careful immune modulation to
balance graft-versus-tumor effects with the risks of immune-related complications59
.
1.11 CAR T-cell therapy for Prostate cancer
Due to the increased risk and number of diagnosed cases of prostate cancer, scientists and
physicians are engaged in rigorous research to discover novel therapeutics with fewer side effects
and greater effectiveness against the Prostate Cancer60. Various treatment options are currently
available for prostate cancer, including androgen receptor (AR) inhibitors, chemotherapy, radiation
therapy, immunotherapy, and others. Among these, this therapy is the most advanced version of
immunotherapy for prostate cancer. Preclinical studies and early-phase clinical trials have also
yielded positive results, demonstrating its efficacy against cancer and its potential as a treatment60
.
However, this therapy not shown promising results for solid tumors, including prostate cancer,
which exhibit heterogeneity in antigen expression and restricted tumor accessibility. Targets such
as PSCA, PSA, AR, and PSMA have demonstrated encouraging results in reducing tumor growth

24
and prolonging survival61. Early-phase clinical trials have indicated the safety, feasibility, and
potential efficacy of CAR T-cell therapy, although certain toxicities and side effects have been
observed. Future developments in fifth-generation CAR T-cell therapy and novel approaches may
lead to highly optimized, improved specificity in targeting, and better clinical outcomes62
.
1.12 Why PSMA is a target?
PSMA, alternatively termed Folate Hydrolase-1 (FOLH1), stands as a pivotal player in prostate
cancer treatment which was discovered in 1986. Originating from the FOLH1 (Folate Hydrolase
1) gene situated on chromosome 11p11.2, PSMA emerges as a type II transmembrane glycoprotein,
showcasing a complex structure delineated into three primary segments: the cytoplasmic domain,
transmembrane domain, and extracellular domain. Within the extracellular domain, PSMA harbors
folate hydrolase and zinc-binding motifs, facilitating its binding capabilities and thereby
establishing its significance in folate metabolism6,63. Additionally, PSMA demonstrates enzymatic
functions encompassing folate hydrolase and N-acetylated-alpha-linked-acidic dipeptidase
(NAALADase), thus contributing to the regulation of intracellular folate levels and cleavage of
neuropeptides. Moreover, PSMA participates in cell signaling, activating MAPK and Akt signaling
cascades to promote cellular proliferation. PSMA expression is highly upregulated in patients with
prostate cancer. Its large extracellular domain makes it an attractive target for therapeutic and
diagnostic purposes64. PSMA is considered a potential target for delivering cytotoxic agents, as it
can help transport cytokines and other cytotoxic molecules, thereby promoting cytotoxicity
signaling. In addition to prostate cancer cells, PSMA receptors are also present in tissues such as
the kidney, brain, salivary glands, and small intestine. However, BBB acts as a shield in the brain,
preventing antibodies or other molecules from passing through and binding to receptors off-target.
Initially, Capromab Pentetide (7E11-C35 ) which was labeled with 111In, was the only FDA-

25
approved antibody targeting PSMA65. However, it was not very effective due to non-specific
binding and lack of sensitivity. Subsequently, scientists developed the humanized J591 antibody,
which exhibited higher levels of specificity and sensitivity. J591 has been used in various clinical
trials. Researchers have also pursued other approaches to develop more specific humanized
antibodies targeting the extracellular domain of PSMA66. PSMA displays highly selective and
upregulated expression in prostate cancer cells, rendering it a potential target and biomarker. This
specificity and differential expression pattern make PSMA an ideal target for CAR-T
immunotherapy66. PSMA expression persists in metastatic lesions, lymph nodes, and organs,
underscoring its relevance for systemic therapy. Upon ligand binding, PSMA undergoes
internalization, facilitating the uptake of therapeutic agents for cytokine or cytotoxic agent
production to target cancer cells. PSMA-targeted CAR imaging agents, such as radiolabeled
antibodies like PSMA-617 and PSMA-11, have been developed. PSMA-targeted strategies,
including the use of PSMA-specific CARs, effectively recognize and eliminate prostate cancer
cells. Overall, PSMA represents a compelling molecular target due to its complex structure,
internalization capabilities, and association with tumor aggressiveness67
.

26
Figure 10 (A): PSMA Expression in Prostate Tissue. (B): Chi-Square Test Result68
.
Figure 11: Representation of PSMA/GCPII transmembrane protein69
.

27
1.13 J591 antibody
The J591 antibody, also known as huJ591, is an anti-PSMA monoclonal antibody designed
specifically to target PSMA. J591's specificity to PSMA is central to its role in prostate cancer
treatment. As an anti-PSMA antibody, J591 binds selectively and with high affinity to PSMA
molecules. This binding specificity enables precise imaging using nuclear medicine techniques
such as PET (Positron Emission Tomography) and SPECT (Single-Photon Emission Computed
Tomography)70. By conjugating J591 with radionuclides like 111In or 89Zr, clinicians can accurately
visualize and localize PSMA-positive prostate cancer lesions throughout the body. This capability
facilitates accurate staging of the disease, monitoring of treatment response, and detection of
recurrent tumors, providing critical information for clinical decision-making71. In addition to its
diagnostic utility, J591 holds significant promise as a therapeutic agent in prostate cancer. When
coupled with cytotoxic payloads such as radioisotopes (177Lu, 225Ac) or potent toxins
(maytansinoids, auristatins), J591 delivers targeted therapy directly to PSMA-expressing cancer
cells72. This approach enhances therapeutic efficacy by concentrating treatment at the tumor site
while minimizing damage to healthy tissues, thereby reducing systemic side effects and improving
patient outcomes73. This targeted therapeutic strategy is particularly advantageous in treating
advanced or recurrent prostate cancers that are resistant to conventional treatments. Clinical trials
have demonstrated J591's efficacy and safety profile in prostate cancer patients, supporting its role
in personalized treatment approaches. These trials explore combination therapies involving J591
with chemotherapy, hormone therapy, and other targeted agents, aiming to optimize treatment
regimens and overcome therapeutic resistance mechanisms74. Ongoing research continues to refine
J591's binding properties, explore novel conjugates and therapeutic combinations, and identify

