University of Southern California Dissertations and Theses

Lym-1 epitope targeted chimeric antigen receptor (CAR) T cells for the immunotherapy of cancer

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Lym-1 epitope targeted chimeric antigen receptor (CAR) T cells for the immunotherapy of cancer

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LYM-1 EPITOPE TARGETED CHIMERIC ANTIGEN RECEPTOR (CAR) T CELLS FOR
THE IMMUNOTHERAPY OF CANCER

By

Long Zheng

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirement of the Degree
DOCTOR OF PHILOSOPHY
(MEDICAL BIOLOGY)

May 2021

Copyright 2021 Long Zheng

ii

Dedication

To my wife Luqing Ren,
and my parents for their constant love and support.

iii
Acknowledgements

Thesis Committee Members:
Josh Neman-Ebrahim, Ph.D. (Chair)
Alan L. Epstein, M.D., Ph.D. (Mentor)
Harvey R. Kaslow, Ph.D.

Medical Biology program Chair:
Martin Kast, Ph.D.

I want to express my sincere thanks to my mentor Dr. Alan L. Epstein for his
intellectual and emotional support of my PhD study for the past four years. His scientific
knowledge and motivation guided me to identify gaps in the current advancement of
immunology and cell therapy. When I was discouraged, he gave me confidence and
encouragement. In addition to academic work, he showed me the skills necessary to lead
a lab and offered me career advice. His continuous dedication to make sure our research
to meet the highest standard inspired my PhD training, and I will definitely carry this
attitude with me as I move forward. I could not express enough the deep gratitude I feel
towards my mentor.
Besides my advisor, I would like to thank my qualifying exam and thesis committee:
Prof. Josh Neman, Prof. Yong Zhang, Prof. Weiming Yuan, Prof. Andre J Ouellette, and
Prof. Harvey Kaslow for their insightful comments and constructive feedback. Their
suggestions improved my science work and presentation skills. I want to especially thank
Dr.Kaslow for his advice and support towards my thesis and my entire PhD study. I really
iv
enjoyed and cherished all the weekly meetings with him in his office and on zoom. His
rigorous science training background greatly helped me to design experiments and
interpret findings.
My dissertation would not be possible without the work from Dr.Peisheng Hu and
Maggie Yun. Their expertise in antibody engineering and molecular biology guided the
CAR construction design and modifications which are the most critical reagent in my
thesis work. In addition to the research, I want to give my deepest gratitude for their
support and care through my entire life at USC. I joined Dr. Epstein’s lab as a visiting
student in 2014, since then, we have been sharing the office for 7 years. They offered
numerous help and advice towards my daily lives and my career development. They act
more like families than colleagues to me. There are no words to express my gratitude to
them.
I also thank my previous colleagues Brandon Wolf and Caryn Gonsalves. I will
never forget the times when we were setting up the procedures of CAR T cell production.
They have been such a delight to work with and their positive attitude helped
troubleshooting in many experiments.
I want to thank Dr. Leslie Khawli for his work to coordinate all aspects in my
research experiments. His work made my research much easier and efficient. I thank his
tremendous contribution to the experimental designs, manuscript editing and publications.
I also would like to thank many friends and colleagues in Drs. Alan Epstein and
Russell Jacobs’s Labs, they have been a pleasure to work with. They are included in the
following: Ryan Graff, Stacy Jaw, Tiffany Jheng, Aida Kouhi, Vyshnavi Pachipulusu,
v
Alison Smith, Chia-Hsin Lin, Julie Jang, Kevin Yu, Joseph Yoo, Gregory Stone, and
Naomi Sta Maria.
I thank Ivetta Vorobyova and Ryan park from the Molecular Imaging Center for
their assistance in animal optical imaging. I would also like to thank Dr.Junji Watanabe
from Translation Research Laboratory trained me using multi-color flow cytometry and
optimal imaging devices.
I acknowledge the following financial support for my graduate training: Commercial
research grant from Cell Biotherapy Inc., and grant (no. 9031415) from the Ming Hsieh
Institute of University of Southern California.
Last but not least, I want to express my deepest gratitude to my wife. My wife
Luqing Ren has been a pillar of support through my whole life in US. She joined our lab
as a research assistant since 2015. She was always there with me in the laboratory on
weekends and occasional nights. Whenever I had troubles in experiments, she was the
first one I would talk to. I will never forget her encouragement when my studies were at
bottle neck. She is the secret person that work closely with me to figure out most of the
technical challenges in experiments. She is the one that helped me with the hundreds of
i.v injections to establish tumor models in mice. This thesis would not have been possible
without her help and support. My thesis and other achievements I had during PhD study
belong much to her as it does to me. Finally, I would like to thank my parents and Ren-
Parents for their loving support and advice on balancing work and life.

vi
Table of Contents
Dedication ........................................................................................................................ ii

Acknowledgements ......................................................................................................... iii

List of Figures ................................................................................................................. viii

Abstract ............................................................................................................................ x

Chapter 1 Introduction ...................................................................................................... 1
1.1 CAR T Cell Progress Timeline ................................................................................ 2
1.2 Current State of B-cell Lymphoma Therapy ............................................................ 4
1.3 The Knowns and Unknowns of Lym-1 Antibody ..................................................... 5
1.4 Summary ................................................................................................................. 8
References .................................................................................................................... 9

Chapter 2 Lym-1 Chimeric Antigen Receptor T Cells Exhibit Potent Anti-Tumor Effects
against B-Cell Lymphoma
1
............................................................................................. 14
2.1 Abstract ................................................................................................................. 14
2.1 Introduction ........................................................................................................... 15
2.2 Materials and Methods .......................................................................................... 16
2.3 Results .................................................................................................................. 21
2.4 Discussion ............................................................................................................. 29
Acknowledgments ....................................................................................................... 32
References .................................................................................................................. 32

Chapter 3 A humanized Lym-1 CAR with Novel DAP10/12 Signaling Domains
Demonstrates Reduced Tonic Signaling and Increased Antitumor Activity in B-Cell
Lymphoma Models
2
........................................................................................................ 35
3.1 Abstract: ................................................................................................................ 35
3.3 Introduction ........................................................................................................... 37
3.4 Materials and Methods .......................................................................................... 40
3.5 Results .................................................................................................................. 47
3.6 Discussion ............................................................................................................. 70
Acknowledgements ..................................................................................................... 74
vii
References .................................................................................................................. 75

Chapter 4 Conclusion and Future directions .................................................................. 79
4.1 Non-impaired generation of huLym-1-B-DAP CAR T cells with DAP12 signaling
domain ........................................................................................................................ 80
4.2 Increase tumor antigen surface retention to improve CAR T cell anti-tumor
efficacy. ....................................................................................................................... 82
4.3 Potential side effects of huLym-1-B-DAP CAR T cells. ......................................... 85
4.4 Conclusion remarks .............................................................................................. 87
References .................................................................................................................. 89

Appendix ........................................................................................................................ 93

Bibliography .................................................................................................................... 95

viii
List of Figures
Figure 1.1: Components of T cell receptor (TCR) complex and the evolution of Chimeric
Antigen Receptor (CAR) design. ...................................................................................... 3
Figure 1.2: Comparison of rituximab and Lym-1 staining patterns shown by indirect
immunofluorescence microscopy. Taken from Nan Zhang et al., 2007(41). ................... 7
Figure 2.1: Lym-1 CAR and CD19 (FMC 63) CAR constructs. ...................................... 21
Figure 2.2: Efficient Lym-1 CAR expression on human primary T cells. ........................ 22
Figure 2.3: Detection of Lym-1 and CD19 epitopes on Daudi and Raji cells but not K562
cells. ............................................................................................................................... 23
Figure 2.4: Epitope-driven upregulation of CD107a on Lym-1 and CD19 CAR T cells. . 24
Figure 2.5: Epitope-driven cytotoxicity of Lym-1 and CD19 CAR T cells. ...................... 25
Figure 2.6: Epitope-driven release of cytokines from Lym-1 and CD19 CAR T cells. .... 26
Figure 2.7: Epitope-driven proliferation of Lym-1 and CD19 CAR T cells. ..................... 27
Figure 2.8: Lym-1 and CD19 CAR T cells eradicate Raji/Luc-GFP xenograft tumors in
vivo. ................................................................................................................................ 29
Figure 3.1: Selection of huLym-1-B as the candidate for CAR development. ................ 48
Figure 3.2: Lym-1 and huLym-1-B CAR T cells with BB3z-CD3z signaling domains are
cytotoxic in culture despite impaired proliferation. .......................................................... 50
Figure 3.3: Both Lym-1 and huLym-1-B CAR with the 4-1BB3z intracellular domain
eradicate Raji tumor in vivo. ........................................................................................... 51
Figure 3.4: The impaired proliferation of huLym-1-B-BB3zCAR T cells is associated with
weakly expressed Lym-1 epitopes on T cells and is mediated by ITAM CD3ζ. ............. 54

ix
Figure 3.5: Ligand-dependent suboptimal CAR signaling is the dominant mechanism for
impaired expansion in huLym-1-B-BB3z CAR T cells. ................................................... 55
Figure 3.6: Replacing BB3z with DAP signaling promotes stimulation-induced
proliferation of huLym-1-B CAR T cells. ......................................................................... 57
Figure 3.7: huLym-1-B-DAP CAR T cells exhibit reduced Activation Induced Cell Death.
........................................................................................................................................ 58
Figure 3.8: Increased endogenous CD3 ζ phosphorylation in huLym-1-B-DAP CAR T
cells. ............................................................................................................................... 59
Figure 3.9: huLym-1-B-DAP CAR T cells mediate superior anti-tumor efficacy than BB3z
counterparts in vivo. ....................................................................................................... 62
Figure 3.10: huLym-1-B-DAP show effector function against a panel of B-cell lines. .... 63
Figure 3.11: Lym-1 epitopes do not significantly downregulate in response to the
presence of huLym-1-B-DAP CAR T cells. .................................................................... 66
Figure 3.12: huLym-1-B-DAP CAR T cells do not induce significant downregulation of
the Lym-1 epitope on lymphoma cell lines. .................................................................... 67
Figure 3.13: CAR signaling domains do not affect antigen modulation. ......................... 68
Figure 3.14: Low-dose huLym-1-B-DAP CAR T cell therapy produces tumor free
survival. .......................................................................................................................... 69
Figure 4.1: Proposed Model of using DAP to improve Lym-1 CAR expansion. ............. 80
Figure 4.2: DAP signaling enable normal Lym-1 CAR expansion. ................................. 82

x
Abstract

The Lym-1 antibody targets a unique, discontinuous epitope (Lym-1 epitope) on
HLA-DR proteins preferentially upregulated in human B-cell lymphomas and leukemias,
without significant expression in normal tissues. It was developed in the late 1970’s by Dr.
Alan Epstein and found to be clinically safe and effective as an I-131
radioimmunoconjugate. Recently, SH7129, a molecule that mimics Lym-1 designed by
SHAL Technologies, was demonstrated to stain a subset of solid tumors suggesting the
application of Lym-1 epitope targeted immunotherapies can go beyond hematological
malignancies. Hence, we leverage the distinct binding profile of Lym-1 antibody with the
goal of developing effective chimeric antigen receptors (CARs) modified T cells to treat
Lym-1 epitope positive cancers, including solid tumors.
CARs are synthetic molecules that are capable of mediating T cell effector
functions upon engaging the target molecules in a major histocompatibility complex (MHC)
independent manner. This dissertation describes the development and evaluation of Lym-
1 epitope targeted CAR T cells generated using parental Lym-1 and a humanized version
(huLym-1-B). Both exhibited impaired expansion and progressive upregulation of
exhaustion markers when the signaling domain consisted of 4-1BB-CD3𝜁(BB3z) which is
the conventional component of 2
nd
generation CAR framework. Here, we identified the
underlying mechanisms of huLym-1-B-BB3z CAR T cells expansion failure and construct
an intracellular DAP10-DAP12 signaling domain-based (huLym-1-B DAP) CAR. This
novel signaling overcomes impaired expansion seen in BB3z based Lym-1 CAR T cells
xi
and mediates significantly better tumor control in a systemic human lymphoma model in
NSG mice. Our results demonstrate that huLym-1-B DAP CAR T cells are a promising
modality to explore in the clinic.

1
Chapter 1 Introduction

In 2017, chimeric antigen receptor (CAR) T cell immunotherapy made its way into
mainstream media with the approval of the US’s first T cell therapy product, Kymriah, a
living drug made of autologous T cells engineered with CD19 targeting CAR molecules
to treat pediatric and young adults with acute lymphoblastic leukemia (ALL). Two months
later, Kite Pharma’s Yescarta became the second CAR T cell therapy drug approved by
the FDA for the treatment of large B-cell lymphoma patients who relapsed after several
lines of conventional chemotherapy and bone marrow transplantation. The concept of
immunotherapy by autologous T cells can be traced back to 1986 when Dr. Steven
Rosenberg published a paper in Science introducing an approach to expand Tumor-
Infiltrating Lymphocytes (TILs) from various type of tumors. The ex vivo expanded TILs
were found to have 50 to 100 times higher anti-tumor efficacy than lymphokine-activated
killer (LAK) cells and led to 100% remission rate in a syngeneic MC-38 colon
adenocarcinoma mouse model(1). With years of expertise in TILs research, Dr.
Rosenberg led a team to take on metastatic tumors by adoptive transfer of TILs(2). A
seminal work published in 1989 by his group reported objective regressions of metastatic
melanoma with TIL therapy, and this treatment achieved significantly higher response
rates compared to previous LAK therapies(3). However, because of the MHC restriction
of T cell receptors (TCR) and the potential for graft versus host disease (GVHD), TILs
generated from tumor biopsies of one patient are unlikely to be useful to treat in other
patients with the same type of cancer. This limitation restricted extensive application of
TIL therapy in cancer immunotherapy(3). Shortly thereafter, a clinical trial was initiated to
investigate the safety and efficacy of human TILs modified by retrovirus mediated gene
2
transfer of neomycin(4). This trial not only allowed examination of the in vivo distribution
and persistence of adoptively transferred TILs, but also demonstrated the feasibility and
safety of introducing exogenous genes into lymphocytes with retroviruses(4). This
seminal work further fueled the development of engineered T cell therapies.
1.1 CAR T Cell Progress Timeline
Preclinical and clinical work in TILs provided indisputable evidence for the
therapeutical potential of T cells and led to a revolution in engineered TCR therapeutics(5).
The costs and long production time of such highly personalized treatments, however,
hindered further large-scale clinical applications. One approach to overcome these
limitations is to develop engineered T cells whose target site recognition is unrestricted
by HLA type (MHC independent). The prototype of first-generation CAR T cells was
developed by Zelig Eshhar et al., at the Weizmann institute in Israel in 1993(6). This
synthetic molecule fused the single chain variable region (scFv) of an antibody to the
signaling domain of either the immunoglobulin Fc receptor 𝛾 chain or the TCR 𝜁 chain(6).
The efficacy of these “first-generation” CAR T cells with signaling domains from both Fc
receptor 𝛾 and TCR 𝜁 has been studied in clinical trials. Though some studies did show
long-term persistence(7-9), first-generation CAR T cells generally exhibited limited
expansion and persistence in vivo and failed to induce meaningful clinical outcomes(10-
13). Of note, among the three clinical trials showed long persistence with first-generation
CAR T cells, two used Epstein-Barr virus specific T cells(7,8) and one used CD4(9) as its
binding domain. In either case, though, not investigated, those CAR T cells may engage
MHCs on activated antigen presenting cells (APCs) through their binding domain and also
receiving co-stimulation which together would promote CAR T cell survival.
3

Figure 1.1: Components of T cell receptor (TCR) complex and the evolution of Chimeric
Antigen Receptor (CAR) design.