28
biomarkers that predict patient response, further advancing its clinical utility and expanding
treatment options for prostate cancer patients75
.
1.14 Molecular Biology of Plasmid of target construct
The process of molecular biology involved in constructing a plasmid target begins with designing
the DNA sequence of interest, typically a gene or regulatory element, and its surrounding regions.
The first step is to isolate a plasmid vector and the DNA insert that contains the target sequence.
Next, the plasmid vector and the DNA insert are subjected to digestion using specific restriction
enzymes. These enzymes cut the DNA at precise recognition sequences, allowing for the removal
of unwanted sequences and ensuring compatibility between the vector and insert ends. Following
digestion, the vector and insert are mixed and ligated using DNA ligase. This enzyme catalyzes
the formation of phosphodiester bonds between the cohesive ends of the vector and insert, resulting
in a recombinant DNA molecule. The ligated product, now containing the target construct, is
introduced into host cells through a process called transformation. Host cells, Escherichia coli (E.
coli), take up the recombinant plasmid DNA. After transformation, bacteria harboring the
recombinant plasmids are selected using antibiotic resistance (Carbenicillin) markers present on
the plasmid vector. This selection ensures that only cells containing the desired plasmid grow and
multiply. Subsequently, bacterial cultures are grown on a larger scale to obtain enough plasmid
DNA. Depending on the required quantity, preparations can be conducted at mini, midi, or maxi
scales. After purification, the plasmid DNA is analyzed to verify the presence and integrity of the
target construct. This is typically achieved through sequencing, where primers are used to initiate
DNA synthesis and confirm the sequence accuracy and fidelity of the inserted DNA fragment.

29
1.15 Lentiviral vectors
A subtype of retroviruses, play a pivotal role in genetic engineering and immunotherapy. Evolving
from the innovation of the human immunodeficiency virus (HIV), lentiviral vectors serve as
carriers for genetic material, offering vital tools for genetic engineering and immunotherapy
endeavors. Leveraging the intricate biology of lentiviruses, researchers have explored their utility
in developing innovative therapies, such as CAR-T therapy, which holds great promise in genetic
modification and targeted cancer immunotherapy. Engineered lentiviral vectors serve as vehicles
for introducing CARs into patient-derived T cells, enabling the expression of tumor recognition
sites essential for targeted cancer immunotherapy. The lentivirus production process involves
sophisticated molecular biology and biotechnology techniques. Plasmids containing lentiviral
genome elements, including gag, pol, and env genes, serve as essential components in lentivirus
production. These plasmids, when transfected into specialized packaging cells, facilitate the
generation of lentivirus. The gag gene encodes the group-specific antigen, contributing to virion
assembly and structural integrity, while the matrix (MA) protein facilitates viral assembly by
directing viral components to the plasma membrane assembly site. Additionally, the capsid (CA)
protein aids in the formation of the viral core and encapsulation of the viral Ribonucleic Acid
(RNA) genome for efficient infection, whereas the nucleocapsid (NC) protein ensures RNA
packaging and the encapsulation of the viral genome in new virions. The pol gene encodes critical
enzymes, including reverse transcriptase (RT) and integrase, which play essential roles in viral
replication and the integration of viral DNA into the host cell genome. Furthermore, the env gene
encodes the viral envelope glycoprotein, facilitating viral entry. Together, these essential genes—
gag, pol, and env—orchestrate the assembly, replication, and transduction characteristic of
lentiviral vectors. Lentiviral vectors are classified into three generations based on safety, with the

30
third generation being the safest. First-generation lentiviral vectors contain a significant amount
of HIV genome, including gag and pol genes, as well as additional viral proteins. In contrast,
second-generation vectors lack accessory genes such as vif, vpr, vpu, and nef, while the thirdgeneration vectors further enhance safety by splitting the viral genome into separate plasmids. The
gag and pol genes are encoded on one plasmid, and the rev gene is encoded on another plasmid.
Tat gene removal and modifications to the 3’LTR in the third generation improve safety by
disrupting the promoter and enhancer activity of LTR. These advancements in lentiviral vector
design contribute to their utility in various genetic medicine applications, including gene editing,
stem cell manipulation, and vaccine development, by facilitating sustained transgene expression
and therapeutic efficacy.
Figure 12: lentivector gene engineering transfer system76
.

31
1.16 Topanga Assay
The Topanga assay stands out as a novel luciferase-based technique renowned for its efficacy,
efficiency, and cost-effectiveness in detecting CARs. This sensitive and robust assay, when
coupled with flow cytometry, facilitates precise CAR identification. Traditional methods often rely
on labor-intensive secondary labeling procedures, consuming considerable time, and resources.
Prior approaches employing firefly luciferase (fluc), characterized by its large molecular weight
(~61 kDa), faced challenges during protein studies. However, recent advancements in marine
luciferase research have unveiled promising alternatives such as Gluc, Nluc, Tluc16, and Mluc7.
These variants, with smaller sizes (~19 kDa) and heightened luminescence, offer enhanced
stability and versatility across various experimental settings. The Topanga assay harnesses
recombinant fusion protein technology for CAR detection, yielding the Topanga reagent through
the fusion of the CAR's extracellular domain with a marine luciferase. This integration forms the
basis of the Topanga assay, leveraging luciferase for both quantitative and qualitative CAR
detection. Furthermore, the inclusion of a flexible linker, Gly-Gly-Ser-Gly, optimizes spatial
orientation and functional synergy between the CAR extracellular domain and the luciferase
component, thereby enhancing binding efficiency and signal transduction upon interaction with
target cells. The inclusion of signal peptides derived from human CD8 in the fusion construct was
observed to facilitate the secretion of the fusion protein, effectively coupling it with the marine
luciferase77. The Topanga assay has been harnessed for the detection of PSMA CARs, employing
a meticulously designed genetic engineering fusion construct comprising the extracellular domain
of PSMA, alongside the signal peptide and Nluc, interconnected by a flexible linker sequence
composed of GLY-GLY-Ser-GLY. This strategic fusion facilitated the incorporation of the Nterminal signal peptide derived from human CD8, thereby enabling the secretion of the Topanga

32
reagent. The development of lentivirus Topanga reagent involved the transfection of the target
construct into HEK 293FT cells, followed by an incubation period of 48 hours, culminating in the
collection of supernatants containing the secreted fusion protein PSMA-ECD-Nluc, commonly
referred to as the Topanga reagent. This pivotal reagent serves as a linchpin for binding to cells
harboring stable CAR expression. Luminescence measurement emerges as a quantitative metric
elucidating the intricate cellular interactions between PSMA-CAR and our Topanga reagent 77
.
1.17 Matador Assay
The Matador assay is a novel, highly sensitive, and specific luciferase-based cytotoxicity assay
designed to measure cell death with high accuracy and rapidity. This assay utilizes the extreme
brightness, stability, and glow-like characteristics of marine luciferases and their engineered
derivatives to quantify cell death 78. The mechanism involves genetically modifying target cells to
express a marine luciferase that lacks its signal peptide, ensuring the luciferase remains within the
cytosol of healthy cells. Upon exposure to cytotoxic agents or effector cells, the target cells
undergo apoptosis or necrosis, resulting in compromised cell membrane integrity. This membrane
disruption leads to the release of the cytosolic luciferase into the extracellular medium.
Additionally, the loss of membrane integrity allows the luciferase substrate to penetrate the cell
more effectively, reacting with any luciferase still present inside the cell. This interaction generates
a luminescent signal that is directly proportional to the extent of cell death. The Matador assay is
performed in a homogeneous, single-step manner, which simplifies the procedure and makes it
amenable to high-throughput screening and automation. The luminescent signal can be detected
directly from the culture medium using a standard plate reader, eliminating the need for specialized
or expensive equipment. This assay has been validated using various types of effector cells and
cytotoxic agents, including antibodies, natural killer (NK) cells, CAR T cells, and bispecific T cell

33
engagers. It can detect cytotoxicity even at very low cell concentrations, highlighting its
exceptional sensitivity and broad applicability 78
.
1.18 ELISA
ELISA is being used in CAR T-cell therapy research for examining the immune response and
cytokine profile linked to this treatment modality. Assessing levels of TNF-alpha, IL-2, and IFNgamma through ELISA provides insights into the mechanisms underlying CAR-T cell therapy.
TNF-alpha, classified as a proinflammatory cytokine, is primarily produced by activated
macrophages and T cells. Upon activation, these cells release TNF-alpha, triggering apoptosis in
target cells and inflammatory responses by inducing the production of additional cytokines and
chemokines. The binding of TNF-alpha to its receptors, TNF-R1 and TNF-R2, initiates
downstream signaling via the NF-kB pathway, leading to apoptosis and inflammation.
IL-2, another cytokine in immune regulation, is synthesized by activated T cells. It functions as a
growth factor for T cell proliferation and differentiation, facilitating the expansion of CAR-T cells
and enhancing their anti-tumor efficacy. Additionally, IL-2 plays a crucial role in modulating
regulatory T cells (Tregs). Binding of IL-2 to its receptor activates the JAK-STAT pathway,
prompting the expression of genes associated with T cell proliferation and effector function.
IFN-gamma, produced by activated T cells and NK cells, significantly contributes to anti-tumor
immune responses. It stimulates the expression of MHC molecules on tumor cells, enhances the
phagocytic activity of macrophages, and regulates various immune cell functions. Upon binding
to its receptor, Interferon Gamma Receptor (IFNGR), IFN-gamma triggers the activation of the
JAK-STAT signaling pathway, leading to the induction of genes linked to anti-tumor activities.

34
Chapter 2: Methods
The methods to develop CAR T-cells incorporating a scFv targeting the PSMA.
2.1 Molecular Biology
We employed a strategy integrating a vector backbone with an insert DNA encoding a PSMAtargeting sequence to construct our CAR. Initially, we identified suitable restriction enzyme sites
for both the vector backbone and the insert DNA, selecting NheI and MluI for precise and efficient
cloning. The scFv targeting PSMA was digested with NheI and MluI to produce cohesive ends
compatible with the vector. This digested PSMA scFv fragment was then ligated into a pre-digested
vector comprising the CAR backbone. Our CAR backbone includes the CD16 transmembrane and
cytosolic domain but lacks any costimulatory domain, specifically designed to evaluate the effects
of excluding costimulatory signals on CRS. Thus, in our target CAR construct, the specificity is
derived from the insert DNA encoding the PSMA-targeting scFv, while the transmembrane and
cytosolic domains are provided by the vector backbone. By eliminating the co-stimulatory domain,
we aim to assess its impact on the overall cytokine release and the therapeutic efficacy of the CAR
construct. The digestion reaction was set up with 1 µg of plasmid DNA, 1 µL each of NheI and
MluI enzymes, 2 µL of 10X reaction buffer, and nuclease-free water to a final volume of 20 µL,
incubating the mixture at 37°C for 2 hours. Following digestion, the DNA fragments were
separated using 1% agarose gel electrophoresis, running at 100V for approximately 1 hour. The
desired bands were visualized under UV light and excised from the gel using a sterile scalpel. The
excised DNA fragments were purified using a gel extraction kit. The gel slices were dissolved in
binding buffer at 55°C, and the DNA was bound to a silica membrane, washed with ethanol-based
wash buffer, and eluted with 30 µL of elution buffer pre-warmed to 65°C. DNA was quantified
using a spectrophotometer. The purified DNA fragments were then ligated using T4 DNA ligase.