To overcome the limitations experienced with first-generation CAR T cells, work
began to integrate a second signal from co-stimulation molecules in the CAR construct
aiming to improve their in vivo longevity and effector functions. One study designed a
synthetic CD28 molecule where the CD28 extracellular binding domain was replaced by
an scFv against GD2, a ganglioside overexpressed on many human tumor types(14).
Transduction of this molecule to human primary T cells selectively enhanced survival and
MHC I/II-antigen complex
!
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P P
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Tumor-specific/associate antigen
ScFv
Hinge
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Signal
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VL
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Signal 1
Signal 1
CD3!
CD3!
CD3!
Signal 1
Signal 1
4-1BB/
CD28
4-1BB&
CD28
TCR complex
Chimeric Antigen Receptor (CAR)
1
st
generation 2
nd
generation 3
rd
generation
Cancer Cell
T Cell
The conventional T cell receptor-CD3 complex comprises of highly variable a and b chains, and
the CD3. The CD3 contains 6 non-covalently associated chains, and each of them bear ITAM(s)
in the intracellular domain. Upon engagement of p-MHC, ITAM(s) are phosphorylated to delivery
signal 1 for T cell activation. The first-generation CAR uses the CD3z as the signaling domain to
activate T cells independent of endogenous TCR. Due to limited expansion and persistence of
first-generation CAR T cells, co-stimulation signal from 4-1BB, CD28, and others is added to the
CAR construct. The second-generation CAR has been actively studied in both pre-clinic and
clinic investigation and demonstrated to induce significantly better outcomes than first-generation
CAR. Third-generation CAR which contains two co-stimulations has also been developed.
However, a clear benefit of using two co-stimulations instead of one in the CAR constructed has
not been confirmed.
4
proliferation of modified cells after culturing with tumor cells expressing both cognate p-
MHC and GD2, suggesting this chimeric CD28 retains its function to co-stimulate T
cells(14). As an extension of this observation, the same group combined signaling
elements from TCR 𝜁 chain and CD28 to one synthetic construct. These human T cells
expressing this novel CAR construct manifested robust proliferation and produced
significant amounts of interleukin-2(IL-2), as well as showing antigen-specific cytolytic
activity in vitro(15). This construct represents the first “second-generation” CAR design
and has been widely used in preclinical and clinical studies. Later, CAR T cells with 4-
1BB co-stimulation were also been developed and proved to be antigen-specific in vitro.
However, none of the above-mentioned studies investigated the in vivo anti-tumor
efficacy of second-generation CAR T cells which was not demonstrated until 2007 when
Brentjens RJ et al. reported complete eradication of systemic acute lymphoblastic
leukemia xenografts by CD19 targeted second-generation CAR armed with the CD28 co-
stimulation (Figure 1.1)(16). Beginning in 2009, second-generation CAR T cells targeting
CD19 (CD19-CAR), with co-stimulatory signaling elements from CD28 or 4-1BB,
emerged as the most efficacious immunotherapy approaches in treating relapsed and
resistant B-cell malignancies (reviewed by (5)).
1.2 Current State of B-cell Lymphoma Therapy
Non-Hodgkin’s Lymphomas (NHL) derived from B-cell lineage (B-NHL) are among
the most common cancers in the United States with increasing incidence. Among all NHL
subtypes, diffuse large B-cell lymphoma (DLBCL) represents the most frequent type of
lymphoma(17). The combination of cyclophosphamide, doxorubicin, vincristine, and
prednisolone (CHOP), including the monoclonal anti-CD20 antibody (R-CHOP) is
5
standard first-line therapy(17). Using R-CHOP and R-CHOP-like regimens,
approximately 30% of treated patients unfortunately ultimately relapse and develop
resistance (R/R)(18). For R/R patients without comorbidity, high-dose chemotherapy
followed by autologous stem cell transplantation is a standard of care. It is estimated,
however, that less than 10% of those patients can be cured(19). Allogenic stem cell
transplant (Allo-SCT) can be used as an alternative strategy for strictly selected patients
and show up to 40% long-term remission; but limited availability of a matched donor and
occurrence of graft-versus-host disease make clear additional therapeutic options are
needed(20,21). Chimeric antigen receptors (CARs) modified autologous T cell therapy
targeting CD19 (CD19-CAR T cells) is now a novel approach to treat such R/R patients.
Multiple clinical trials of CD19-CAR T cells have demonstrated over 80% initial response
rates with ongoing complete response rate of 30-40% after 6 months(22-25). Despite of
its remarkable success, CD19 low/loss is a prominent mechanism of resistance in CD19
redirected T cell therapies(26,27). Thus, optimal immunotherapies for B-cell malignancies
will likely require targeting alternative epitopes on other antigens. Under consideration
are epitopes found on CD20(7), CD22(8), CD123(9) and others (10, 11). This dissertation
tested the hypothesis that the antibody Lym-1 could recognize another epitope for the
immunotherapy of B-cell lymphoma.
1.3 The Knowns and Unknowns of Lym-1 Antibody
Lym-1 is a murine IgG2a antibody which was developed by Alan Epstein in
1987(28). Unlike the immunogen for many other antibodies, the Lym-1 antibody was
isolated from mice hyper-immunized with nuclei from Raji Burkitt’s Lymphoma cells(28).
The Lym-1 antibody was initially thought to bind a conformational epitope on HLA-DR10
6
beta chain when the alpha chain and beta chain are intact(28). Although HLA-DR10 is a
relatively rare subtype found in only 2% of the American population, greater than 80% of
the lymphoma biopsies screened in clinical trials were found to be positive for Lym-1(29).
This observation prompted efforts to identify of the epitope bound by Lym-1 antibody.
Rose et al., identified four critical residues present on several HLA-DRs for Lym-1 binding,
and reasoned that both epitope density and the presence of critical residues are required
for optimal Lym-1 binding(29). This research clearly demonstrated that the conformational
epitope recognized by Lym-1 is not exclusively in HLA-DR10 but are HLA-DR molecules
that share at least the four critical residues.
An early Phase 1a clinical trials of the murine Lym-1 antibody demonstrated safety
but only a minor response in patients with B-cell lymphomas, most likely due to fast
clearance, low penetration to the tumor, and limited efficacy as an unmodified
antibody(30). Thus, three forms of modifications were applied: 1) construction of a
chimeric Lym-1(31), 2) conjugation of radionuclides for radio immunotherapy (32-37), 3)
and the addition of vasoactive moieties using genetic engineering methods to enhance
antibody and drug uptake in tumors(38,39). Among those improvements, isotope
conjugated Lym-1 was actively studied for both imaging and therapeutic reagents in
patients with B-cell lymphomas and chronic lymphocytic leukemias in several clinical trials.
Overall response rates of Iodine-131(
131
I) labeled Lym-1 (
131
I-Lym-1) in 30 patients was
57%. No severe toxicity was observed except those commonly seen in radio labeled
antibody therapeutics such as decreased white blood cells and platelets which are
attributed to non-specific radiation exposures (reviewed by(40) ). In addition, studies also
found that a significantly less amount of
131
I-Lym-1 (<20mg) was sufficient for optimal
7
tumor targeting than
131
I-tositumomab, a radio labeled anti-CD20 monoclonal antibody(7).
This result is consistent with prior work that showed Lym-1 epitopes to be upregulated in
B-cell malignancies, whereas CD20 is equally expressed in both normal and malignant
B-cells at high levels(28,29).

Figure 1.2: Comparison of rituximab and Lym-1 staining patterns shown by indirect
immunofluorescence microscopy. Taken from Nan Zhang et al., 2007(41).

Previous work from our lab found that CD20 staining on B-cell lymphoma cells gave an
evenly distributed pattern compared to Lym-1 which produced a unique speckled,
segregated pattern of immunofluorescence indictive of Lym-1 epitope presence in lipid
raft clusters (Figure 1.2) (41). Furthermore, Lym-1 binding alone is sufficient to induce
apoptosis in several B-cell lymphoma cell lines which is likely associated with loss of
mitochondrial potential and cytochrome C release(41). Those two features of Lym-1
antibody offer additional improvements in the targeting Lym-1 epitopes in the context of
CAR T cells, namely, that antigens present in lymphoma cells clustered in lipid rafts
provide an excellent docking sites for CAR T cells, and also, that binding of the Lym-1
epitopes by Lym-1 CARs will directly induce cell apoptosis independent of T cell
cytotoxicity.
8
1.4 Summary
The response of T cell receptors (TCR) to peptide-MHC (p-MHC) complexes
shows a high degree of sensitivity and specificity. In particular, TCR is able to sense as
few as 1-10 p-MHC molecules and produce sufficient stimulatory signaling that promotes
T cell activation and cytotoxic effector functions(42). CAR T cells, however, are more
sensitive to antigen level variation on target cell surface and require at least one
magnitude higher antigen density than TCR in order to be fully activated and effective(43).
Therefore, antigen downregulation in tumor cells could allow tumor immune escape from
CAR T cell therapy. In fact, antigen-low escape has emerged as a barrier to CAR-T cell
therapy success when used to target B cell differentiation antigens such as CD19, CD20,
BCMA and CD22(27,44). A common way to overcome antigen-low escape is to deploy
multi-targeting CAR(45,46) or increase the sensitivity of CAR T cells(47). Alternatively,
targeting antigens that are less prone to downmodulation may induce a higher rate of
sustained remission due to reduce antigen-low escape.
CD19 tends to downregulate in the presence of CD19 targeted therapies such as
CD19 CAR T cells or antibodies(48,49), the underlying mechanisms remain to be
investigated, but likely involve crosslinking induced endocytosis(49). Indeed, endocytosis
inhibition prolongs CD19 targeted antibodies retention on the tumor cell surface and
enhances ADCC effects(50). In contrast, HLA-DR antigens bearing the Lym-1 epitope,
like conventional MHC II molecules, do not significant downregulate upon crosslinking
and thus provide an attractive target for CAR T cells(30). In this thesis, my aim was to
develop effective Lym-1 CAR T cells for clinical use, as well as investigating Lym-1
epitope modulation in the presence of Lym-1 CAR T cells. The subsequent chapters of
9
this dissertation describe novel Lym-1 CAR construct design, in vitro and in vivo CAR T
cell phenotype characterization, and effector function evaluation. Lastly, the new findings
in these studies that can be exciting directions for the better understanding of CAR T cell
biology and basic immunology will be discussed.

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14
Chapter 2 Lym-1 Chimeric Antigen Receptor T Cells Exhibit Potent
Anti-Tumor Effects against B-Cell Lymphoma
1

2.1 Abstract
T cells expressing chimeric antigen receptors (CARs) recognizing CD19 epitopes have
produced remarkable anti-tumor effects in patients with B-cell malignancies. However,
cancer cells lacking recognized epitopes can emerge, leading to relapse and death. Thus,
CAR T cells targeting different epitopes on different antigens could improve
immunotherapy. The Lym-1 antibody targets a conformational epitope of Human
Leukocyte Antigen-antigen D Related (HLA-DR) on the surface of human B-cell
lymphomas. Lym-1 CAR T cells were thus generated for evaluation of cytotoxic activity
towards lymphoma cells in vitro and in vivo. Human T cells from healthy donors were
transduced to express a Lym-1 CAR and assessed for epitope-driven function in culture
and towards Raji xenografts in NOD-scidIL2Rgammanull (NSG) mice. Lym-1 CAR T cells
exhibited epitope-driven activation and lytic function against human B-cell lymphoma cell
lines in culture and mediated complete regression of Raji/Luciferase-Green fluorescent
protein (Raji/Luc-GFP) in NSG mice with similar or better reactivity than CD19 CAR T
cells. Lym-1 CAR transduction of T cells is a promising immunotherapy for patients with
Lym-1 epitope positive B-cell malignancies.

1
This chapter is taken from my first author paper in International journal of molecular science.
(Zheng L, et al. Lym-1 Chimeric Antigen Receptor T Cells Exhibit Potent Anti-Tumor Effects
against B-Cell Lymphoma. International journal of molecular sciences 2017;18(12) doi
10.3390/ijms18122773.)

15
2.1 Introduction
B-cell non-Hodgkin lymphomas (B-NHL) encompass a heterogeneous group of
cancers with increasing incidence(17,51). R-CHOP (Rituximab, cyclophosphamide,
Adriamycin, vincristine, and prednisone) is the frontline treatment regimen with a high
initial response rate(52). However, 30%-60% of responding patients relapse and are
refractory to subsequent R-CHOP treatment(52). Patients may be eligible for allogenic
stem cell transplantation (ASCT) but the prognosis is dismal with 9.9 months median
overall survival (OS)(53). Thus, new modalities are needed to address this unmet medical
need.
Chimeric antigen receptors (CARs) are synthetic molecules containing 3 distinct
modules: an extracellular antibody-based recognition site, a transmembrane module that
anchors the molecule into the cell membrane, and a chimeric intracellular signaling
domain that transmits the activation signal(54). Upon engagement of an epitope on a
tumor cell, CAR-directed T cells can induce tumor cell apoptosis or death through
cytotoxic T cell machinery in an MHC-independent manner(55). In the past few years,
CAR T cells targeting CD19 have achieved impressive outcomes in the treatment of
patients with relapsed or refractory (R/R) acute lymphoblastic leukemia (ALL)(56-58).
Although CD19 CAR T cell treatment of R/R B-NHL has been less successful, it has
improved objective response rates (ORR) from 20%-30% to 79% with complete remission
rates of 30%-50% which is 7-fold higher than historical results(59-62). The successful
treatment of B-cell malignancies with CD19 CAR T cell therapy provides direct evidence
for the effectiveness of this strategy.
16
Lym-1, a murine IgG2a monoclonal antibody, was generated by immunizing mice
with nuclei isolated from Raji lymphoma cells(28). Lym-1 binds to a discontinuous
conformational epitope on several HLA-DR subtypes with a greater binding affinity for
malignant B-cells than normal B-cells(29). In clinical studies, 80% of patients with Non-
Hodgkin’s Lymphoma (NHL) and 40% of patients with chronic lymphocytic leukemia (CLL)
were found to be Lym-1 positive(35). A clinical trial performed several decades ago using
murine Lym-1 resulted in limited therapeutic effects in 10 patients with refractory B-cell
lymphoma, perhaps due to the short half-life and immunogenicity of the murine
antibody(30). Subsequently, a chimeric version of Lym-1 was tested clinically after
labeling with 131I or 67Cu-2IT-BAT to treat resistant intermediate and high-grade
lymphoma where it was found to achieve significant efficacy in the treatment of
chemotherapy-resistant tumors(32,37). This record makes Lym-1 an attractive antibody
to test using CAR T cell technology.
This chapter reports the design and evaluation of a second generation Lym-1 CAR
using human primary T cells. The results demonstrate that Lym-1 CAR T cells display
epitope-driven cytokine release, proliferation, and cytotoxicity against Lym-1 epitope-
positive cell lines, and complete eradication of Lym-1 epitope-positive Raji xenograft
tumors in NSG mice.
2.2 Materials and Methods
2.2.1 Antibodies
PE-Anti-huCD107a (Clone: H4A3, Biolegend), PE-Anti-huCD3 (Clone: UCHT1, BD),
APC-Anti-huCD45 (Clone: H130, BD), Lym-1 and chLym-1 (lot: 100226) were produced
17
in our laboratory(31), IL-7-Fc and IL-15-Fc were produced through mammalian
expression system and purified in-house.
2.2.2 Cell Lines
K562, Daudi, Jurkat, and Raji cell lines were obtained from American Type Culture
Collection (ATCC, Manassas, VA). Raji/Luc-GFP cells were a gift from Dr Yvonne Y.
Chen at the University of California, Los Angeles. All cell lines were cultured in RPMI-
1640 supplemented with 10% dialyzed FCS (dFCS, Hyclone, Logan, UT), 2% Glutamine
and 1% Pen/Strep (Gemini Bio-Products, West Sacramento, CA). HEK-293 LTV cells
(Cell Biolabs Inc, San Diego, CA) cultured in DMEM (Corning, Manassas, VA)
supplemented with 10% dFCS, 2% Glutamine and 1% Pen/Strep were used for lentivirus
production.
2.2.3 Vector Construction and Preparation of Lentivirus
For comparison purposes, a second generation CD19 CAR comprised of a CD19 specific
targeting scFv derived from antibody FMC63, a CD8α leader sequence, a (G4S)3 linker
between the VH and the VL domains, a CD8α hinge, a CD8α transmembrane domain
followed by 4-1BB co-stimulatory domain, and intact intracellular CD3ζ were cloned
between the EcoRI and MluI restriction sites into the lentiviral vector pLVX-EF1α-IRES-
Zsgreen (Clontech, Mountain View, CA). For this study, the IRES-ZsGreen moiety was
removed by restriction enzyme digestion with EcoRI and MluI (NEB, Ipswich, MA) prior
to insertion of the CAR encoding gene. The pLVX-EF1α-Lym-1 construct was made by
substituting a scFv derived from Lym-1 for the FMC63 scFv in the CD19 vector (described
above). The whole amino acid sequences for all CAR constructs are provided in the
appendix. Lentivirus was produced by transient co-transfection of the CAR transfer
18
vectors (pLVX-EF1α-CD19 or pLVX-EF1α-Lym1) with packaging plasmids, psPAX2 and
pMD2.G (Addgene) using HEK-293LTV cells. Supernatants containing viral particles
were collected at 24 and 48 hours after transfection and were combined, filtered, and
concentrated by ultracentrifugation. Pelleted virus was then resuspended in PBS
supplemented with 1% BSA and 7% trehalose, aliquoted, and stored at -80°C. Viral titers
were measured by transducing 10
6
Jurkat T cells with 10-fold serial dilutions of virus
vector. Forty-eight hours after transduction, cells were labeled with biotinylated Protein-L
(Genscript, Piscatawat, NJ) and detected by APC-labeled Streptavidin (BD, Franklin
Lakes, NJ). Positively transduced cells at a range of 10~20% were used to calculate the
virus transducing units (TU) by the following formula: TU/mL = (10
6
seeded cells × %
positive cells × 1,000)/ μl of virus vector.
2.2.4 Primary T Cell Isolation and Transduction
Blood from healthy donors was obtained using heparin collection tubes in accordance
with IRB regulations at the University of Southern California (Protocol #11606). Primary
blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque (Life Technologies,
Inc.) as per the manufacturer's protocol. T cells were then isolated using a T cell negative
selection kit (Stem Cell Technologies, Seattle, WA) and cultured in T cell medium (43%
Clicks, 43% RPMI 1640, 2% Glutamax (Life Technologies, Inc.), 10% dFCS, 1% non-
essential amino acid solution, 1% Pen/Strep solution, 50ng/ml IL-7-Fc, 50ng/mL IL-15-
Fc). Three days prior to transduction, T cells were activated by adding CD3/CD28 beads
(Life Technologies, Inc.) at a 1:1 ratio, and then transduced by centrifugation at 800g for
90 min with lentivirus (MOI = 15) and Lentiblast (OZ bioscience, San Diego, CA).
Transduction was performed twice, followed by a media change after 24 hours, after
19
which cells were transferred to 24-well G-Rex plates (Wilson Wolf, St Paul, MN)
supplemented with fresh T cell medium. T cells were used for assays at day 10 after
transduction. CAR virus transduction efficiency was evaluated by flow cytometry at day
10 using biotinylated Protein L followed by detection with APC-labeled Streptavidin. Mock
transductions were conducted as negative controls as described above bit in the absence
of a viable virus vector.
2.2.5 Cytotoxicity
Transduced effector CAR T cell preparations were adjusted to be 30% positive for CAR
T cells by addition of Mock T cells. These adjusted preparations were incubated with
target K562, Daudi, and Raji cells with ratios of CAR T cells to targets of 20:1, 10:1, 5:1,
and 1:1 each in flat-bottom 96 well plates for 24 hours in the absence of added cytokines.
Supernatants were collected and subjected to measurement of LDH activity (Pierce,
Waltham, MA) according to the manufacturer’s instructions.
2.2.6. Cytokine Production and Degranulation Assays
One x 10
5
target cells and 2 × 10
5
CAR T cells (30% CAR positive) were co-cultured in
200 µL T cell medium without addition of cytokines. After 24 hours, supernatants were
collected and analyzed for levels of INF-γ and IL-2 by ELISA using in-house antibodies.
For the CD107a degranulation assay, CAR T cells (30% CARs positive) and target cells
were mixed at defined ratios as described above. Five µl of APC conjugated anti-
huCD107a antibodies and 1X Monensin (BioLegend, San Diego, CA) were added directly
to each well. After incubation for 5 hours at 37°C, cells were labeled with 5 µl PE-anti-
huCD3 antibody and subjected to flow cytometry using an Attune Acoustic Focusing
Cytometer (Life Technologies, Carlsbad, CA) to detect CD107a expression.
20
2.2.7 CFSE Proliferation Assay
One x 10
6
T cells transduced either with the pLVX-EF1a-Lym-1 (30% CAR positive) or
pLVX-EF1a-CD19 (30% CAR positive) were labeled with CFSE-Far-red as per
manufacturer’s instructions (Invitrogen, Carlsbad, CA) and co-cultured with either
irradiated K562, Raji, or Daudi cells at a ratio of 1:1 in 24 well G-Rex plates, in the absence
of cytokines for 5 days. A flow cytometer was used to measure the cell proliferation profile.
Irradiated cells were prepared by exposing 10
7
cells to a total dose of 100Gy using an X-
RAD 320 (PXI, North Branford, CT). After irradiation, cells were washed and
cryopreserved by using a freezing solution (Cryostor CS10, Bothell, WA), and stored at -
80°C.
2.2.8. Raji/Luc-GFP Xenograft Studies in NOD Scid-IL2Rgammanull (NSG) Mice
All mouse experiments were approved by the USC Animal Care and Use Committee
(Protocol #20585). Eight-week old male NSG mice purchased from Jackson Laboratories
(JAX #3877458) were housed in the USC vivarium in sterile cages. 10
6
Raji/Luc-GFP
cells in 100 µl PBS were injected intravenously (i.v.) via the lateral tail vein using an insulin
syringe (designated as day 0). Luciferase activity was measured on day 6 via
bioluminescence imaging to assess tumor burden. On day 7, 10
7
Mock transduced T cells,
CD19 CAR T cells (50% CAR positive), or Lym-1 CAR T cells (50% CAR positive) were
prepared in 100 µl PBS and injected i.v. using an insulin syringe. A chimeric Lym-1
antibody control group was included in which 100 μg chLym-1 in 100 µl PBS was injected
i.v. on days 7, 9, and 11. Tumor progression was monitored by bioluminescence imaging
using an IVIS imaging system at the USC Molecular Imaging Center. At day 60, surviving
mice were euthanized, spleen and bone marrow cells harvested and re-suspended in a
21
total volume of 2 mL of FACS buffer (PBS, supplemented with 2% FCS). Two hundred µl
of the cell suspension were then labeled with PE-ani-huCD3 and APC-anti-huCD45
antibodies and analyzed by flow cytometry to determine the percentage of human T cells.
2.2.9. Statistical Analysis
All results were expressed as means ± standard deviation (SD) or standard error of the
mean (SEM), as indicated. Statistical analyses were performed using unpaired 2-tailed
Student t test by Graphpad Prism 6.
2.3 Results
2.3.1 Efficient Expression of Lym-1 CAR on Transduced Primary Human T cells
The Lym-1 CAR construct used in this study was a second-generation construct
with a CD8α leader sequence, a Lym-1 specific scFv moiety fused to a CD8α hinge and
transmembrane domain, followed by a 4-1BB co-stimulation and CD3ζ signaling domains
(Figure 2.1).