35
The ligation reaction, set up with a 1:3 molar ratio of vector to insert DNA, included 1 µL of T4
DNA ligase enzyme, 2 µL of 10X ligation buffer, and nuclease-free water to a final volume of 20
µL. The ligated DNA was introduced into competent E.coli Stbl3 cells through heat shock
transformation. This process involved thawing 30 µL of competent cells on ice, adding 5 µL of the
ligation mixture, incubating on ice for 30 minutes, heat shocking at 42°C for 45 seconds,
recovering on ice for 2 minutes, adding 500 µL of SOC (Super Optimal broth with Catabolite
repression) medium, and incubating at 37°C for 1 hour with shaking. Transformed cells were
plated on LB agar plates containing carbenicillin (100 µg/mL) and incubated at 37°C overnight.
The following day, individual colonies were picked and inoculated in 7 mL of LB medium with
carbenicillin, grown overnight at 37°C with vigorous shaking. Plasmid DNA was extracted using
a miniprep kit, involving steps of cell lysis, neutralization, binding, washing, and elution. The
purified plasmid DNA was sent to Genewiz for sequencing to confirm the integrity of the 3' and 5'
ends of the construct, and the sequences were analyzed to ensure correct insertion and orientation
of the target DNA. Upon confirmation of the correct sequence, a larger scale preparation
(midiprep) of the plasmid DNA was performed, and the DNA was used for lentivirus production
following standard protocols. This specific design choice aims to potentially mitigate the risks of
cytokine release syndrome (CRS) by excluding costimulatory signals. The ligation product was
subsequently transformed into E.coli (Escherichia coli.) Stbl3 for amplification. Positive clones
were identified through colony PCR (polymerase chain reaction) and confirmed by sequencing.

36
2.2 In Vitro Experiments:
• In vitro experiments were conducted to generate lentivirus using HEK 293FT cells via transient
transfection.
• JNG cells were infected with the lentiviral supernatant generated with cloned CAR constructs
to induce CAR expression. Co-culture assays were performed by co-culturing CAR-infected
JNG cells with the LNCaP cell line to assess their engagement and response.
• T cells isolated from a healthy donor were infected with the PSMA-CAR virus supernatant,
and their cell surface expression was evaluated using the Topanga assay. The cytotoxic activity
of CAR-infected T cells against target cells was assessed using the Matador assay.
Furthermore, ELISA was employed to measure the production of IL-2, TNF-alpha, and INFgamma following the engagement of CAR-T cells with target cells.
2.2.1 Lentiviral Vector Construction: The lentiviral vector used for the construction of PSMAscFv-CAR is PSMA scFv is pCCL-c-MNDU3-X, obtained as a gift from Donald Kohn. The vector
backbone, 021321-BBdG2, was previously cloned, and the insert plasmid, designated as 061219-
WYKA2, was utilized to clone PBC2 (anti-PSMA construct) employing advanced molecular
biology techniques. Restriction enzymes Nhe and Mlu, procured online from Thermo Fisher, were
employed for digestion. The inserted DNA was added at the Nhe and Mlul sites, followed by
transformation of the ligated product into Stbl3 competent cells. Subsequently, the transformed
cells were cultured on agar plates supplemented with carbenicillin antibiotic. Two colonies were
selected for mini prep, and purification of the mini prep was performed to extract DNA. The
extracted DNA was then sent to Genewiz along with primers for sequencing analysis.
Construct Description: The constructed vector contains the hinge transmembrane domain of
human CD8 but lacks 4-1BB as a co-stimulatory domain. This construct was designated as

37
071423-PBC2, and sequencing analysis confirmed that the sequence matched the desired target
sequence.
071423-PBC2- pCCLc-MNDU3-EcoRI-Nhe-CD8SP-hu-PSMA-J591-scFv-Mlu-CD16A-v158-
S197P-FL-v3-ter-Sal
2.2.2 Lentivirus Production
HEK-293FT cells were selected as the transfection host to generate the CAR-encoding lentivirus.
These cells were ordered from Invitrogen. The lentiviral vectors were constructed to encapsulate
the CAR gene, designed to confer specific targeting properties against the PSMA- a pivotal target
in prostate cancer immunotherapy. The psPAX2 vector, an essential component in lentiviral vector
production, having critical genes including Gag, Pol, Rev, and Tat, which helps at various stages
of viral replication and packaging. Additionally, the pLP/VSVG vector, sourced from Invitrogen,
facilitated the expression of the viral envelope glycoprotein (G protein), for virus entry into host
cells. Notably, the psPAX2 vector used as virus packaging. To monitor transfection efficiency, an
GFP vector was concurrently transfected alongside the lentiviral components. This served as a tool
for assessing the success of the transfection process, enabling the visualization and quantification
of transfected cells. The transfection procedure, adhering to the standard calcium phosphate
method, involved the addition of 10μg of CAR lentiviral plasmid, 7.5μg of psPAX2 plasmid, 3μg
of pLP/VSVG plasmid, and 0.25μg of GFP encoding plasmid to confluent HEK-293FT cell
cultures in 100mm culture dishes with Dulbecco's Modified Eagle (DMEM) media. Posttransfection, supernatants were collected at 72 hours for optimal virus production. These
supernatant with lentiviral particles, underwent filtration through a 0.45µm filter to eliminate
cellular debris and contaminants, thus ensuring the purity and integrity of the viral stock.
Subsequently, the concentrated viral particles were harvested via ultracentrifugation at 18,500 rpm