Figure 2.1: Lym-1 CAR and CD19 (FMC 63) CAR constructs.
To transduce T cells to express the CAR, the CAR cDNA was inserted into a
modified self-inactivating pLVX lentiviral vector, between the EcoRI and MluI sites. The
replication-incompetent lentiviruses encoding the CARs or pLVX- EF1α-IRES-ZsGreen
(as a control) were used to transduce human primary T cells three days after activation
(day 0). Compared to mock transduced T cells (T cells processed without addition of a
lentiviral transfer vector), Lym-1 CAR and CD19 CAR transduced T cells showed similar
22
rates of proliferation up to day 7. After day 7 CD19 CAR T cells proliferated more rapidly
than Lym-1 CAR T cells (Figure 2.2 A). Lym-1 and CD19 CAR T cells showed consistent

Figure 2.2: Efficient Lym-1 CAR expression on human primary T cells.

(A) Cell numbers were recorded after mock or virus transduction. (n = 3 replicates per point;
representative of three donors). (B) At day 10, 10
6
T cells were labeled with 2 ug Biotin-Protein
L, followed by detection with APC-streptavidin. Mock-transduced T cells served as a negative
control. (n= 6). (C) After expansion, the CD4/CD8 ratio of the T cell preparations shown in Panel
B were analyzed for CD4 and CD8 expression (representative of three donors).
23
transduction efficiencies ranging from 30% to 80%, as determined by flow cytometry using
protein L (Figure 2.2 B). At day 12, the T cell preparations consisted of a mixture of CD4+
(~65%) and CD8+ (~20%) T cells (Figure 2.2 C).
2.3.2. Epitope-Driven Expression of CD107a and Epitope-Dependent Cytotoxicity
of Lym-1 and CD19 CAR T Cells
Three cell lines were used to assess epitope-driven functions of Lym-1 and CD19
CAR T cells. Flow cytometry using chLym-1 and anti-CD19 antibodies identified two cell
lines expressing Lym-1 and CD19 epitopes, Raji and Daudi, and one that expressed
neither, K562 (Figure 2.3). pLVX-EF1α-IRES-ZsGreen transduced T cells and mock
transduced T were used to detect T cell activity independent of either the Lym-1 or CD19
CAR. Both Lym-1 and CD19 CAR T cells significantly up-regulated CD107a in response
to co-culture with Raji and Daudi (p<0.01) but not K562 (Figure 2.4). Similarly, the

Figure 2.3: Detection of Lym-1 and CD19 epitopes on Daudi and Raji cells but not K562
cells.
Cell surface epitope intensity was detected by incubation with Dylight 650 conjugated chLym-1
antibody or APC conjugated anti-CD19 antibody.
24

Figure 2.4: Epitope-driven upregulation of CD107a on Lym-1 and CD19 CAR T cells.

(continued) Lym-1 and CD19 CAR T cells efficiently lysed the epitope-expressing Raji
and Daudi cell lines but not the epitope-negative K562 cell line. Mock transduced T cells
and pLVX-EF1α- IRES-ZsGreen transduced T cells did not show a significant level of cell
lysis at any of the target:effector cell ratios tested (Figure 2.5).
2.3.3 Epitope-Driven Release of Cytokines from Lym-1 and CD19 CAR T Cells
Lym-1 and CD19 CAR T cells were detected by protein L and APC-streptavidin flow cytometry.
Mock transduced T cells were added to each preparation to adjust the CAR T cell fraction to
30%. Two × 10
5
T cells were then incubated with 10
5
Raji or Daudi cells. Mock transduced T cells
alone and CAR transduced T cells incubated with epitope-negative K562 cells served as negative
controls. An anti-CD107a antibody and monensin were then added to the wells soon after. After
a 5 hr incubation, cells were labeled with PE-anti-CD3 antibody to distinguish tumor and T cells
using flow cytometry. Top panel: examples of data. Bottom panel: data from n = 3 (ns, not
significant; **=P<0.01; compared to CD107a level when co-incubated with K562).
25
Lym-1 and CD19 CAR T cells were incubated with tumor cell lines at a ratio of 2:1
as described above. T cell preparations comprised of either Lym-1 CAR (30% CAR
positive) or CD19 CAR (30% CAR positive) T cells released IFN-γ and IL-2 when co-
cultured overnight with epitope-positive Raji and Daudi cells, but not with K562 or in the
absence of a target tumor cell line.

Figure 2.5: Epitope-driven cytotoxicity of Lym-1 and CD19 CAR T cells.

Neither Zsgreen or Mock transduced T cells released IFN-γ or IL-2 when cultured with
any of the three tumor cell lines (Figure 2.6). Therefore, release of these cytokines by
Lym-1 and CD19 CAR T cells appears to be due to recognition of a Lym-1 or CD19
epitope.
2.3.4. Epitope-Driven Proliferation of Lym-1 and CD19 CAR T Cells
Lym-1 CAR and CD19 CAR T cells labeled with CFSE-Far-red cell proliferation
trace dye were cultured alone or with irradiated tumor cell lines with no cytokines added.
After 5 days, the CFSE signal was measured and overlap histograms prepared to
represent the fluorescence intensity of CAR T cells alone compared to CAR T cells with
T cells (control or 30% CAR positive) were cultured overnight with 2 × 10
4
K562, Raji, or Daudi
cells at indicated ratio. Supernatants were processed to measure cytotoxicity. Data from one
donor is shown; similar results were obtained from a second donor. For each donor, three
independent transductions were each assessed using triplicate determinations. **= P<0.01; ****=
P<0.001 compared to % lysis in Mock transduced T cells at the same E/T ratio.

26
indicated target T cells after five days of co-culture. When co-cultured with Raji and Daudi
cells, Lym-1 and CD19 CAR T cells showed a significant shift to the left, indicating cell
division and proliferation. However, little or no shift was observed when the CAR T cells
were co-cultured with epitope negative K562 cells (Figure 2.7). These findings
demonstrate Lym-1 and CD19 CAR T cells proliferate when cultured with cells expressing
recognized epitopes.

Figure 2.6: Epitope-driven release of cytokines from Lym-1 and CD19 CAR T cells.

2.3.5. Lym-1 and CD19 CAR T Cells Eradicate Raji/Luc-GFP Xenograft Tumors in
NSG Mice
The anti-tumor effects of CAR T cells were evaluated using an NSG mouse
leukemic Raji/Luc-GFP xenograft model. Luciferase activity of Raji/Luc-GFP was
measured and titered in vitro to confirm the feasibility of in vivo optical imaging (data not
shown). Tumor burden was assessed by serial bioluminescent imaging of luciferase
The percentage of CAR-transduced T cells was adjusted to 30%. Two × 10
5
cells were then
incubated with 10
5
K562, Raji, Daudi, or no target cells. Representative cytokine release levels
from two donors are shown.

27
expression six days after tumor inoculation. Equal tumor burden was verified in each
group before treatment as evidenced by the luciferase signal (Figure 2.8 A). On day 7, a
single dose of 10
7
Mock- transduced T cells and Lym-1 CAR and CD19 CAR T cells (50%
CAR positive cells as measured by Protein L) were injected i.v. When the Lym-1 antibody
binds to a Lym-1 epitope it can induce target cell apoptosis or autophagy in addition to
ADCC or CDC effects(41,63). To determine if this effect of Lym-1 was sufficient to
eradicate Raji cells in this in vivo model, a group of mice received 100 µg human chimeric
Lym-1 (chLym-1) on days 7, 9, and 11.

Figure 2.7: Epitope-driven proliferation of Lym-1 and CD19 CAR T cells.

One million Lym-1 or CD19 CAR T cells were labeled with CSFE-Far-red dye and co-cultured
with 10
6
irradiated K562, Daudi, or Raji cells in the absence of exogenous cytokines. CAR T cells
cultured alone served as a negative control. After five days of co-culture, cells were labeled with
PE-anti CD3 and analyzed by flow cytometry.
28
In mice treated with Lym-1 CAR T cells, only background bioluminescence was detected,
and the antitumor effect was durable throughout the 60-day experiment (Figure 2.8 A-C).
Essentially equal tumor eradication was achieved in the CD19 CAR T cell group, except
that one mouse relapsed 30 days after treatment and died on day 53 (Figure 2.8C). In
contrast, treatment with the chLym-1 antibody controlled tumor progression only up to
day 13, whereupon tumor growth began (Figure 2.8 A-B). These mice were sacrificed on
days 25 and 30 due to paralysis of the hind legs and severe systemic tumor burden. In
the five mice receiving mock transduced T cells, tumor burden progressed until day 20
and then modestly regressed with tumor resurgence in three mice causing death (Figure
8 A,C). This result was likely due to an allogeneic reaction of the human T cells to Raji
cells(64).
29

Figure 2.8: Lym-1 and CD19 CAR T cells eradicate Raji/Luc-GFP xenograft tumors in vivo.

2.4 Discussion
Lym-1 is a mouse IgG2a antibody developed in the early 1980’s(28). Clinical
studies confirmed its safety and potential benefit in the treatment of B-cell
malignancies(34). In addition, when bound to polymorphic variants of HLA-DR subtypes,
the Lym-1 antibody has not been found to cause antigen shedding or modulation,
(A) Ventral and dorsal bioluminescence imaging of tumor burden in control and treated mice. 10
6

Raji/Luc-GFP cells were injected i.v. into 8-10 weeks old male NSG mice (day 0). Luciferase
bioluminescence was measured at day 6 to assess pre-treatment tumor burden. On day 7, 10
7

Mock transduced T cells, CD19 CAR T cells, or Lym-1 CAR T cells in 100ul PBS were injected
i.v (n=5). For the chLym-1 antibody group, 100 ug chLym-1 was injected i.v. in 100ul PBS on
days 7, 9, and 11. (B) Quantification of bioluminescence shown in “A.” (C) Kaplan-Meier plot of
survival of mice. *=P<0.01, compared to Mock T cells group.
30
important characteristics for a CAR T cell therapy. Therefore, a second generation Lym-
1 CAR was designed for evaluation as a potential therapeutic for Lym-1 epitope positive
B-cell malignancies(30).
This report demonstrates a Lym-1 CAR construct can be efficiently expressed in
human primary T cells from healthy donors with transduction efficiencies ranging from 30-
-80%. The CD8/CD4 ratio of the preparation was approximately 2:5 after expansion
(Figure 2.2 B,C). During expansion, Lym-1 CAR T cells proliferated at a lower rate than
Mock-transduced T cells (Figure 2.2 A). Since significant Lym-1 epitope expression was
not detected on the T cells from the 3 different donors used in this study (data not shown),
the reduced proliferation rate may arise from tonic signaling from chimeric antigen
receptors induced by the clustering of scFv(65). 4-1BB as a co-stimulation domain was
reported to enhance persistence of engineered T cells in vivo and resulted in an improved
survival rate in CD19 CAR clinical studies(57,66). In addition, 4-1BB signal activation can
ameliorate T cell exhaustion, induced by the tonic signaling of chimeric antigen receptors,
which is a common phenomenon in most of the CARs products except the highly
functional CD19 CAR(65). Therefore, a 4-1BB co-stimulation domain was chosen for this
Lym-1 CAR construct.
The modest increase in CD107a expression on Lym-1 transduced T cells (10% vs
3%) before encountering the Lym-1 epitope positive tumor cell lines (Figure 2.4) may
also arise from tonic signaling. Nonetheless, this increase did not affect epitope-driven
activity of Lym-1 CAR T cells as monitored by secretion of IFN-γ and IL-2 (Figure 2.6) or
lysis of Lym-1 epitope negative K562 cells (Figure 2.5). Importantly, the observed lower
levels of expansion of Lym-1 CAR T cells during manufacture did not reduce proliferation
31
upon recognition of the Lym-1 epitope: Lym-1 and CD19 CAR T cells proliferated at the
same rate when cultured with irradiated tumor cell lines expressing the Lym-1 and CD19
epitopes in the absence of exogenous cytokines (Figure 2.7).
The antitumor effects of Lym-1 CAR T cells were evaluated in a metastatic
Raji/Luc-GFP xenograft model. A single dose of Lym-1 CAR T cells induced sustained
tumor suppression in the NSG Raji xenograft model and a successful engraftment of
human T cells in the NSG mice. In addition to ADCC and/or CDC effector mechanisms,
chLym-1 is also able to induce cell apoptosis directly upon binding antigen(41). This effect
is caspase independent and partially attributed to a rapid loss of mitochondrial membrane
potential and cell autophagy(41,63). To determine if this effect is sufficient to eradicate
the Raji tumor cells in this model three doses of chLym-1 were administered. Although
Raji tumor growth was initially checked, dramatic tumor progression was observed after
day 20 (Figure 2.8). This limited suppression may arise because the half-life of the
chimeric antibody is less than that of the CAR T cells that proliferate upon recognizing a
target. Nonetheless, the apoptotic effect of Lym-1 binding to lymphoma cells may confer
on Lym-1 CAR T cells enhanced target killing compared to CAR T cells based on an
antibody without this effect.
The data presented here demonstrate that primary human T cells can be efficiently
transduced with a Lym-1 CAR. In culture systems, Lym-1 CAR T cells recognize human
lymphoma cell lines in an epitope-driven manner to secrete cytokines, proliferate, and kill
tumor cells. Lym-1 CAR T cells eradicated Lym-1 epitope positive tumors in NSG mice.
Lym-1 CAR T cells thus hold promise for the treatment of lymphomas in patients.

32
Acknowledgments
This work was supported in part by Cell Biotherapy, Inc., a company focused on
the development of novel CAR T cell products for the treatment of hematopoietic
malignancies and solid tumors. The authors acknowledge the technical contributions of
Ryan Graff and Stacey Jaw.

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35
Chapter 3 A humanized Lym-1 CAR with Novel DAP10/12 Signaling
Domains Demonstrates Reduced Tonic Signaling and Increased
Antitumor Activity in B-Cell Lymphoma Models
2

3.1 Abstract:
Purpose: The murine Lym-1 monoclonal antibody targets a discontinuous epitope (Lym-
1 epitope) on several subtypes of HLA-DR, which is upregulated in a majority of human
B-cell lymphomas and leukemias. Unlike CD19, the Lym-1 epitope does not downregulate
upon crosslinking, which may provide an advantage as a target for CAR T cell therapy.
Lym-1 CAR T cells with a conventional 4-1BB and CD3z (BB3z) signaling domain
exhibited impaired ex vivo expansion. This study aimed to identify the underlying
mechanisms and develop strategies to overcome this effect. Experimental: Design: A
functional humanized Lym-1 antibody (huLym-1-B) was identified and its scFv form was
used for CAR design. To overcome observed impaired expansion in vitro, a huLym-1-B
CAR using DAP10 and DAP12 (DAP) signaling domains was evaluated for ex vivo
expansion and in vivo function. Results: Impaired expansion in huLym-1-B-BB3z CAR T

2
This chapter is taken from my first author paper in Clinical Cancer Research. (Zheng L, et al., A
humanized Lym-1 CAR with novel DAP10/DAP12 signaling domains demonstrates reduced tonic
signaling and increased anti-tumor activity in B-cell Lymphoma models. Clinical cancer research:
an official journal of the American Association for Cancer Research 2020 doi 10.1158/1078-
0432.Ccr-19-3417.)

36
cells were shown to be due to ligand-dependent suboptimal CAR signaling caused by
interaction of the CAR binding domain and the surface of human T cells. Using the novel
DAP signaling domain construct, the effects of suboptimal CAR signaling were overcome
to produce huLym-1-B CAR T cells with improved expansion ex vivo and function in vivo.
In addition, the Lym-1 epitope does not significantly downregulate in response to huLym-
1-B-DAP CAR T cells both ex vivo and in vivo. Conclusions: DAP intracellular domains
can serve as signaling motifs for CAR, and this new construct enables non-impaired
production of huLym-1-B CAR T cells with potent in vivo anti-tumor efficacy.

37
3.3 Introduction
Non-Hodgkin’s Lymphomas (NHL) derived from B-cell lineage (B-NHL) are among
the most common cancers in the United States with increasing incidence(17) . Autologous
T cells engineered to express chimeric antigen receptors (CARs) targeting CD19 (CD19-
CAR T cells) is a recently FDA-approved approach to treat patients that have relapsed
and are resistant (R/R) to traditional therapy. Clinical trials of CD19-CAR T cells have
achieved over 80% initial response rates falling to 30-40% after 6 months(22-25). Loss of
CD19 epitopes is a prominent mechanism of resistance in CD19-CAR T cell
therapies(26,27). Thus, optimal immunotherapies for B-cell malignancies will likely
require targeting additional epitopes on multiple antigens. Under consideration are
epitopes found on CD20(67), CD22(68), CD123(69), and others(70,71). The epitope
recognized by the antibody Lym-1 is also a promising candidate for the immunotherapy
of B-cell lymphomas. The murine Lym-1 antibody binds to a discontinuous epitope (Lym-
1 epitope) on several subtypes of HLA-DR with a higher binding avidity for malignant B-
cells than normal B-cells(28). Thus far, four amino acid residues have been identified as
binding sites for Lym-1 but additional studies may be wanted to determine if other sites
also contribute to Lym-1 binding(29). However, in an extensive screen only HLA-DR
positive cells can be recognized by Lym-1 antibodies(28). Critical amino acid sequences
and sufficient antigen density are both required for Lym-1 binding(29). Based on previous
histology data, cancer cells from 80% and 40% of patients with B-NHL and chronic
lymphocytic leukemia (CLL), respectively, were positive for the Lym-1 epitope(29,35). In
addition, the epitopes recognized by Lym-1 are not shed nor internalized enabling stable
binding to human lymphoma cells(28,30). Lym-1 binding is also enhanced since antigens
38
bound by Lym-1 are concentrated in surface lipid rafts to produce a speckled membrane
pattern by indirect immunofluorescence staining compared to CD19, CD20, and CD22
which produce a ring pattern indicating that, unlike the Lym-1 antigen, these other B-cell
antigens are more evenly distributed over the surface of tumor cells(41). The Lym-1
antibody also has been extensively studied in patients as an I-131 radiolabeled antibody
for the imaging and treatment of Lym-1 positive tumors and shown to be safe at
therapeutic doses without causing B-cell aplasia(30,32,35). Thus, developing CAR T cells
targeting a Lym-1 epitope provides a promising strategy to treat R/R B-cell lymphoma
patients, and would potentially decrease relapse rates if targeted concurrently with other
lymphoma associated antigens.
During development of Lym-1CAR T cells, we observed impaired ex vivo
expansion and exhausted phenotypes when the intracellular signaling domain (ICD)
contained 4-1BB3z (BB3z), an ICD commonly used in the construction of CAR T cells.
To reduce potential immunogenicity, a humanized Lym-1 antibody was generated to
construct huLym-1-B-BB3zCAR T cells which also exhibited the aberrant phenotypes.
Although high doses of Lym-1-BB3z(72) and huLym-1-B-BB3z CAR T cells (this report)
eradicated metastatic Raji tumors in vivo, limited ex vivo expansion of the CAR T cells
would pose a challenge for the manufacture of these CAR T cells. Using huLym-1-B-
BB3zCAR as a model, we now demonstrate that the adverse effects of huLym-1-B-BB3z
CAR on T cells is associated with ligand-dependent CAR signaling mediated by the
interaction of CAR molecules with weakly expressed Lym-1 epitopes on human T cells.
There is increasing recognition that CAR tonic signaling compromises anti-tumor
efficacy of CAR T cells(65,73). CAR tonic signaling can arise from sustained non-
39
coordinated and weak CD3ζ motif activation caused by ligand-independent mechanisms
which result in impaired T cell proliferation, accelerated T cell exhaustion, enhanced
activation induced cell death (AICD), and poor efficacy in vivo(73). Ligand-independent
tonic signaling can be reduced by using appropriate promoter(74),
hinge(75),transmembrane domain(76), and signaling domains(65). Aberrant phenotypes
caused by ligand-dependent CAR signaling have been attributed to fratricide(77). We
report here that the dominant cause of impaired expansion in huLym-1-B-BB3z CAR T
cells is due to suboptimal ligand-dependent signaling rather than fratricide and propose
a strategy using DAP10 and DAP12 (DAP) ICD to address this issue.
DAP12, an adaptor protein with one immunoreceptor tyrosine-based activation
motif (ITAM) in its cytoplasmic domain, is expressed in a broad range of hematopoietic
cells including macrophages, natural killer cells, and some subsets of T cells(78). A
unique feature of DAP12’s ITAM is that the domain also contains an immunoreceptor
tyrosine-based inhibitory motif (ITIM) which is not present in any of the ITAMs from
CD3ζ(79). In osteoclasts, weak stimulation of DAP12 associated receptor-TREM2
recruits Src homology 2 (SH2)-containing inositol phosphatases (SHIP-1) to the ITIM
motif to blunt tonic signaling, whereas a strong receptor stimulation prevents SHIP-1
recruitment(78). In addition, antigen-specific activation of ectopically expressed DAP12
is sufficient to drive T cell cytotoxicity(80-82) suggesting that the signaling domain from
DAP12 can be used for CAR construction. DAP10, another signaling subunit, contributes
to DAP12-dependent phosphatidylinositol 3-kinase (PI3K) activation(78), a pathway that
is critical for T cell expansion and differentiation(83). Thus, we hypothesized a CAR
constructed with a DAP10-DAP12 signaling domain would possess a threshold that blunts
40
weak epitope recognition from causing the deleterious effects of suboptimal CAR
signaling but allows stronger signals to promote cytotoxic functions. Here, we report that
the replacement of ICD in huLym-1-B-BB3zCAR with the cytoplasmic domains from DAP
addresses the CAR T cell expansion issue ex vivo and leads to significantly better tumor
control in vivo than its BB3z counterpart. In addition, we also demonstrate that the epitope
recognized by Lym-1 on human B lymphoma cell lines does not significantly downregulate
in the presence of huLym-1-B-DAP CAR T cells, whereas detection of CD19 antigen
diminishes in response to CD19-CAR T cells both in vitro and in vivo. Our studies suggest
that the DAP signaling domain described here offers a new strategy to circumvent
adverse effects of suboptimal signaling from BB3z-based CARs and thus huLym-1-B-
DAP CAR T cells provide a promising modality for patients with lymphomas and
leukemias recognized by Lym-1.
3.4 Materials and Methods
3.4.1 Mice
Mouse experiments were approved by the USC Animal Care and Use Committee (IACUC
20585) and involved 8 to 13-week-old NSG mice (female and male) purchased from
Jackson Laboratories or bred in the USC animal facility under IACUC 20697.
3.4.2 Cytokines and Antibodies
Chimeric Lym-1 (chLym-1) a chimeric analog of the original Lym-1 antibody, IL-7-Fc, IL-
15-Fc, and Dylight 650 anti-261tag antibodies were developed and prepared in our
laboratory. Commercial antibodies used were: Alexa 488 Goat anti-human IgG(H+L)
(ThermoFisher, CAT#A-11013), Alexa Fluor 647 anti-human IgG Fc (Biolegend,
CAT#409320), Phycoerythrin (PE) anti-human phosphorylated CD3ζ (pY142) (BD
41
Sciences, CAT#558448), PE mouse anti-human CD22 (Biolegend, CAT#302506), PE
mouse anti-human CD19 (Biolegend, CAT#302254), PE mouse anti-human PD-1
(Biolegend, CAT#329906 ), PE mouse anti-human LAG-3 (Biolegend, CAT#369306),
Mouse anti-CD247(pY142) ( BD, CAT#558402), Mouse anti-human CD247 (BD,
CAT#551033).
3.4.3 Reagents
Reagents used to conduct these studies included: RPMI-1640 (Genesee Scientific,
Lot#0519108), DMEM (Genesee Scientific, Lot#05191016), Dialysed fetal calf serum
(dFCS) (Hyclone, Cat#SH30079.03), GlutaMAX (ThermoFisher, CAT#35050-061),
Penicillin/Streptomycin (Corning, CAT#30-002-CI), non-essential amino acids (Genesee
Scientific, CAT#25-536), Click’s medium (SIGMA, CAT#C5572-500ML), EcoRI (NEB,
CAT#R3101M), MluI (NEB, CAT#R3198L), psPAX2 (Addgene,CAT#12260), pMD2.G
(Addgene, CAT#12259), Xfect (Clontech, CAT#631418), Ficoll-Paque (Life Technologies,
CAT#GE17-1440-02), EasySep Human T cell isolation Kit (STEMCELL, CAT#19051), D-
(+)-Trehalose dihydrate (SIGMA, CAT#90210-50G), Lentiblast (OZBiosciences,
CAT#LB01500), 24-well G-Rex plates (Wilson Wolf ,CAT#80240M), Dynabeads human
T-activator CD3/CD28 (ThermoFisher, CAT#11131D), ImmunoCult human
CD3/CD28/CD2 T cell activator (STEM CELL, CAT#10970), IL-2 ELISA kit
(ThermoFisher, CAT#EH2IL2 ), GM-CSF ELISA kit (ThermoFisher, CAT#EHGMCSF),
and INF-gamma ELISA kit (ThermoFisher, CAT#EHIFN), Intracellular staining
permeabilization wash buffer (Biolegend, CAT#421002), and FluoroFix Buffer (Biolegend,
CAT#422101), Sytox Green (ThermoFisher, CAT#S7020), CountBright Absolute
counting beads (ThermoFisher, CAT#C36950).
42
3.4.4 Cells
Jurkat, K562, Daudi, Karpas-299, B35M, BALL-1, Chevallier, and Raji cell lines were
obtained from American Type Culture Collection (ATCC). The SU-DHL-6(84), SU-DHL-
10(84), and NU-DHL-1(85) human lymphoma cell lines were developed by Alan Epstein
in-house. Raji-eGFP/Luc cells were a gift from Dr Yvonne Y. Chen at the University of
California, Los Angeles. All lymphoma lines were cultured in RPMI-1640 supplemented
with 10% dialyzed fetal calf serum (dFCS), 1% GlutaMAX, and 1% Penicillin/Streptomycin.
HEK-293 LTV cells (Cell Biolabs, CAT# LTV-100) were cultured in DMEM supplemented
with 10% dFCS, 1% GlutaMax, 1% non-essential amino acids, and 1%
Penicillin/Streptomycin. Primary human T cells were enriched from human buffy coats
(Zen-Bio, CAR#SER-BC-SDS) and cultured in T cell medium (43% Click’s medium, 43%
RPMI-1640, 10% dFCS, 2% GlutaMAX, 1% non-essential amino acids, 1%
Penicillin/Streptomycin) supplemented with 50ng/mL IL-7-Fc and 100ng/mL IL-15-Fc. All
cell lines used were routinely tested for mycoplasma contamination using MycoFluor
Mycoplasma Detection kit (ThermoFisher, CAT#M7006).
3.4.5 Humanized Lym-1 Binding Studies
Figs.3.1a, 1b, and 3.3b: Antibodies at concentrations ranging from 0.013nM to 1300nM
in 100µl were incubated with 0.2 million Raji cells at 4°C for 30mins, followed by three
washes with washing buffer (2%FBS in PBS). Bound antibodies were then incubated for
30 minutes with Alexa Fluor (AF) 488 conjugated goat anti-human IgG(H+L) secondary
antibody at a concentration of 5ug/mL or by AF 647 conjugated anti-human IgG Fc at 5μl
per sample. Cells were washed twice and subjected to flow cytometry analysis. Mean
fluorescence intensity (MFI) was recorded and plotted to evaluate antibody binding. For
43
staining in Figs.1c and 3c, 10 μg of the antibodies was incubated with 0.2 million cells in
100μl at 4°C for 30mins. After washing as above, 5μl AF-647 conjugated anti-human IgG
Fc was added to the cells in wash buffer residuals for detection. Samples were evaluated
using an Attune flow cytometer (ThermoFisher) and analyzed using Flowjo software (BD).
3.4.6 Vectors Construction and Preparation of Lentivirus
The coding genes for CAR were synthesized by Integrated DNA Technologies (IDT) and
ligated into the lentiviral vector pLVX-EF1α-IRES-Zsgreen (Clontech) through EcoRI and
MluI restriction sites. For all CAR constructs, a 10 amino acids epitope ‘‘AVPPQQWALS’’
(261-tag) derived from human placenta growth factor was inserted directly after the scFv
sequence. The whole amino acid sequences for all CAR constructs are provided in the
appendix. Lentivirus was produced by transient transfection of HEK-293LTV following a
Xfect protocol as previously described(72). Briefly, transfer vectors alone with psPAX2
and pMD2.G (molar ratio 2:1:1) were mixed and co-transfected to HEK-293LTV cells.
Supernatants containing viral particles were collected at 24 and 48 hours after
transfection and were combined, filtered, and concentrated by ultracentrifugation at
20,000g for 2h. Pelleted virus was then resuspended in PBS supplemented with 1% BSA
and 7% trehalose, aliquoted, and stored at -80°C. Viral titers were measured by
transducing 10
6
Jurkat T-cells with 10-fold serial dilutions of virus vector. Forty-eight
hours after transduction, Jurkat cells were washed and analyzed for transgene expression
by flow cytometry. Positively transduced cells at a range of 10~20% were used to
calculate the virus transducing units (TU) via the following formula: TU/mL = (10
6
seeded
cells × % positive cells × 1,000)/μl of virus vector.
3.4.7 Primary T Cell Isolation, Transduction, Expansion, and Analysis
44
Human buffy coat preparations were purchased from Zenbio Inc. and used to obtain
peripheral blood mononuclear cells (PBMCs) for T cell enrichment. PBMCs were isolated
using Ficoll-Paque followed by T cell isolation using the EasySep Human T Cell Isolation
Kit as per the manufacturers’ protocols. Isolated cells were then cultured in T cell medium.
On day 0, T cells were activated by adding Dynabeads human T-activator CD3/CD28 at
a 1:1 ratio, and on day 3 transduced by centrifugation at 1200g for 45 min with lentivirus
(MOI = 10) and Lentiblast. Transduction was performed once, followed by a media
change after 24 hours, after which cells were transferred to 24-well G-Rex plates
supplemented with fresh T cell medium. Transduction efficiency was evaluated by flow
cytometry at day 7 using Dylight 650 conjugated anti-261tag antibody. For re-stimulation,
on day 7, 20μl ImmunoCult human CD3/CD28/CD2 T cell activator was added with one
million T cells in 2ml T cell medium and on day 9, 5ml T cell medium was added. On day
12, approximately 5ml medium above the settled cells was removed and 5 ml fresh T cell
medium added. On day 14, for some preparations, re-stimulation was performed with the
same procedure on day 7. ImmunoCult was used for re-stimulation because, in our hands,
Dynabeads CD3/CD28 stimulator promoted the expansion of mostly CD4+ T cells
whereas ImmunoCult human CD3/CD28/CD2 T cell activator led to a balanced expansion
of CD4+ and CD8+ T cells. For all cell counting, the Countess automated cell counter
(Invitrogen) was used. PD-1 and LAG-3 expression on Mock or CAR T cells were
assessed on days 7 and 14 before re-stimulation. Half million cells were incubated with
Dylight 650 conjugated anti-261tag antibody and PE conjugated anti-human PD-1 or anti-
LAG-3. Labeled cells were then subjected to flow analysis. “Mock T cells” refers to T cells
45
that were carried through the above procedures except no virus was added at the
transduction step on day 3.
3.4.8 Cytotoxicity Assays
Luminescent-based cytotoxicity assays: CAR T cells from day 9, not re-stimulated on day
7, were adjusted to 50% positive for CAR T cells by the addition of Mock T cells. These
adjusted preparations were incubated with 0.1 million target cells at various ratios of CAR
T cells in flat-bottom 96 well plates for 24h without the addition of cytokines. Luminescent
reads from target cells without effector cells were used as controls. Two-fold serial
dilutions of 0.2 million target cells were used to generate a standard curve to correlate
live cells to luminescent reads. Live target cell number after 24h incubations was
calculated by correlating the luminescent signal reads to the standard curve. The
percentage of cell lysis was calculated by following formula:
Lysis% = ((#Raji in control-#Raji in effector)/(#Raji in control))×100%.
3.4.9 Flow cytometry-based cytotoxicity assays
Two x 10
5
CAR T cells were incubated with target cells at a 1:1 ratio in 24-well plates.
The percentage of live target cells at 1h and 48h after mixing was recorded and used to
calculate Lysis % by the following formula:
Lysis% = ((% live target cells at 1h-% live target cells at 48h)/(% live target cells at
1h))×100%.
3.4.10 Cytokine Secretion Assays
Mock or CAR T cells from day 9, not re-stimulated on day 7, were used for cytokine
secretion assays. Two x 10
5
effector cells and target cells were co-cultured in the
absence of added cytokines at a ratio of 1:1 in 96-well plates for 24 hours. Supernatants
46
were collected and subjected to enzyme-linked immunosorbent assay (ELISA)
measurement per manufacturer’s instructions.
3.4.11 CD3-ζ Phosphorylation Assay
One half million Mock or CAR T cells from day 9, not stimulated on day 7, were fixed and
then stained with 2μg Dylight-conjugated anti-261 tag antibody. Labeled cells were
permeabilizated per manufacturer’s instructions. Permeabilized cells were then stained
with PE conjugated anti-human phosphorylated CD3ζ antibody at 5μl per sample.
Samples were washed and subjected to flow cytometry analysis.
3.4.12 Antigen Down Regulation Experiments
Ex vivo experiments: Mock or CAR T cells from day 9, not re-stimulated on day 7, were
used for these experiments. Tumor cells (4x10
5
) were co-cultured with 2x10
5
CAR T cells
at an E:T ratio of 1:2 in 2ml T cell medium without cytokine supplement for 24h. One
hundred µl medium with cells were collected and stained for CD22, Lym-1, or CD19. For
Raji-eGFP/Luc, instead of using CD22, GFP was used to identify tumor cells. After
incubation at room temperature for 20min, 400 µl PBS and 25 µl counting beads were
added to each tube. Samples were then subject to flow cytometry analysis. Mean
fluorescence intensity was quantified by Flowjo software.
3.4.13 Raji/Luc-eGFP xenograft studies in NOD Scid-IL2Rgammanull (NSG) mice
One million Raji/Luc-GFP cells in 100 µl PBS were injected i.v. via the lateral tail vein
(designated as day 0). Luciferase activity was measured on day 6 via bioluminescence
imaging (BLI) to assess tumor burden. On the same day, five million Mock T or CAR T
cells were prepared in 100 µl PBS and injected i.v. using insulin syringes. Tumor
progression was monitored by bioluminescence at indicated days by using an Xenogen
47
IVIS 200 at the USC Molecular Imaging Center or IVIS Lumina Series III at the USC
Translational Research Laboratory. Mice were anesthetized with vaporized isoflurane
and administrated D-luciferin (50mg/kg) via intraperitoneal injection before imaging. In
some experiments, as specified in figure legends, the first BLI measurement was
performed on day 7 followed by injection with variable amount of Mock or CAR T cells on
day 8. In all studies, Mock or CAR T cells from day 9 (no re-stimulation on day 7) were
used and hind-leg paralysis was used as end point for euthanasia. Survival data are
shown by Kaplan-Meier plots and analyzed by the log-rank test.
3.4.14 Statistical Analysis
Graphs were plotted using GraphPad Prism Software. Data were analyzed using SPSS
software (IBM). The statistical analysis method is indicated in the figure legends. Unless
otherwise stated, data are presented as mean ± SD, and p < 0.05 was considered as
significant.
3.5 Results
3.5.1 Selection of humanized Lym-1 antibody
In previous studies, we demonstrated that Lym-1-B-BB3z CAR T cells induced
complete remission in mice with disseminated Raji tumors(72). Because of these
promising results, antibody humanization was performed to reduce potential
immunogenicity of the Lym-1 ScFv when used in the CAR T cells for future patient studies.
Humanization was out-sourced to Oak BioScience and a panel of 12 humanized
antibodies were supplied to us (Figure 3.1 a). The binding ability of those antibodies on
Raji cells was measured by flow cytometry and the huLym-1-B antibody which showed
the highest binding at each tested concentration was chosen for the development of CAR
48
T cells (Figure 3.1 a). We then produced the huLym-1-B antibody in-house using
transient expression with the provided sequence for subsequent evaluations. Compared
to chLym-1, huLym-1-B binds to Raji cells with reduced MFI and an approximately 2-fold
higher EC50 (Figure 3.1 b). Similar to its binding to Raji cells, huLym-1-B shows slightly
lower MFI than chLym-1 in most Lym-1 positive cell lines (Figure 3.1 c). Both chLym-1
and huLym-1-B did not bind to K562 cells indicating that huLym-1-B retains specificity
similar to that of the parent Lym-1 antibody (Figure 3.1 c).