38
at 4°C overnight, to sediment viral particles while discarding non-essential debris. The resulting
viral pellet was gently resuspended in RPMI or XVIVO medium, a culture medium ideal for
maintaining JNG cells and T cells, respectively. Finally, the concentrated lentiviral suspension was
aliquoted and stored at -80°C for preservation.
2.2.3 Viral Transduction
The JNG cell line with a NFAT-dependent GFP reporter gene, was used. This cell line was
generously provided by Dr. Arthur Weiss from the University of California, San Francisco (UCSF)
and was cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS). A
total of 2 million JNG cells were suspended in 2 ml of media and plated into individual wells of a
6-well plate. Subsequently, each well containing 2 million JNG cells was infected with 150 μl of
concentrated virus supernatant (comprising anti-PSMA scFv CAR) along with 20μl of polybrene
(Polybrene stock 800μg/mL) . As a control, one well containing 2 million JNG cells was left
uninfected. Following infection, the cells were centrifuged at 1,800 rpm at 32°C for 45 minutes
and were then incubated undisturbed at 37°C with 5% CO2 overnight. After the overnight
incubation, the medium containing the viral supernatant and polybrene was replaced with fresh
RPMI medium supplemented with 10% FBS. Subsequently, the cells were transferred to a T25
flask and allowed to proliferate under standard incubation conditions at 37°C with 5% CO2.
2.2.4 Co-Culture Assay
To assessing the interaction between CAR-expressing Jurkat cells and target cells, a co-culture
assay was conducted by incubating them together overnight. The Jurkat cell line utilized in this
assay harbors an NFAT-dependent GFP reporter gene, enabling the production of GFP upon
activation, which can be quantified via flow cytometric analysis. For each reaction, approximately

39
1 million CAR-JNG cells and 1 million target cells were combined. The target cell lines employed
included the LNCaP prostate cancer cell line. Both the CAR-JNG cells and target cells were
resuspended in 1ml of media and co-cultured in a 24-well plate, which was then incubated
overnight at 37°C with 5% CO2. Following the incubation period, the cells were subjected to flow
cytometric analysis using a BD FACSVerseTM instrument.
2.2.5 Infection of primary human T-cells with PSMA-CAR virus
PSMA-CAR concentrated lentivirus was used to infect primary human T cells. In general, primary
T cells were infected using spin-infection at 32°C at 2,800 rpm for 90 minutes where 5 × 106
cells/2 ml were incubated with 500 μl of concentrated virus in the presence of polybrene in
morning. The media was changed in the evening and the infection was repeated for two more days
for a total of 3 infections. After the 3rd infection, the cells were pelleted and re-suspended in
complete T-cell XVIVO medium supplemented with 100 IU/ml IL2 and 5% human AB serum for
expansion.
2.3 Topanga Assay
Synthesis and Production of Luciferase Fusion Proteins: The genetic sequences encoding the
extracellular domains (ECDs) of human PSMA were linked together with a signal peptide-deleted
form of Nluc, a marine luciferase, using a short Glycine-Serine flexible linker. Additionally, a
signal peptide from human CD8 was incorporated at the N-terminus to facilitate secretion of the
fusion protein. The Topanga fusion proteins were synthesized in HEK 293FT cells through
transfection of the PSMA-ECD-Nluc plasmids using the calcium phosphate method. For a 100mm
culture plate containing fully confluent HEK 293FT cells, 10μg of fusion protein expression
plasmid and 0.25μg of GFP encoding plasmid were utilized. Approximately 48 hours post-

40
transfection, the supernatants containing the fusion proteins were harvested and filtered through a
0.45μm filter. This resulting product, referred to as the Topanga reagent, was then aliquoted and
stored at -80°C for subsequent use.
2.4 Binding Assay and Luminescence Detection: For each reaction, approximately 2x105 CAR
infected Jurkat/T cells were used. These cells were centrifuged at 1300 rpm for 5 minutes and then
suspended in 100μl of Topanga reagent, which consisted of the filtered supernatant containing the
fusion protein. Following this, the cells were incubated on ice for 45 minutes. After the incubation
period, the cells underwent four washes with 1 ml of wash buffer (0.5% FBS in PBS). Upon
completion of the final wash, the cell pellet was resuspended in 30μl of wash buffer and transferred
to a white 384-well Lumitrac plate. A substrate solution consisting of 20μm Coelenterazine (CTZ)
in Phosphate-Buffered Saline (PBS) was added to the plate to facilitate luminescence
measurement. The luminescent signal was measured using a BioTek Synergy H4 hybrid microplate
reader in a well mode using injectors for a duration of 10 seconds.
2.5 Matador assay
For this assay T cells expressing PSMA-CAR were used as Effector cells and LNCaP cells
expressing Luc-PPE were generated as target cells. Effector (PSMA-CAR-T-cells) and target
(LNCaP-Luc-PPE) cells were mixed at various an Effector-to-Target (E:T) ratios of 3:1, 1:1 and
0.3:1. This co-culture experiment was performed in a white 384-well Lumitrac plate, with each
well containing effector and target cells in XVIVO media without any additional supplements for
24 and 48 hours. Post-incubation, the Matador assay was conducted by adding 15 μl of 1X
Luciferase assay buffer containing DTT (Dithiothreitol) and ATP (Adenosine Triphosphate) to
each well of the 384-well plate, followed by luminescence measurement using a luminometer