Figure 3.1: Selection of huLym-1-B as the candidate for CAR development.

(a) Binding of 12 humanized IgG1 isoform Lym-1 antibodies to Raji cells. huLym-1-60 has the
same sequence as the parent antibody chLym-1. hu51 is an IgG1 isotype control produced in-
house. Raji cells were incubated with indicated concentration of antibodies for 30 mins followed
by detection with AF-488-conjugated Goat anti-human IgG. Mean fluorescence intensity (MFI)
was assessed by flow cytometry. (b) Binding of in-house produced huLym-1-B and chLym-1 to
Raji cells assessed using flow cytometry and a secondary APC-conjugated mouse anti-human
IgG monoclonal antibody. (c) Binding of chLym-1 and huLym-1-B to a panel of chLym-1 positive
cell lines and the negative cell line K562 using a secondary APC-conjugated mouse anti-human
IgG monoclonal antibody.
49
3.5.2 huLym-1-B-BB3zCAR T cells are Highly Functional Yet Have Impaired
Expansion
To generate CAR against Lym-1 epitope, the ScFvs derived from Lym-1 or huLym-
1-B were fused to a conventional 2nd generation CAR framework with 4-1BB and CD3ζ
signaling domains (Figure 3.2 a). A 10 amino acid epitope ‘‘AVPPQQWALS’’ (261-tag)
derived from human placenta growth factor was inserted between the ScFv and CD8a
hinge to enable CAR detection by using an in-house antibody (Dylight 650 conjugated
anti-261 tag antibody) (Figure 3.2 a). Both constructs were successfully expressed in
human primary T cells with comparable transduction efficiency (Figure 3.2 c). T cells
expressing Lym-1- or huLym-1-B-BB3z CAR T lysed Lym-1 positive Raji cells efficiently
after overnight co-culture, whereas increased cytotoxicity was not observed when co-
cultured with Lym-1 negative K562 cells, indicating similar epitope specific cytotoxicity of
the two CAR T cells (Figure 3.2 d). In addition, a dose of 5 million Lym-1- and huLym-1-
B-BB3z CAR T cells eradicated disseminated Raji-eGFP/Luc tumors in NSG mice and
led to tumor free survival for at least 60 days (Figure 3.3). However, in response to α-
CD2/CD3/CD28 stimulation, both Lym-1- and huLym-1-B-BB3z CAR T cells exhibited
impaired expansion (Figure 3.2 e). When re-stimulated on day 7, Mock T cells increased
approximately 30-fold by day 14, compared to less than an average of 4-fold increase for
Lym-1- and huLym-1-B-BB3z CAR T cells (Figure 3.2 e). Furthermore, increased CD3ζ
phosphorylation was observed in Lym-1- and huLym-1-B-BB3z CAR positive cells, but
not in untransduced T cells in the same preparation indicating sustained activation of CAR
transduced cells (Figure 3.2 f, g). Consistent with these results, CAR positive T cells also
manifested increased expression of inhibitory receptors PD-1 and LAG-3 (Figure 3.2 h,
50
i). Enhanced CD3ζ phosphorylation and inhibitory receptors expression in CAR T cells
suggested that the impaired proliferation arose from increased basal level activation.
Although huLym-1-B-BB3z CAR T cells showed strong activity in vitro and in vivo, limited
proliferation would challenge the production of huLym-1-B-BB3z CAR T cells for clinical
application since extensive ex vivo expansion is required to generate optimal therapeutic
doses.

Figure 3.2: Lym-1 and huLym-1-B CAR T cells with BB3z-CD3z signaling domains are
cytotoxic in culture despite impaired proliferation.
51

Figure 3.3: Both Lym-1 and huLym-1-B CAR with the 4-1BB3z intracellular domain
eradicate Raji tumor in vivo.

(a) Schematic representation of CAR constructs. 261 tag is a 10 amino acids linear epitope
derived from human placenta growth factor. (b) Schedule of CAR T production and expansion.
(c) Flow cytometry analysis of CAR expression on Mock or transduced primary human T cells on
day 7; CAR expression was measured using a Dylight 650-conjugated antibody against the 261
tag. (d) Cytotoxicity of CAR T cells against Lym-1 epitope negative (K562) and positive (Raji) cell
lines at indicated effector to target (E: T) ratios; Mock and CAR T cells from day 9 not stimulated
on day 7 were used to measure cytotoxicity (n = 3 technical replicates, representative from 3
donors). (e) Expansion of in vitro cultured Mock or CAR positive T cells from days 7 to14 (re-
stimulated on day 7, pooled results from 6 donors. ns-not significant, * P < 0.001 by Student’s t-
test). (f) Representative plots showing phosphorylation of CD3ζ (p- CD3ζ) on day 9 (no
stimulation on day 7; n = 3 donors). (g) Quantification of p-CD3ζ in Mock and CAR positive T cell
(n = 3 donors). (h) Representative CAR and inhibitory receptor expression on day 7 and day 14
before re-stimulation. (i) Quantification of the percentage of inhibitory receptors on Mock or CAR
positive T cells was calculated by: % Positive in CAR = (Q2/(Q1+Q2)) *100, pooled from 3 donors
(* P < 0.05 by Student’s t-test. Scatterplots and Bar graphs show mean ± SD).

(a) schematic representation of in vivo study schedule. (b) Bioluminescence images of NSG mice
inoculated i.v. with 10
6
Raji-eGFP/Luc on day 0, followed by treatment of 5x10
6
Mock T cells,
Lym-1-BB3z CAR T cells, huLym-1-B-BB3z CAR T cells, or 100ul PBS on day 6. (n=5
mice/group). (c) Kaplan-Meier curve of survival (* p< 0.001 by log-rank test.)
52
3.5.3 Impaired Ex Vivo Expansion of huLym-1-B-BB3z CAR T cells is Antigen
Dependent
CD19-BB3z CAR T cells consisting of the anti-CD19 FMC63 ScFv generated in
our laboratory, a construct with the same CAR framework as huLym-1-B-BB3z, did not
show the impaired expansion seen with huLym-1-B-BB3z CAR T cell preparations(72).
This difference suggested that the cause involves the huLym-1-B ScFv. The Lym-1
antibody recognizes a conformational epitope in several subtypes of HLA-DR(29), but
Lym-1 binding on human T cells has not been reported. Thus, previously unreported
sparse Lym-1 epitope expression on activated T cells might be sufficient to induce ligand-
dependent suboptimal CAR signaling or lead to CAR-mediated fratricide either of which
could cause impaired expansion of huLym-1-B-BB3z CAR T cells. To test this hypothesis,
Lym-1 and huLym-1-B binding to activated T cells was carefully assessed and a small but
real amount of binding was detected (Figure 3.4 c). To further test this hypothesis, two
CAR constructs were generated. In one construct, two point-mutations were introduced
in the CDR3 region of the variable heavy chain to disrupt the binding ability of huLym-1-
B and to construct huLym-1-Bmut-BB3z CAR (Figure 3.4 a). The antibody version of
huLym-1-B with these two mutations in CDR3 (huLym-1-Bmut) was also produced. The
huLym-1-Bmut antibody has approximately a 25-fold greater EC50 than huLym-1-B
(EC50, 1.4x10
-7
vs 5.9x10
-9
, Figure 3.4 b) when measured against Raji cells and showed
no enhanced binding to T cells compared to isotype (Figure 3.4 c). Although CD3ζ
phosphorylation was found in about 5% of huLym-1-Bmut-BB3z CAR T cells and PD-1
and LAG-3 were transiently upregulated on day 7 (Figure 3.4 e-h), huLym-1-Bmut-BB3z
53
CAR T cell preparations did not exhibit impaired expansion suggesting epitope
recognition was required for the impairment (Figure 3.4 d-h).
To determine if CAR signaling was required to impair expansion, a second
construct, huLym-1-B-BB3zY-F, was generated wherein all 6 tyrosines in the three ITAMs
of the CAR-CD3ζ domain were converted to phenylalanines (Figure 3.4 a). HuLym-1-B-
BB3zY-F CAR T cells had neither increased CD3ζ phosphorylation nor upregulation of
PD-1 and LAG-3 and expanded as efficiently as Mock T cells, indicating signaling
involving CAR-CD3ζ was required for the impairment (Figure 3.4 d-h).
We next investigated whether fratricide is a substantial cause of the diminished
expansion of huLym-1-B-BB3z CAR T cells. huLym-1-B-BB3z CAR T cells showed
dramatically enhanced spontaneous apoptosis (~56%) compared to the CAR negative
population (~10%) in the same preparation (Figure 3.5 a); In addition, we did not observe
markedly increased apoptosis of CD19-BB3z CAR T cells when they were co-cultured
overnight with huLym-1-B-BB3z CAR T cells (Figure 3.5 b). Taken together, these results
suggest that in this time frame the dominant cause of impaired ex vivo proliferation of
huLym-1-B-BB3zCAR-T cell is ligand-dependent suboptimal CAR signaling and not
fratricide. It is possible that fratricide could occur later and was not detected in this
experiment.

54

Figure 3.4: The impaired proliferation of huLym-1-B-BB3zCAR T cells is associated with
weakly expressed Lym-1 epitopes on T cells and is mediated by ITAM CD3ζ.