41
reader to read for 10 seconds. This comprehensive approach allowed for the assessment of the
cytotoxic activity of PSMA expressing T cells against LNCaP target cells.
2.6 ELISA
To conduct the ELISA for human interferon-gamma (hIFNg), human tumor necrosis hTNF- alpha,
and IL2, the following method was employed. The target cells (LNCaP-Luc-PPE-RO1) were
plated at a density of 50,000 cells per well in 100 μl of media in the 96-well plate. The target cells
were incubated either alone or with the effector cells (either T-UI or T-CAR) at an E:T ratio of 1:1
in 100 μl of media. Both the target and effector cells were co-cultured in the 96-well plate for a
duration of 48 hours, allowing for interactions between the cells to occur. Following the incubation
period, the supernatants were collected from each well for subsequent ELISA analysis. This ELISA
assay facilitated the quantification of hIFN gamma, hTNF alpha, and IL2 levels in the co-culture
supernatants, providing valuable insights into the immune response and cytokine profile associated
with the CAR T-cell therapy.

42
2.7 In Vivo Experiment: In vivo preclinical experiments were conducted to evaluate the efficacy
of CAR T-cells against prostate cancer using an NSG xenograft model.
2.7.1 In Vivo Testing
Fifteen male NSG 572 mice (Charles River), aged 7 weeks were used for the study. Each mouse
received a subcutaneous injection of 1 million LNCaP-Luc PPE cells suspended in Matrigel
(50:50%). Optical live imaging was performed post-injection to verify successful tumor cell
engraftment. The PSMA CAR-T cells and T-uninfected (T-UI) were administered intravenously
(i.v.) at a dose of 5 million cells per mouse. Regular imaging were done weekly to monitor tumor
burden and growth progression along with the weekly tumor measurements. This imaging
approach allowed for assessment of tumor size and volume, facilitating the evaluation of
therapeutic efficacy and anti-tumor response elicited by the PSMA CAR T-cell therapy in the NSG
572 mouse model.

43
Chapter 3: RESULTS
Results for all molecular cloning, In Vitro and In Vivo experiments to develop PSMA specific
CAR T-cells.
3.1 CAR plasmid construction
We have engineered an PSMA CAR construct designated as 071423-PBC2, with a scFv targeting
the PSMA antigen. This construct represents a CAR. Notably, our CAR design excludes the 4-1BB
co-stimulatory domain, a component commonly associated with cytokine release syndrome (CRS)
adverse effects. In this study, we aim to assess the potential of our modified CAR to induce
sustainable levels of TNF-alpha, IFN-gamma, and IL-2 production, thereby mitigating CRS risks.
The construction of 071423-PBC2 involved the use of a vector backbone and insert DNA
fragments, which were subjected to restriction enzyme digestion using NheI and MluI enzymes
for precise assembly.
Vector backbone021321-BBdG2- pCCLc-MNDU3-EcoRI-Nhe-CD8SP-CD19-hu-mROO5-1-scFv-Mlu-CD16Av158-S197P-FL-v3-ter-Sal (013021-BBaH1)
Insert DNA061219-WYKA2-pLenti-EF1-Nhe-CD8SP-hu-PSMA-J591-scFv-Mlu-GGS-Nluc-Xho-Flagx4-
Streptag-GGS-8XHis-T2A-Pac
Target construct- 071423-PBC2- pCCLc-MNDU3-EcoRI-Nhe-CD8SP-hu-PSMA-J591-scFv-MluCD16A-v158-S197P-FL-v3-ter-Sal

44
After the development of our targeted construct 071423-PBC2, we proceeded with sequencing
conducted by GENEWIZ.
3.2 Jurkat infection
The next procedure we conducted was infecting JNG cells with CAR lentivirus supernatant which
involved several steps. Initially, to generate virus carrying our CAR constructs, a combination of
plasmids was transfected into HEK-293FT cells. These plasmids included the CAR plasmids
themselves, along with other essential packaging plasmids such as pLP/VSVG and psPAX2.
Additionally, we also included GFP plasmid in the transfection mix as a marker for assessing the
efficiency of transfection. The transfection process was conducted using the calcium phosphate
method. Following transfection, the HEK-293FT cells were allowed to produce viral particles
containing the CAR constructs. After 72 hours of transfection, the supernatants were harvested. To
concentrate the viral particles, the collected supernatants were concentrated by ultracentrifugation
at 18,000 rpm (revolutions per minute) for overnight, at a low temperature of 4°C. The aim of this
process was to make pellet of the viral particles, thereby concentrating them into a smaller volume
and remove cell debris. Subsequently, JNG cells were used to confirm that our PSMA-CAR is
functionally active. These cells were chosen as they produce GFP upon NFAT activation.
3.3 JNG-CAR co-culture with LNCaP cell line
The co-culture experiment was conducted in which we did co-culture by combining PSMA-CARJNG with PSMA-positive LNCaP target cell line. For co-culture experiment, we added one million
Jurkat cells expressing our CAR with one million target cells for 24 hours. The Jurkat cell line
have NFAT-dependent GFP reporter gene, which induce GFP expression when they are activated,
and can be detected by flow cytometric analysis. Controls included culturing CAR-expressing