55

Figure 3.5: Ligand-dependent suboptimal CAR signaling is the dominant mechanism for
impaired expansion in huLym-1-B-BB3z CAR T cells.
47.6 46.8
Q1
0.022
Q2
0.33
Q3
10.0
Q4
89.6
Q1
0
Q2
0.30
Q3
5.25
Q4
94.4
43.6 50.5
Q1
0.019
Q2
0.58
Q3
56.9
Q4
42.5
Q1
0
Q2
0.45
Q3
11.8
Q4
87.8
84.8 0.040
Q1
0
Q2
0.12
Q3
2.02
Q4
97.9
Mock huLym-1-B-BB3z huLym-1-B-DAP
Annexin V
Sytox-Green
a
b
CAR+ CAR- CAR+ CAR- CAR+ CAR-
Q1
0.054
Q2
0.24
Q3
3.23
Q4
96.5
Q1
0.061
Q2
0.59
Q3
4.11
Q4
95.2
Q1
0.042
Q2
0.90
Q3
7.68
Q4
91.4
Q1
0.11
Q2
0.44
Q3
4.85
Q4
94.6
Co-culture
CD19-BB3z with
Medium Mock
huLym-1-B-
BB3z
huLym-1-B-
DAP
Annexin V
Sytox-Green
(Figure 3.4 continued) (a) Introducing two amino acid mutations in the CDR3 of variable heavy
chain of huLym-1-B to generate huLym-1-Bmut antibody or huLym-1-Bmut-BB3zCAR; Tyrosine
(Y) to Phenylalanine (F) mutation in all three ITAMs of CD3 ζ moiety in CAR to eliminate huLym-
1-B-BB3z activity (huLym-1-B-BB3zY-F). (b) Binding of chLym-1, huLym-1-B, and huLym-1-Bmut
to Raji cells. AF-488-conjugated Goat anti-human IgG used as secondary antibody; EC50 is
annotated on each curve. (c) One half million activated T cells were stained with 10 μg/100μl of
indicated antibodies followed by detection with APC-anti-huIgG. Mean fluorescence intensity was
calculated and plotted; scatterplots show median of MFI. The color of the dot represents a donor
(* P < 0.05; ns, not significant by Kruskal-Wallis test). (d) Fold expansion of in vitro cultured Mock
T, huLym-1-B-BB3zY-F, and huLym-1-Bmut-BB3z CAR positive T cells from days 7 to 14 (re-
stimulated on day 7, pooled results from 3 donors. ns, not significant by Student’s t-test,
scatterplot show mean ± SD). (e) Representative plots showing phosphorylation of CD3ζ on day
9; CAR expression was detected by Dylight 650 conjugated anti-261tag antibody. (f)
Quantification of p-CD3ζ in Mock and CAR positive T cell (n = 3 donors). (g) Representative plot
of CAR and PD-1 and LAG-3 expression on days 7 and 14 before re-stimulation. (h)
Quantification of the percentage of PD-1 and LAG-3 in Mock or CAR positive T cells was
calculated by: % Positive in CAR = (Q2/(Q1+Q2)) *100, pooled from three donors ( * P < 0.05 by
Student’s t-test. Bar graphs show mean ± SD).
56

3.5.4 Replacing BB3z with DAP Enables Efficient Ex Vivo Expansion of huLym-1-B
CAR T cells
Next, we used DAP signaling domains to construct huLym-1-B-DAP CAR (Figure
3.6 a, b). HuLym-1-B-DAP CARs were expressed on human primary T cells with
equivalent transduction efficiency as huLym-1-BB3z CAR (Figure 3.6 d). Importantly, ex
vivo expansion of huLym-1-B-DAP CAR T cells was not impaired (Figure 3.6 c). In
addition, compared to huLym-1-B-BB3z CAR T cells, huLym-1-B-DAPCAR T cells
showed no enhanced spontaneous Annexin V staining in culture (Figure 3.5 a) and
exhibited less AICD when cultured with Raji cells (
Figure 3.7).
Interestingly, phosphorylation of the endogenous CD3ζ was increased in huLym-
1-B-DAP CAR T cells compared to mock T cells (Figure 3.6 d, e; Figure 3.8). Consistent
with the basal levels of CD3ζ phosphorylation in huLym-1-B-DAP CAR T cells, PD-1 and
LAG-3 expression were also higher than Mock T cells but were significantly lower than
huLym-1-B-BB3z CAR T cells on day 14 (Figure 3.6 f, g). Together, these data
demonstrate that DAP signaling domains circumvent adverse effects caused by
suboptimal CAR-CD3ζ signaling and enable non-impaired production of huLym-1-B-CAR
T cells.
(Figure 3.5 continued) (a) Mock or CAR transduced T cells from day 9 (no re-stimulation on day
7) were assessed for Annexin V and dead cell (Sytox-Green) staining (Representative results
from two donors). (b) CD19-BB3z CAR T cells were pre-labeled with Cell trace far-red dye and
then co-cultured overnight with Mock or indicated CAR T cells at 1:1 ratio. CD19-BB3z CAR T
cells were then subjected to Annexin V and Sytox-Green staining to measure potential fratricide
by huLym-1-B-BB3z or huLym-1-B-DAP CAR T cells (Representative results from two donors).

57

Figure 3.6: Replacing BB3z with DAP signaling promotes stimulation-induced proliferation
of huLym-1-B CAR T cells.

(a) Left column illustrates the consensus amino acids (AA) sequence of ITIMs and ITAMs, and
the right column shows the AA sequences of ITAM(s) in DAP12 and CD3ζ; Shade highlights the
presence of ITIM consensus in the ITAM of DAP12. (b) Schematic representation of huLym-1-B
CAR with BB3z or DAP as intracellular signaling domain. (c) In vitro expansion of Mock, huLym-
1-B-DAP, and huLym-1-B-BB3z CAR positive T cells from days 7-14 (re-stimulated on day 7,
pooled results from 5 donors. ns, not significant by Student’s t-test). (d) Representative plots
showing CD3ζphosphorylation (p-CD3ζ) on day 9 (representative of three donors). (e)
Quantification of p-CD3ζ in Mock and CAR positive T cell (n = 3 donors). (f) Representative CAR
and inhibitory receptor expression on days 7 and 14 before re-stimulation (n = 3 donors, * P <
0.05 by Student’s t-test). (g) Quantification of the percentage of inhibitory receptors in Mock and
CAR positive T cells was calculated by: % Positive in CAR = (Q2/(Q1+Q2)) *100, pooled from
three donors (ns, not significant; * P < 0.05 by Student’s t-test. Scatterplots and bar graphs show
mean ± SD).
58

Figure 3.7: huLym-1-B-DAP CAR T cells exhibit reduced Activation Induced Cell Death.

Mock, huLym-1-B-BB3z and huLym-1-B-DAP CAR T cells were cultured in the presence of a-
CD2/3/28 stimulator, or with K562 or Raji cells overnight. Mock and CAR T cells were then
subjected to Annexin V and Sytox Green (dead cell dye) staining. (a) representative plot from one
of the three donors. (b) Percentage of Annexin V positive cells in live cells were quantified by the
following formula: [Q3/(Q3+Q4)] *100 (pooled from three donors, * P < 0.05 by Student’s t-test.
Bar graphs show mean ± SD).
59

Figure 3.8: Increased endogenous CD3ζ phosphorylation in huLym-1-B-DAP CAR T cells.

3.5.5 huLym-1-B-DAPCAR T cells are Highly Functional Both In Vitro and In Vivo
To evaluate the effector function of huLym-1-B-DAP CAR T cells in vitro,
cytotoxicity and cytokine release in response to Lym-1 epitope negative (K562) and
positive (Raji) cell lines were assessed. HuLym-1-B-DAP CAR T cells lysed Raji cells in
proportion to increased effector to target ratio, reaching about 80% killing at 2:1 ratio after
overnight co-culture (Figure 3.9). No enhanced cytotoxicity was evident when K562 cells
were used as target cells (Figure 3.9). Consistent with these findings, huLym-1-B-DAP
CAR T cells also secreted multiple cytokines when co-cultured with Raji but not K562
cells (Figure 3.9). In addition, we assessed the function of huLym-1-B-DAP CAR T cells
against a panel of human lymphoma and leukemia B-cell lines with variable Lym-1
epitope expression. Despite highly variable cytokine release, huLym-1-B-DAP CAR T
Cell lysates of Mock-resting, Mock activated with a-CD3/CD28 beads, and huLym-1-B-DAP CAR
T cells from day 9 (no re-stimulation on day 7) were probed for (a) CD3 ζ and (b) CD3ζ-pY142
(representative results from 2 donors).
60
cells exhibited equivalent cytotoxicity (Figure 3.10). These data demonstrated huLym-1-
B-DAP CAR T cells retained the specificity of the parent Lym-1 antibody.
We next examined the in vivo anti-tumor efficacy of huLym-1-B-DAP CAR T cells
against disseminated Raji tumors in NSG mice. To better reveal if the DAP signaling
domains confer improved function in vivo, only 1 million CAR T cells were injected instead
of 5 million cells and to increase the tumor burden challenge treatment was given on day
8 after injecting Raji cells rather than on day 6. Using this modified protocol, one million
huLym-1-B-BB3z CAR T cells were unable to eliminate Raji tumors and all mice
succumbed to tumor progression by day 51 (Figure 3.9 d, e, f). In contrast, treatment
with one million huLym-1-B-DAP CAR T cells led to durable tumor control and significantly
better survival (Figure 3.9 f). Moreover, surviving mice re-challenged with tumor cells
showed delayed tumor progression and prolonged survival, indicating huLym-1-B-DAP
CAR T cells persisted and conferred resistance in those NSG mice (Figure 3.9 g, h).
61

62

Figure 3.9: huLym-1-B-DAP CAR T cells mediate superior anti-tumor efficacy than BB3z
counterparts in vivo.

(a) Cytotoxicity of Mock, huLym-1-B-DAP or huLym-1-B-BB3z against Lym-1 positive (Raji, left
panel) or negative (K562, right panel) cells. (b) Cytokines released by Mock, huLym-1-B-DAP or
huLym-1-B-BB3z when co-cultured overnight with K562 or Raji at 1:1 effector to target ratio. (n =
3 technical replicates. Representative results from three donors). (c) Schematic representation of
the in vivo study with modified protocol. NSG mice inoculated with 10
6
Raji-eGFP/Luc on day 0,
followed by treatment of 10
6
Mock, huLym-1-B-DAP, or huLym-1-B-BB3z CAR T cells on day 8
(n = 5 mice per group). (d,e) Bioluminescence images from two independent studies with different
donors. (f) Kaplan-Meier curve of survival (P = 0.000325 by log-rank (Mantel-Cox) test. Pooled
from two independent studies.). (g) Surviving mice from (d) were re-challenged with 10
6
Raji-
eGFP/Luc on day 61. Bioluminescence images on indicated days are shown. (h) Kaplan-Meier
curve of survival (P = 0.01 by log-rank (Mantel-Cox) test).
63

Figure 3.10: huLym-1-B-DAP show effector function against a panel of B-cell lines.
B35M Daudi BALL-1 Chevallier
Mock T
1h
48h
huLym-1-B-DAP
huLym-1-B-BB3z
1h
48h
1h
48h
B35M Daudi BALL-1 Chevallier
B35M Daudi BALL-1 Chevallier
Chevallier
BALL-1
Daudi
B35M
-50
0
50
100
Lysis%
Tumor Cells
47.0
10
0
10
2
10
4
10
6
RL1-A :: RL1-A
0
2.0M
4.0M
6.0M
8.0M
SSC-A :: SSC-A
Tumor Cells
56.8
10
0
10
2
10
4
10
6
RL1-A :: RL1-A
0
2.0M
4.0M
6.0M
8.0M
SSC-A :: SSC-A
Tumor Cells
72.1
10
0
10
2
10
4
10
6
RL1-A :: RL1-A
0
2.0M
4.0M
6.0M
8.0M
SSC-A :: SSC-A
Tumor Cells
69.2
10
0
10
2
10
4
10
6
RL1-A :: RL1-A
0
2.0M
4.0M
6.0M
8.0M
SSC-A :: SSC-A
Tumor Cells
53.0
10
0
10
2
10
4
10
6
RL1-A :: RL1-A
0
2.0M
4.0M
6.0M
8.0M
SSC-A :: SSC-A
Tumor Cells
71.5
10
0
10
2
10
4
10
6
RL1-A :: RL1-A
0
2.0M
4.0M
6.0M
8.0M
SSC-A :: SSC-A
Tumor Cells
50.4
10
0
10
2
10
4
10
6
RL1-A :: RL1-A
0
2.0M
4.0M
6.0M
8.0M
SSC-A :: SSC-A
Tumor Cells
52.1
10
0
10
2
10
4
10
6
RL1-A :: RL1-A
0
2.0M
4.0M
6.0M
8.0M
SSC-A :: SSC-A
Tumor Cells
41.2
10
0
10
2
10
4
10
6
RL1-A :: RL1-A
0
2.0M
4.0M
6.0M
8.0M
SSC-A :: SSC-A
Tumor Cells
55.1
10
0
10
2
10
4
10
6
RL1-A :: RL1-A
0
2.0M
4.0M
6.0M
8.0M
SSC-A :: SSC-A
Tumor Cells
10.7
10
0
10
2
10
4
10
6
RL1-A :: RL1-A
0
2.0M
4.0M
6.0M
8.0M
SSC-A :: SSC-A
Tumor Cells
8.10
10
0
10
2
10
4
10
6
RL1-A :: RL1-A
0
2.0M
4.0M
6.0M
8.0M
SSC-A :: SSC-A
Tumor Cells
5.31
10
0
10
2
10
4
10
6
RL1-A :: RL1-A
0
2.0M
4.0M
6.0M
8.0M
SSC-A :: SSC-A
Tumor Cells
12.9
10
0
10
2
10
4
10
6
RL1-A :: RL1-A
0
2.0M
4.0M
6.0M
8.0M
SSC-A :: SSC-A
Tumor Cells
53.1
10
0
10
2
10
4
10
6
RL1-A :: RL1-A
0
2.0M
4.0M
6.0M
8.0M
SSC-A :: SSC-A
Tumor Cells
57.2
10
0
10
2
10
4
10
6
RL1-A :: RL1-A
0
2.0M
4.0M
6.0M
8.0M
SSC-A :: SSC-A
Tumor Cells
51.7
10
0
10
2
10
4
10
6
RL1-A :: RL1-A
0
2.0M
4.0M
6.0M
8.0M
SSC-A :: SSC-A
Tumor Cells
56.1
10
0
10
2
10
4
10
6
RL1-A :: RL1-A
0
2.0M
4.0M
6.0M
8.0M
SSC-A :: SSC-A
Tumor Cells
11.4
10
0
10
2
10
4
10
6
RL1-A :: RL1-A
0
2.0M
4.0M
6.0M
8.0M
SSC-A :: SSC-A
Tumor Cells
10.3
10
0
10
2
10
4
10
6
RL1-A :: RL1-A
0
2.0M
4.0M
6.0M
8.0M
SSC-A :: SSC-A
Tumor Cells
12.5
10
0
10
2
10
4
10
6
RL1-A :: RL1-A
0
2.0M
4.0M
6.0M
8.0M
SSC-A :: SSC-A
Tumor Cells
16.7
10
0
10
2
10
4
10
6
RL1-A :: RL1-A
0
2.0M
4.0M
6.0M
8.0M
SSC-A :: SSC-A
c
Chevallier
BALL-1
Daudi
B35M
0
200
400
600
800
pg per 10000 cells
GM-CSF
Mock T
hulym-1-B-DAP
huLym-1-B-BB3z
Chevallier
BALL-1
Daudi
B35M
0
200
400
600
800
1000
1200
ng per 10000 cells
INF-g
Chevallier
BALL-1
Daudi
B35M
0
20
40
60
80
100
ng per 10000 cells
IL-2
Mock T
hulym-1-B-DAP
huLym-1-B-BB3z
Tumor Cells
52.9
10
0
10
2
10
4
10
6
RL1-A :: RL1-A
0
2.0M
4.0M
6.0M
8.0M
SSC-A :: SSC-A
Tumor Cells
58.7
10
0
10
2
10
4
10
6
RL1-A :: RL1-A
0
2.0M
4.0M
6.0M
8.0M
SSC-A :: SSC-A
CFSE CFSE
CFSE
CFSE
a
b
64

3.5.6 huLym-1-B-DAP CAR T Cells Do Not Cause Significant Downregulation of
the Lym-1 epitope
Relapse is often observed in the treatment of B cell malignancies by CAR T cells
targeting CD19 (CD19-CAR) and this downregulation represents an important
mechanism enabling resistance to CD19-CAR therapy(86). CAR T cells directed against
antigens that are less prone to downregulate could reduce antigen escape and improve
therapeutic efficacy. To determine if huLym-1-B-DAP CAR T cells promote
downregulation of the Lym-1 epitopes, we co-cultured Raji cells with Mock, huLym-1-B-
DAP CAR, or CD19-BB3z CAR T cells. Within 24 hours, both huLym-1-B-DAP CAR and
CD19-BB3z CAR T cells inhibited Raji expansion (Figure 3.11 c, d). However, there was
marked CD19 antigen downregulation when Raji cells were co-cultured with CD19-BB3z
CAR T cells, whereas neither CD19 nor Lym-1 epitope downregulation was evident when
co-cultured with huLym-1-B-DAP CAR T cells (Figure 3.11 a, b). Similar results were
obtained from a panel of human B lymphoma cell lines (Figure 3.12). To determine if the
difference was due to the use of the DAP signaling domain instead of the BB3z domain,
CD19-DAP CAR T cells were generated and co-cultured with Raji cells. Downregulation
of CD19 antigen on Raji cells still occurred (Figure 3.13). In addition, significant Lym-1
epitope downregulation was not observed on Raji cells when they were co-cultured with
(Figure 3.10 continued) (a) Cytokines released from Mock, huLym-1-B-DAP, and huLym-1-B-
BB3z when co-cultured overnight with a panel of human lymphoma B-cell lines at 1:1 effector to
target ratio. Quantification of secreted cytokines (n = 3 technical replicates. Representative
results from two donors). (b) To measure the cytotoxicity of Mock, huLym-1-B-DAP, and huLym-
1-B-BB3z, tumor cells were pre-labeled with Cell trace far-red dye and then co-culture with
effector cells at 1:1 ratio; Live tumor cells at each time point were gated. (c) Lysis percent is
calculated as (%tumor at 1h - %tumor at 48h) / (%tumor at 1h) (n = 3 technical replicates and
representative results from two donors are shown. Bar graphs show mean ± SD).
65
either huLym-1-B-BB3z or huLym-1-B-DAP CAR T cells (Figure 3.13). These results
indicate that target downregulation is attributed to a property of the antigen rather than
the signaling domain of the CAR construct.
To assess epitope downregulation in vivo, CD19 and Lym-1 epitope expression
were measured on Raji cells obtained from bone marrow of mice undergoing CAR T cell
therapy. As seen ex vivo, treating NSG mice bearing Raji cells with CD19-BB3z CAR led
to significant CD19 antigen downregulation (Figure 3.11 e, f). Importantly, detection of
neither Lym-1 epitope nor CD19 antigen was downregulated during huLym-1-B-DAP CAR
T cell treatment (Figure 3.11 e, f). The in vivo anti-tumor efficacy of huLym-1-B-DAP,
CD19-BB3z, and CD19-DAP CAR T cells in disseminated Raji bearing NSG mice was
also characterized in a protocol wherein on day 0, 10
6
Raji cells were injected
intravenously followed by various doses of Mock or CAR T cells on day 8. One dose of 2
million huLym-1-B-DAP CAR T cells led to tumor free survival for at least 90 days (Figure
3.14 b, c). In contrast, high tumor burden existed in most of the NSG mice treated with
CD19-BB3z or CD19-DAP CAR T cells and all mice succumbed to tumor progression by
day 79 at the dose of 2 million cells (Figure 3.14 b, c). Moreover, in this experimental
model, increasing the CD19-CAR T cell dose to 5 million cells still failed to achieve tumor
free survival (Figure 3.14 b, c). In summary, the Lym-1 epitope was not found to
downregulate under the pressure of huLym-1-B-DAP CAR T cell treatment and this
property of Lym-1 epitope may have contributed to the superior in vivo efficacy of huLym-
1-DAP CAR T cells compared to CD19-CAR T cells.

66

Figure 3.11: Lym-1 epitopes do not significantly downregulate in response to the presence
of huLym-1-B-DAP CAR T cells.

67

Figure 3.12: huLym-1-B-DAP CAR T cells do not induce significant downregulation of the
Lym-1 epitope on lymphoma cell lines.

Lymphocytes
44.7
0 2.0M 4.0M 6.0M 8.0M
FSC-A :: FSC-A
0
2.0M
4.0M
6.0M
8.0M
SSC-A :: SSC-A
Single Cells
96.3
0 2.0M 4.0M 6.0M 8.0M
FSC-A :: FSC-A
0
2.0M
4.0M
6.0M
8.0M
FSC-H :: FSC-H
BL1-A, FSC-H subset
56.1
10
0
10
2
10
4
10
6
CD22
0
2.0M
4.0M
6.0M
8.0M
FSC-H :: FSC-H
Mock T
BALM-2 Chevallier SU-DHL-6 SU-DHL-10 BALL-1
huLym-1-B
-DAP
CD19-BB3z
Co-culture
CD19
Lym-1
a
Q1
0.57
Q2
88.7
Q3
9.82
Q4
0.89
Q1
0.032
Q2
94.2
Q3
5.71
Q4
0.064
Q1
0.99
Q2
69.9
Q3
28.6
Q4
0.53
Q1
0.29
Q2
94.4
Q3
5.11
Q4
0.22
Q1
45.3
Q2
51.6
Q3
0.38
Q4
2.35
Q1
42.1
Q2
30.0
Q3
2.29
Q4
25.6
Q1
54.6
Q2
42.8
Q3
0.46
Q4
2.19
Q1
36.6
Q2
61.3
Q3
0.69
Q4
1.38
Q1
37.0
Q2
60.1
Q3
0.84
Q4
1.99
Q1
0.027
Q2
96.1
Q3
3.84
Q4
0.027
Q1
0.26
Q2
89.5
Q3
9.97
Q4
0.29
Q1
0
Q2
99.1
Q3
0.86
Q4
0
Q1
0
Q2
90.8
Q3
9.25
Q4
0
Q1
0
Q2
86.5
Q3
13.5
Q4
0.059
Q1
0.24
Q2
94.8
Q3
4.82
Q4
0.15
(Figure 3.11 continued) (a) Mock T, CD19-BB3z, and huLym-1-B-DAP CAR T cells (~50%
positive in all CAR preparations) were co-cultured overnight with Raji-eGFP/Luc cells at 1:2 ratio
(CAR versus Raji), CD19 antigen and Lym-1 epitope expression in the residual live Raji cells
were then measured by fluorophore conjugated monoclonal antibodies via flow cytometry. (b)
Scatterplots of mean fluorescence intensity (MFI) of Lym-1 epitope and CD19 antigen expression
in the residual Raji cells. (c) Percentage and (d) Concentration of live Raji cells after overnight
co-culture (n = 3 technical replicates, representative from two donors). (e) NSG mice inoculated
with 10
6
Raji-eGFP/Luc on day 0, followed by treatment of 1x10
6
Mock, CD19-BB3z, or huLym-
1-B-DAP CAR T cells on day 8. On day 16, bone marrow samples were collected and Raji cells
were assessed for Lym-1 epitopes and CD19 antigen expression by fluorophore-conjugated
monoclonal antibodies via flow cytometry. (f) Scatterplot of mean fluorescence intensity (MFI) of
Lym-1 epitopes and CD19 antigen expression on Raji. (n = 5 mice per group, * P < 0.001; ns, not
significant by Kruskal-Wallis test. Scatterplots show mean ± SD).

Mock T, huLym-1-B-DAP, and CD19-BB3z were co-cultured with a panel of B lymphoma and
leukemia cell lines at 1:2 ratio. After overnight co-culture, B-cell lines were labeled with antibodies
against CD22, Lym-1, and CD19.
68

Figure 3.13: CAR signaling domains do not affect antigen modulation.
d
CD19
Lym-1
Mock T
huLym-1-B-DAP
CD19-BB3z
a
Medium
Raji cells
97.8
Pre
Overnight
GFP
SSC-A
c
CD19-DAP
huLym-1-B-BB3z
Q1
0.10
Q2
98.8
Q3
1.07
Q4
0.051
Q1
0.18
Q2
97.7
Q3
2.05
Q4
0.059
Q1
45.3
Q2
54.4
Q3
0
Q4
0.29
Q1
47.4
Q2
52.4
Q3
0.036
Q4
0.15
Q1
0.047
Q2
99.1
Q3
0.81
Q4
0
Q1
0
Q2
98.7
Q3
1.30
Q4
0
Mock T
huLym-1-B-DAP
CD19-BB3z Medium CD19-DAP
huLym-1-B-BB3z
Raji cells
64.8
Raji cells
57.5
Raji cells
60.7
Raji cells
61.5
Raji cells
66.8
Raji cells
71.0
Raji cells
76.1
Raji cells
73.2
Raji cells
67.0
Raji cells
97.8
Raji cells
67.5
Medium
Mock T
CD19-BB3z
CD19-DAP
huLym-1-B-DAP
huLym-1-B-BB3z
0
5000
10000
15000
20000
25000
MFI
CD19 Antigen
Medium
Mock T
CD19-BB3z
CD19-DAP
huLym-1-B-DAP
huLym-1-B-BB3z
0
15000
30000
45000
60000
MFI
Lym-1 epitopes
Treatment
Medium
Mock T
CD19-BB3z
CD19-DAP
huLym-1-B-BB3z
huLym-1-B-DAP
0
50
100
150
200
250
Raji cell concentration
counts perµl
medium
Pre
Overnight
b
(a) Mock T, CD19-BB3z, CD19-DAP, huLym-1-B-BB3z, and huLym-1-B-DAP CAR T cells (~50%
positive in all CAR preparations) were co-cultured overnight with Raji-eGFP/Luc cells. CD19
antigen and Lym-1 epitope expression in the residual live Raji cells was then measured by
fluorophore conjugated monoclonal antibodies via flow cytometry. (b) Scatterplots of mean
fluorescence intensity (MFI) of Lym-1 epitope and CD19 antigen expression in the residual Raji
cells. (c) Percentage and (d) Concentration of live Raji cells after overnight co-culture (n = 3
technical replicates, representative from two donors).
69

Figure 3.14: Low-dose huLym-1-B-DAP CAR T cell therapy produces tumor free survival.

Mock T
huLym-1-B-DAP
CD19-DAP-2M
CD19-DAP-5M
CD19-BB3z-2M
CD19-BB3z-5M
Mock T
CD19-BB3z
CD19-DAP
Day
7
22
27
34
Radiance
p/sec/cm
2
/sr
Color Scale
Min = 2.00e
5
Max = 2.00e
7
48
1 M
a
c
b
Day
1M Raji
0 60/90 7 8
Imaging
Mock/CAR-T treatment
Imaging and monitoring for
hind leg paralysis
Radiance
p/sec/cm
2
/sr
Color Scale
Max = 2.00e
5
Min = 6.00e
3
D 7
D14
D 21
D 31
Radiance
p/sec/cm
2
/sr
Color Scale
Min = 2.00e
5
Max = 2.00e
7
Radiance
p/sec/cm
2
/sr
Color Scale
Max = 2.00e
5
Min = 6.00e
3
D 43
D 57
huLym-1-B-DAP
Mock T
CD19-DAP
CD19-BB3z
CD19-BB3z
CD19-DAP
Day
Dose
5 M 2 M 5 M
e
ns
ns
d
0 10 20 30 40 50 60
0
20
40
60
80
100
Days after tumor inuculation
Percent survival
CD19-DAP
CD19-BB3z
Mock T
p = 0.002
Percent survival
Days post tumor inoculation
0 15 30 45 60 75 90
0
20
40
60
80
100
70

3.6 Discussion
Clinical trials of CD19-CAR T cells to treat B-cell malignancies have produced high
initial complete response rates. Unfortunately, a substantial fraction of treated patients
relapse with CD19-negative/low tumors(86), indicating the need to identify additional
effective targets. Here, we describe the design and development of human CAR T cells
directed to the Lym-1 epitope, which is highly expressed on most human B cell
lymphomas and leukemias(35,87). By substituting the conventional 4-1BB3z signaling
domain with DAP, we were able to circumvent impaired ex vivo proliferation of huLym-1-
B-CAR T cells induced by sustained interaction of huLym-1-B-CAR with weakly
expressed Lym-1 epitope on T cells. Moreover, huLym-1-B-DAP CAR T cells exhibited
epitope driven effector functions as evidenced by increased in vitro cytotoxicity, cytokines
release, as well as potent in vivo tumor control even with reduced doses of CAR T cells.
Furthermore, neither the Lym-1 epitope nor CD19 antigen on B cell lines downregulated
in the presence of huLym-1-B-DAP CAR T cells. These findings indicate huLym-1-B-DAP
CAR T cells appear to be a promising cell therapy product to explore in the clinic.
During the course of these studies, we observed impaired expansion of huLym-1-
B-BB3z CAR T cells that targets the Lym-1 epitope, but such limited expansion was not
found in huLym-1-B-BB3z CAR T cells with crippled binding ability (huLym-1-Bmut-BB3z)
(Figure 3.14 continued) (a) Schematic protocol for the in vivo study. (b) Bioluminescence images
of NSG mice inoculated i.v. with 10
6
Raji-eGFP/Luc on day 0, followed by treatment of Mock,
huLym-1-B-DAP, CD19-BB3z, or CD19-DAP CAR T cells at indicated dose on day 8 (n = 5 mice
per group). (c) Kaplan-Meier curve of survival (ns = not significant by log-rank (Mantel-Cox) test).
(d) Bioluminescence images of NSG mice inoculated i.v. with 10
6
Raji-eGFP/Luc on day 0,
followed by treatment of 10
6
Mock, CD19-BB3z, or CD19-DAP CAR T cells on day 8 (n = 5 mice
per group). This experiment was done with the one in Fig. 5e, to enable the same Mock T group
to be used. (e) Kaplan-Meier curve of survival (P=0.002 by log-rank (Mantel-Cox) test).
71
nor with ablated CD3ζ activity (huLym-1-B-BB3zY-F). These data suggested that limited
expansion was mediated by ligand-dependent activation of the CD3ζ ITAMs signaling
moiety in the huLym-1-B-BB3z CAR construct. This observation is consistent with
previous reports of second generation CAR with CD28 co-stimulation domain redirected
against GD2(65) and ErbB2(77) where limited expansion, activation induced cell death
(AICD), progressive exhaustion, and poor in vivo efficacy were attributed to unconstrained
CAR-CD3ζ activation. In both cases, replacing the CD28 co-stimulation domain with the
signaling domain from 4-1BB mitigated adverse effects induced by chronic CAR-CD3ζ
signaling through incompletely understood mechanisms. In our hands, however, using a
4-1BB co-stimulation domain still resulted in a preparation with poor in vitro expansion of
huLym-1-B-BB3z CAR T cells.
Qualitatively different functions of ITAMs were first documented by Combadiere et
al.,(88) who reported that phosphorylation of the first and third ITAMs in CD3ζ stimulated
greater apoptosis than phosphorylation of the second ITAM in T cells. Consistent with this
observation, in a murine B-cell lymphoma model, Kochenderfer et al., demonstrated that
anti-murine CD19-CAR T cells with mutated first and third CAR-CD3𝜁 ITAMs are resistant
to apoptosis and could mediate anti-lymphoma efficacy better than CD19-CAR with 3
functional ITAMs.(89) A recent study by Feucht et al.,(90) found ablating the function of
the second and third ITAMs in CD3ζ moiety of CD19-CAR resulted in preferential central
memory differentiation, decreased T cell exhaustion, and increased persistence in vivo.
These data suggest that the function of each ITAM can be qualitatively different and the
selection of ITAM(s) in CAR signaling domains is an approach to improve the efficacy of
CAR T cells.
72
Evidence from others(65,77) and this report supports the hypothesis that chronic
suboptimal activation of CAR-CD3ζ ITAMs is a cause of aberrant phenotypes of CAR T
cells. We therefore sought to mitigate these adverse effects by using other ITAM-
containing motifs to substitute CAR-CD3ζ moiety while retaining T cell activation potential.
We chose DAP12 because there is an ITIM motif embedded in its ITAM sequence, this
property may provide distinct signal outputs of DAP12 in response to stimuli with
differential strength(78). DAP12 does not have target recognition domains in the
extracellular region. Instead, DAP12 associated proteins, such as KIR2DS2(81) and
NKp44(91), are responsible for target recognition, resulting in DAP12 activation to initiate
cytotoxic functions. Teng et al.,(80) and Wang et al.,(81) demonstrated that ectopic co-
expression of DAP12 along with its associated scFv-modified receptors in either murine
or human T cells mediated antigen-specific tumor eradiation, indicating that activation of
DAP12 in T cells is sufficient to drive T cell cytotoxicity. In our CAR construct design,
instead of using a multi-chain format, we directly substituted the CD3ζ signaling domain
with DAP12 and added DAP10 for co-stimulation. The resulting huLym-1-B-DAP CAR
addressed the in vitro expansion problem and mediated significantly better in vivo efficacy
than huLym-1-B-BB3z CAR T cells, even though the two showed equivalent cytotoxicity
in vitro (Figure 3.9 a). These results further support the finding that in vitro cytotoxicity is
insufficient to predict relative in vivo efficacy(65) and highlights the impact of the signaling
domain has on in vivo function of CAR T cells. Additional studies are required to identify
the mechanisms by which the DAP signaling domain circumvents ligand-dependent
suboptimal CAR signaling which caused huLym-1-B-BB3z CAR T cells expansion failure.
73
Our results demonstrate that, unlike CD19, the Lym-1 epitope does not
significantly downregulate upon CAR engagement. This lack of downregulation of the
Lym-1 epitope downregulation was replicated in a panel of human B lymphoma cell lines
when co-cultured with huLym-1-DAP CAR T cells (Figure 3.12). Although CAR T cell
trogocytosis may play a role in antigen downregulation(92), we did not observe equivalent
Lym-1 epitope downregulation, suggesting that markedly CD19 antigen downregulation
may involve other mechanisms (Figure 3.11; Figure 3.12). Crosslinking with anti-CD19
antibodies could induce CD19 antigen downregulation through receptor-mediated
endocytosis(49), indicating that interaction between CD19-CAR and CD19 antigen
interaction may contribute to surface CD19 antigen downregulation. Though CD19
downregulation in tumor cells under the pressure of CD19-CAR T cells is a reversable
process(93), transient antigen downregulation could diminish CD19-CAR T cells’ anti-
tumor efficacy and allow tumor immune escape(92). Consistent with this hypothesis,
neither CD19-BB3z nor CD19-DAP CAR T cells were able to induce tumor-free survival
at a dose up to 5 million in the modified animal protocol, whereas huLym-1-B-DAP CAR
T cells mediated a rapid and sustained tumor control leading to tumor free survival at the
lower dose of 2 million cells (Figure 3.14 b, c). Interestingly, treatment with CD19-DAP
CAR T cells induced a significantly better survival than CD19-BB3z T cells at the 1 million
cell dose level (Figure 3.14 d, e). In addition, regardless of the CAR T cell dose, no hind-
leg paralysis, the criterial for euthanasia, was observed in CD19-DAP CAR before day 41
(Figure 3.14 8b-e). In contrast, earlier development of hind-leg paralysis (between days
20-30) was repeatedly seen in CD19-BB3z CAR treated mice (Figure 3.14 b-e). These
observations suggest using the DAP ICD instead of the BB3z ICD may improve the
74
efficacy of CAR T cell preparations that do not exhibit impaired function due to suboptimal
CAR signaling. The underlying mechanisms and the significance of this difference remain
to be investigated.
In summary, our work indicates that huLym-1-B-DAP CAR T cells hold promise for
treating Lym-1 positive B-cell lymphomas. The observation that the DAP signaling domain
can circumvent impaired proliferation induced by ligand-dependent signaling in CD3ζ-
based CAR while retaining equivalent or higher anti-tumor efficacy, highlights the
importance of the stimulation domain selection for CAR design and identifies a new CAR
structure format to address the adverse effects of suboptimal CAR signaling on T cells.
Furthermore, DAP signaling domains may also improve the function of other CAR T cell
preparations even if there is no evidence of adverse CAR signaling. Finally, our report
suggests that targeting an epitope that does not significantly downregulate upon CAR
engagement may also contribute to sustained CAR T cell efficacy.

Acknowledgements
We gratefully thank Ivetta Vorobyova and Ryan Park from the Molecular Imaging Center
(USC) and Dr. Junji Watanabe from the Translation Research Laboratory (USC) for the
mouse Bioluminescence imaging and data processing. We are also grateful to the
USC animal facility for providing animal support and breeding protocols. This work was
supported in part by Cell Biotherapy, Inc., and grant #9031415 from the Ming Hsieh
Institute.