45
Jurkat cells alone with media and uninfected Jurkat cells (Jurkat parental) with the target cells. Coculture of LNCaP cells and JNG-PSMA CAR cells exhibited high GFP expression, indicating
engagement with LNCaP cells. Conversely, the control JNG cells showed no GFP expression upon
incubation with LNCaP cells.
3.4 T cell infection
Primary T cells were isolated from blood collected from a healthy donor at CHLA using Cd3
microbeads from Miltenyi Biotec. PSMA-CAR concentrated lentivirus was used to infect primary
human T cells using spin-infection at 32°C at 2,800 rpm for 90 minutes where 5 × 106 cells/2 ml
were incubated with 500 μl of concentrated virus in the presence of polybrene in morning. The
media was changed in the evening and the infection was repeated for two more days for a total of
3 infections. After the 3rd infection, the cells were pelleted and re-suspended in complete T-cell
XVIVO medium supplemented with 100 IU/ml IL2 and 5% human AB serum for expansion.
3.5 Topanga assay
The Topanga assay was conducted to assess the expression of CAR on T-cells and its capability to
bind to the Topanga reagent. The Topanga fusion protein comprises the extracellular domain
(ECD) of the target antigen PSMA in addition to Nluc, connected by a short glycine-serine flexible
linker. After incubation of PSMA-CAR-T-cells with the PSMA-ECD-Topanga fusion proteins and
washing steps, luminescence was measured using a BioTek Synergy H4 hybrid microplate reader
for a duration of 10 seconds. A significant higher relative luciferase value confirmed the binding
of PSMA scFv-CAR-T cells with the PSMA-ECD Topanga reagent. Conversely, T-UI incubated
with the PSMA-ECD-Topanga fusion protein did not show any binding.

46
3.6 Matador assay
To measure the CAR-induced cell death, the PSMA-CAR-T cells were co-cultured with LNCaP
cells expressing Luc-PPE at various effector-to-target (E:T) ratios of 3:1, 1:1, and 0.3:1 in a 384-
well plate. The cells were co-cultured overnight in XVIVO media without any supplements. Target
cells were plated at a density of 5,000 cells per well in 30 μl of media. Subsequently, a Matador
assay was performed by adding 15 μl of 1X Luciferase assay buffer containing DTT and ATP to
each well using a dispenser. Luminescence was then measured for 10 seconds to assess the
cytotoxic activity of the T cells against the target cells. As shown in Figures 13-15, PSMA-CART cells showed significant higher cytotoxicity as compared to control UI-T cells at all the three
Effector: Target ratios.
Med T-UI (1) T-UI (2) T-PSMA-PBC2
Series1 7579 8529 7795 25960
0
5000
10000
15000
20000
25000
30000
Relative Luminescence
LNCap E:T :: 3:1
Matador assay-T_PSMA-LNCaP-082223
Figure 13: Matador assay (E:T::3:1)

47
3.7 ELISA
We used ELISA assay to quantify the levels of tumor necrosis factor alpha (TNF alpha), interferon
gamma (IFN gamma), and interleukin-2 (IL2) secretion. These cytokines play crucial roles in the
Med T-UI (1) T-UI (2) T-PSMA-PBC2
Series1 6912 7392 6810 12096
0
2000
4000
6000
8000
10000
12000
14000
Relative Luminescence
LNCap E:T :: 0.3:1
Matador assay-T_PSMA-LNCaP-082223
Figure 15: Matador assay (E:T::0.3:1)
Med T-UI (1) T-UI (2) T-PSMA-PBC2
Series1 7571 8196 6959 22005
0
5000
10000
15000
20000
25000
Relative Luminescence
LNCap E:T :: 1:1
Matador assay-T_PSMA-LNCaP-082223
Figure 14: Matador assay (E:T::1:1)

48
immune response and are associated with cytokine release syndrome (CRS), a potentially severe
side effect of CAR-T cell therapy. Our objective was to investigate whether the presence of the 4-
1BB co-stimulatory domain in our CAR constructs influenced the secretion of these cytokines and
contributed to CRS. To address this, we compared the cytokine release profiles of CAR-T cells
engineered with and without the 4-1BB co-stimulatory domain. As shown in Figures 16-18, our
results revealed that CAR-T cells lacking the 4-1BB co-stimulatory domain exhibited an adequate
release of TNF alpha, IFN gamma, and IL2, indicating effective immune activation required for
tumor killing. While CAR-T cells with the 4-1BB co-stimulatory domain displayed significantly
elevated levels of these cytokines, suggesting an exaggerated immune response. Through statistical
analysis and graphical representation of our findings, we demonstrated cytokine secretion
differences between the two CAR constructs along with negative controls.
Figure 16: ELISA Results of TNF-alpha Comparison between CAR's and CAR with 4-1BB
Med T-UI (1) T-UI (2) T-PSMAPBC2
T-PSMA4-1BB
Series1 0.02 0.02 0.03 0.16 0.53
0
0.1
0.2
0.3
0.4
0.5
0.6
hTNFa secretion OD 650nm
LNCap
hTNFa-T-PSMA-E:T 1:1-082323

49
Figure 14: ELISA Results of IFN-Gamma Comparison between CAR's and CAR with 4-1BB
Figure 18: ELISA Results of IL2 Comparison between CAR's and CAR with 4-1BB
Med T-UI (1) T-UI (2) T-PSMAPBC2
T-PSMA-4-
1BB
Series1 0.02 0.02 0.02 0.05 0.16
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
hIFNg secretion OD 650nm
LNCap
hIFNg-T-PSMA-E:T 1:1-082323
Med T-UI (1) T-UI (2) T-PSMAPBC2
T-PSMA4-1BB
Series1 0.04 0.03 0.04 0.03 0.2
0
0.05
0.1
0.15
0.2
0.25
hIL2 secretion OD 650nm
Axis Title
hIL2-T-PSMA-E:T 1:1-48h-082323

50
3.8 In Vivo testing
We conducted in vivo experiments of PSMA-CAR-T cells (PBC2) using NSG mice injected with
prostate cancer. Initially, LNCaP cells were injected to 3 groups to induce prostate cancer
development. Subsequently, UI-T cells and CAR-T cells targeting PSMA were administered to
assess the efficacy of our treatment approach. Throughout the experiment, we performed regular
bioluminescence imaging to monitor tumor progression and growth. Additionally, tumor size was
measured to evaluate treatment outcomes. Our findings revealed promising results, with the CART group demonstrating prolonged survival as compared to control groups. Specifically, when
compared to negative control mice receiving no treatment and mice infused with uninfected T
cells, the mice treated with our CAR-T cells exhibited extended survival. However, we faced
challenges related to graft-versus-host disease (GVHD), which necessitated more research and
optimization of our therapy. Overall, the results of our study support potential effectiveness of our
treatment approach in preclinical models of prostate cancer.
Cage
Loc.
(CSC)
Ear tag # Group CAR construct
R3A10
9946, 9950,
9951, 9953,
9954
G1: LNCaP
alone: 5
mice
No treatment, only tumor cells
R3B10
9936, 9942,
9943, 9945,
9988
G2: T-UI: 5
mice Only T uninfected cells
R3O5
9977, 9979,
9980, 9982,
9983
G9: TPSMACD16-
PBC2: 5
mice
071423-PBC2- pCCLc-MNDU3-EcoRI-Nhe-CD8SPhu-PSMA-J591-scFv-Mlu-CD16A-v158-S197P-FLv3-ter-Sal-PBC2 (021321-BBdG2)
Table 1: In Vivo testing Plan

51
Figure 19: BLI imaging depicting Tumor burden in mice, Survival days and any conditions.

52
Figure 20: Survival curve of 15 NSG mice included in experiments.

53
Chapter 4: Discussion
My thesis focused on the development and evaluation of CAR T-cell therapy targeting the PSMA
that has a potential to treat prostate cancer. Initially, we successfully engineered a novel CAR
(scFv) to target PSMA. Through in vitro experiments involving JNG cells, we demonstrated the
effective activation of CAR construct in the presence of PSMA-positive target cells (LNCaP),
indicating its potential for tumor recognition. Subsequent characterization assays involving
PSMA-CAR-T cells also confirmed the cytotoxicity potential of PSMA-CAR-T cells.
Furthermore, we demonstrated the role of the co-stimulatory domain 4-1-BB in cytokine release
syndrome (CRS). By eliminating 4-1BB, we achieved a more balanced immune response with
adequate levels of TNF alpha, IL-2, and INF-gamma factors necessary for cytotoxicity. In
addition, in vivo testing of PSMA-CAR-T cells in NSG mice revealed promising results by
prolonged survival in mice administered with PSMA-CAR-T cells as compared to control
groups. However, the GvHD highlighted a challenge which requires further research and
strategy.

54
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1

Abstract (if available)

Abstract Chimeric Antigen Receptor (CAR) T-cell therapy has emerged as a promising treatment for various cancers, including prostate cancer. In this study, we developed CAR T-cells specifically targeting Prostate-Specific Membrane Antigen (PSMA) to treat prostate cancer. We designed PSMA-specific next generation CAR comprising a single-chain variable fragment (scFv), derived from the humanized J591 antibody, into a CAR vector backbone lacking the 4-1BB co-stimulatory domain, instead incorporating the CD16 co-stimulatory domain. The J591 antibody was selected for its high specificity and reliability in targeting PSMA, previously utilized in radio conjugation studies. We hypothesized that the exclusion of the 4-1BB co-stimulatory domain would mitigate cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) by reducing the excessive release of cytokines such as TNF-α, IFN-γ, and IL-2. Following restriction enzyme digestion and ligation of the insert DNA (Deoxyribonucleic Acid) into the vector backbone (4-1BB excluded), we developed lentivirus to transduce Jurkat cells and performed co-culture experiments to evaluate Nuclear factor of activated T cells (NFAT) induction against cancer cell lines. Flow cytometry confirmed NFAT-induced Green fluorescent protein (GFP) expression, indicating successful transduction. Peripheral blood mononuclear cells (PBMCs) and T cells were isolated from healthy donors, activated, and transduced with the CAR construct. Specificity and efficacy assays, including the Topanga assay, Matador assay, and Enzyme-Linked Immunosorbent Assay (ELISA), were conducted to assess scFv presence, cytotoxicity, and cytokine release, respectively. Our results demonstrated a significant reduction in cytokine release for PSMA-PBC2-CAR-T cells compared to hu-PSMA-scFv-BBz-CAR with 4-1BB costimulatory domain, confirming our hypothesis. In vivo studies involved engrafting NSG mice with the LNCaP prostate cancer cell line, followed by administration of the engineered PSMA-PBC2-CAR-T. Tumor regression, survival curves, and tumor burden analysis indicated enhanced survival in treated subjects, although some experienced graft-versus-host disease (GvHD), indicating the need for further optimization. In conclusion, our study provides evidence that CAR T cells targeting PSMA without the 4-1BB co-stimulatory domain can effectively reduce cytokine release and demonstrate potential in treating prostate cancer, with in vivo results showing increased survival rates. Further research is necessary to address GvHD and enhance the therapeutic efficacy of these engineered CAR T-cells.

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Creator Barot, Parv (author)

Core Title Chimeric Antigen Receptor targeting Prostate Specific Membrane Antigen (PSMA)

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Degree Conferral Date 2024-08

Publication Date 07/31/2024

Defense Date 07/30/2024

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

Tag allogenic CAR-T cell therapy,antigen specificity,cancer immunotherapy,CAR-T cell therapy,cell-based therapy,clinical trials,gene therapy,immunotherapy,next-generation CAR-T,OAI-PMH Harvest,oncology,prostate cancer biomarkers,prostate cancer treatment,PSMA-targeted therapy,tumor immunology,tumor targeting

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Advisor Chaudhary, Preet (committee chair), Haworth, Ian (committee chair), Culty, Martine (committee member)

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