75
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79
Chapter 4 Conclusion and Future directions

In the previous chapters, we demonstrated epitope specific tumor lysis of Lym-1
CAR T cells in vitro. However, both murine and humanized Lym-1 ScFv based CAR T
cells with conventional 4-1BB and CD3 intracellular signaling manifested impaired
expansion. We have identified that this expansion failure is attributed to the interaction
between CAR molecules and weakly expressed Lym-1 epitopes on T cells which leads
to suboptimal CAR ITAMs phosphorylation in the absence of epitope positive tumor cells.
With the hypothesis that DAP12 signaling domain would recruit the SH-2 containing
inositol 5’ polyphosphatase 1 (SHIP1) to counteract weak ITAM signaling while allow
strong stimulation to fully activate T cells, I developed a novel DAP10 and DAP12
signaling domains-based CAR construct that retained epitope specific effector function
(Figure 4.1). In a systemic administered Raji Burkitt’s Lymphoma xenograft model in NSG
mice, huLym-1-B-DAP CAR T cells eradicated tumor and conferred resistance to
rechallenge. In addition, unlike CD19, the Lym-1 epitopes do not significantly
downregulate in the presence of huLym-1-B-DAP CAR T cells both in vitro and in vivo.
This stable Lym-1 epitope surface retention is likely contributed to the improved anti-
tumor efficacy of huLym-1-B-DAP CAR T cells in the Raji xenograft model enabling the
use of significantly lower doses required to eradicate metastatic tumors. Capitalizing on
these findings, we propose that huLym-1-B-DAP CAR T cells is a promising candidate to
explore in clinical trials. This chapter reviews my thesis work with primarily focus on
discussing the limitations of our studies and the potential directions for research in huLym-
1-B-DAP and other CAR T cell therapeutics.

80

Figure 4.1: Proposed Model of using DAP to improve Lym-1 CAR expansion.

4.1 Non-impaired generation of huLym-1-B-DAP CAR T cells with
DAP12 signaling domain
Precursor T lymphocytes emerge in bone marrow and then migrate to thymus
where they undergo positive and negative selection. As a result of positive selection, all
TCRs are capable of engaging with self-MHC at an optimal affinity to avoid activation
induced cell apoptosis, and this self-MHC and TCR interaction induces low-level,
constitutive TCR signaling at basal state which plays a critical role for T lymphocyte
persistence and peripheral tolerance. This basal TCR signaling is often termed “tonic
signaling” [reviewed by(94)]. In contrast, tonic signaling from CAR molecules is implicated
as a cause of impaired T cell proliferation, accelerated exhaustion, and poor efficacy in
vivo [reviewed by(73)]. The potential mechanisms why tonic signaling from a TCR
promotes T cell survival but from a CAR induces T cell dysfunction remains an open
question.
Hypothesis: A CAR constructed with DAP10-DAP12 signaling domain would possess a threshold
that blunt tonic signaling from causing the deleterious effects in T cells but allows stronger signals
to promote cytotoxic function.
81
In Chapter 3, we designed two constructions to address the impaired expansion
issue seen in huLym-1-B-BB3z CAR T cells. In one construct, we mutated two amino
acids in the variable heavy chain to cripple scFv binding. In the second construct, we
mutated all tyrosine residues in CAR CD3z moiety to phenylalanine so that the stimulatory
domain became non-functional. Both mutants showed non-impaired expansion
suggesting cognate epitope binding and CAR signaling are required for the aberrant
phenotypes. We then further demonstrated that ligand-dependent CAR tonic signaling is
the dominant cause of impaired expansion. Building on this observation, we hypothesized
that a signaling domain that blunts tonic signaling induced by weak stimulation but allows
high affinity/avidity binding to activate T cells, would address the expansion issue seen in
Lym-1 CAR T cells. In light of the work by Qisheng et al., which describes how DAP12
recruits SHIP1 to dampen tonic signaling when TREM2, a receptor associated with
DAP12, are bound by low affinity/avidity ligands which blocks SHIP1 recruitment in the
presence of high affinity/avidity ligands and promotes the transcription machinery for
macrophages and osteoclast fusion(78). Together with previous findings that activation
of ectopically expressed DAP12 is able to activate human T cells to execute effector
function(81), the DAP10-DAP12 (DAP) signaling domain for huLym-1-B CAR T cells were
designed.
Although the DAP signaling domain enabled non-impaired expansion of huLym-1-
B CAR T cells, further work is needed to investigate whether recruitment of SHIP1 to
DAP12 is the underlying mechanism of addressing tonic signaling. Our preliminary data
found that DAP signaling also enables normal expansion of Lym-1 CAR with parental
82
murine Lym-1 scFv indicating the use of DAP signaling can be extended to any Lym-1
epitopes targeting domains (Figure 4.2).

Figure 4.2: DAP signaling enable normal Lym-1 CAR expansion.

4.2 Increase tumor antigen surface retention to improve CAR T cell
anti-tumor efficacy.
A lesson learned from clinical trials involving first-generation CAR T cells is that
persistence of administered CAR T cells is crucial for an effective therapeutic
outcome(7,10). Adding co-stimulation to the CAR construct was the seminal step tipping
the scale to cause effective and long-lasting cancer regression in patients (14). Following
this demonstration, many studies incorporated different co-stimulation domains in CAR
constructs seeking to identify signaling domains producing enhanced persistence and
better effector function [Reviewed by(95)]. Other approaches such as adding cytokine
receptor signaling to the CAR construct or introducing mutations in the co-stimulation or
CD3z have shown improved CAR T cell efficacy in preclinical tests [Reviewed by(95)].
How those novel CARs will perform in the clinic is under active investigation.
Recent clinical studies have not shown a significant correlation has been shown
Mock T
Lym-1-BB3z
Lym-1-DAP
0
10
20
30
40
50
Fold increase from
D7 to D14
Expansion of in vitro cultured Mock or CAR-positive T cells from days 7 to14 (restimulated on day
7).
83
between CD19-CAR T cell persistence and disease-free remission, whereas disease
burden and peak CD19-CAR T cell numbers are highly correlated with effective cancer
eradication and complete remission(96). It is conceivable that when leukemia is at low
burden and sufficient number of CD19-CAR T cells are infused, those antigen specific
effector cells will readily clear most of the cancer cells and induce sustained remission.
However, when patients have a relatively high tumor burden and infused CAR T cells are
not able to achieve an optimal effector to target ratio, some tumor cells may survive,
develop resistance, and cause relapse. Therefore, increase CAR T cell persistence and
in vivo expansion is a very promising strategy to improve current CAR T cell therapy
practice.
In Chapter 3, we report Lym-1 epitopes on tumor cells do not significantly
downregulate in the presence of huLym-1-B-DAP CAR T cells whereas CD19 antigen
expression significantly diminishes when bound by CD19-CAR T cells both in vitro and in
vivo. Decreased surface CD19 expression in tumor cells is independent of the signaling
domains used in CD19 CAR construct suggests binding alone is sufficient to induce this
downregulation effect. In fact, sequential CD19 loss/downregulation was also reported in
a high percentage of patients during CD19-CAR T cell treatment(27,97). Our observation,
thus, raises an unexplored question in CAR T cell therapy, namely, how the degree of
antigen retention on tumor cells after CAR engagement affect CAR T cell anti-tumor
efficacy. Upon engagement by endogenous ligands or antibodies, most cell receptors will
downregulate to prevent excessive activation/inhibition. Likewise, CAR molecules also
able to downregulate after cognate antigen recognition, leading to desensitization and
improved selection(98). However, this trade-off may contribute to tumor escape, as CAR
84
requires significant numbers of available targets in order to fully activate T cells. Wentao
et al., recently discovered that tumor antigen encounter induces CARs ubiquitination
followed by surface downregulation through lysosomal degradation pathway(99). To
inhibit ubiquitination, they mutated all cytoplasmic lysines to arginines in the CAR
construct and this new construct ameliorated surface CARs loss upon antigen
engagement to promote enhanced tumor control in vivo(99). This study highlights the
crucial role of the number of productive CAR-antigen complexes and provides a simple
and effective strategy to improve CAR T cell efficacy. However, if the targeted antigens
rapidly downregulate due to CAR-antigen engagement, the above-mentioned strategy will
become meaningless. Essentially, most of the tumor associated antigens, such as
Her2(100), EGFR(101), CD19(49), and BCMA(102) used for CAR T cell therapy are pro-
survival receptors and are able to trigger receptor-mediated endocytosis when clustered
by corresponding ligands, though the mechanism of receptor downregulation may be
distinct across different receptors. To the best of my knowledge, approaches to prevent
tumor antigen downregulation while retaining CAR function are still lacking. Future
research in this field is highly desired.
Alternatively, targeting tumor associated antigens that resist down modulation
upon CAR binding is another potential option. The mechanism as to why Lym-1 epitopes
have better retention than CD19 is unknown. One possible mechanism is that the
peptides bound by Lym-1 epitope bearing HLA-DRs are very abundant in B cell
lymphomas and high enough to saturate available specific HLA-DRs. In summary, novel
approaches to improve antigen surface retention or identifying potential targets that
85
resistant to downregulation after CAR binding can be promising research directions to
further improve CAR T cell efficacy.
4.3 Potential side effects of huLym-1-B-DAP CAR T cells.
While we have demonstrated the therapeutic activity of huLym-1-B-DAP CAR T
cells in Chapter 3, we did not address the potential adverse effects that may arise from
on-target off-tumor effects of targeting Lym-1 epitopes and the clinically unexplored DAP
signaling when deployed in T cells. In this thesis, we found that activated T cells weakly
express Lym-1 epitopes which is responsible for huLym-1-B-BB3z CAR T cells expansion
failure. Though numerous studies with Lym-1 found that it selectively binds to the B cell
lineage and clinical trials with naked or radio labeled Lym-1 showed no serious side
effects including normal B cell aplasia(87), studies have yet to examine other potential
Lym-1 positive cells systemically. This is because the composition and position of critical
amino acid residues of Lym-1 epitope are unknown. An alternative way to evaluate Lym-
1 epitope expression in healthy tissues is to identify the sequence of bound peptides in
the HLA-DR groove. With this information, we might be able to identify the proteins that
generates the specific peptide after degradation. Instead of examining the Lym-1 epitope
bearing HLA-DRs, we can screen the expression of those putative proteins with
proteomics and single cell RNA sequencing technologies. Knowing the location of
potential Lym-1 epitopes in healthy tissues, we will be able to predict the potential side
effects and design intervention regiments to prevent side effects of huLym-1-B-DAP CAR
T cells. It may be, however, that Lym-1 positive antigen presenting dendritic cell
populations in lymph node tissue regenerate quickly, thereby preventing significant side
effects such as antigen amnesia.
86
Perhaps the most under explored area is transcription differences in DAP CAR T
cells compared to other conventional CD3z based CAR constructs. Such variation may
associate with lower/higher clinical toxicities observed in CAR T cell therapy. Sara et al.,
generated a CD19-CAR with lower affinity than parental FMC-63 and explored its efficacy
in the clinic(103). They found lower affinity binding domains give rise to CD19-CAR T cells
with enhanced expansion and persistence, and better disease control. When tested in
pediatric patients with ALL, this low-affinity CD19-CAR mediated comparable complete
remission, and most importantly did not cause severe cytokine release syndrome (CRS)
in any of 14 patients(103). Two independent groups lead by James N. Kochenderfer and
Si-Yi Chen, respectively, tested their hypothesis that modifications in hinge and
transmembrane domains generating CD19-CAR T cells with lower cytokine release but
equivalent cytotoxic function(104,105). James N. Kochenderfer et al., replaced CD28
transmembrane domain with CD8a Hinge, while Si-Yi Chen et al., screened a panel of
CAR constructs with different sizes and compositions in the hinge, transmembrane and
part of the intracellular domain. Clinical trial results from the novel anti-CD19 CAR T cells
developed by Si-Yi Chen group showed unprecedented safety in treating patients with B-
cell lymphomas with no patients developing neurological toxicity or CRS (greater than
grade 1)(106). Work form James N. Kochenderfer et al., also showed an improved safety
profile when compared to CD19-CAR with CD28 transmembrane domain(104). Although
using different approaches, both studies demonstrate that alterations in hinge and
transmembrane domains can have an immediate effect on CAR T cell safety.
Regardless of the modifications made in the CAR structure, absence of severe
grade CRS and neurotoxicity in patients is highly correlated to the lower-level cytokine
87
secretion by activated CAR T cells. By the time we published our discovery, no clinical
studies reported the effect of DAP12 based CAR signaling on T cells, so it is unknown
how the DAP signaling will influence cytokine secretion in huLym-1-B-DAP CAR T cells.
Recently, one group reported a CAR constructed with NK receptor 2D (NKG2D) binding
domain and used 4-1BB and DAP12 as the signaling domain(107). They found this
DAP12 based construct secreted significantly lower IL-2, TNF-a, and IFN-g than CD3z
CARs when co-cultured with a panel of NKG2D ligands positive tumor cells(107). In
contrast, the relative amount of cytokine secretion from huLym-1-B-DAP CAR T cells do
not show consistently lower levels when compared to huLym-1-B-BB3z CAR T cells
(Figure 3.9 Figure 3.10). One possible mechanism of this disparity is because
malignancies from B cell lineage(108) usually expresses a variety of co-stimulation
ligands which may work in concert with DAP signaling to enhance cytokine release, and
this synergistic effect might be absent in BB3z based CAR T cells. Additional studies are
required to investigate the effect of using DAP signaling on CRS development in pre-
clinical models.
4.4 Conclusion remarks
In this dissertation, I described the design and development of a humanized Lym-
1 CAR with DAP10/DAP12 signaling for the treatment of Lym-1 epitope positive cancers.
My work comprised of two major contributions: 1) development of a CAR against a novel
epitope preferentially upregulated in human B cell malignancies. 2) design of a DAP10/12
based CAR construct that enables non-impaired production of Lym-1 CAR T cells. The
Lym-1 antibody has been extensively examined in the clinical either as a naked or isotope
conjugated antibody and proved to be safe and effective(87). Importantly, the epitope
88
bound by Lym-1 do not significant down regulate or shed after Lym-1 antibody binding
making it an ideal target for CAR T cell therapy(28).
When developing Lym-1 epitope targeted CAR T cells, we first adopted 4-1BB-
CD3z based CAR frame. This design showed impaired expansion and exhaustion in vitro.
As we did not observe a significant binding of Lym-1 on activated T cells, we hypothesized
that some sequence in the murine scFv may induce CAR cluster and, therefore, mediate
CAR tonic signaling(65). However, such impaired expansion still occurs after scFv
humanization. Next, we stepped back and reasoned that a weak but real interaction may
exist between scFv and Lym-1 epitopes on T cells, and this consistent interaction
mediated suboptimal CAR signaling which resulted in Lym-1 CAR T cell expansion failure.
We demonstrated our reasoning by constructing two Lym-1 CAR mutants. In one
construction, we ablated single chain binding, and in the second construction, we
constructed a non-functional CAR by deactivating all ITAMs in the CD3z moiety. Both
CAR constructs showed normal expansion which suggested that scFv and T cell
interactions and suboptimal CD3z signaling are both required for the impaired expansion
seen in huLym-1-B-BB3z CAR T cells.
To address such issue, our approach is to design a signaling moiety that would
blunt tonic signaling by recruiting phosphatases upon weak stimulation and induce T cell
activation when given a strong stimulation. We chose DAP12 signaling because one
report showed this molecule could recruit phosphatase when the two tyrosines in the
ITAM is mono phosphorylated(78). In addition, activation of ectopically expressed DAP12
is sufficient to induce T cell activation and effector function(81,82). Our new construct with
89
DAP10/DAP12 showed normal expansion and exhibited enhanced in vivo tumor control
compared to 4-1BB/CD3z based huLym-1-B CAR T cells.
Co-stimulation domains in CAR constructs have been demonstrated to be crucial
in generating improved CAR T cells with enhanced persistence and effector functions. As
we have not comprehensively explored which co-stimulation is the best choice for DAP
in this thesis, replacing DAP10 signaling domain with the ones from 4-1BB, CD28, OX40,
and mutated CD28 may found to be useful in the generation of a new class of Lym-1 CAR
T cells with improved efficacy. In addition to structure modifications, future clinical trials
need to answer whether combining Lym-1 epitope with other antigens such as CD19,
CD20 would provide superior cover of a broader range of cancer cells. Thus, developing
a dual or tri-specific CAR may be beneficial to reduce tumor escape.
In highlighting the unanswered questions and limitations from my studies, this
chapter discusses future research directions of huLym-1-B-DAP CAR T cell and
illuminates potential application of DAP12 signaling as the stimulation moiety in other
CARs. By exploring the limitations and full potential of huLym-1-B-DAP CAR T cell
therapy, I hope to develop an effective and safe CAR T cell product for patients with Lym-
1 epitope positive cancers.

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90
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93
Appendix

CAR
construct
Amino Acid Sequences
Lym-1-BB3z
MALPVTALLLPLALLLHAARPQVQLKESGPGLVAPSQSLSITCTISGFSLTSYGVHWVR
QPPGKGLEWLVVIWSDGSTTYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTAIYYCA
SHYGSTLAFASWGHGTLVTVSAASGGGGSGGGGSGGGGSDIQMTQSPASLSASVGE
TVTIICRASVNIYSYLAWYQQKQGKSPQLLVYNAKILAEGVPSRFSGSGSGTQFSLKINS
LQPEDFGSYYCQHHYGTFTFGSGTKLEIKRTTTTPAPRPPTPAPTIASQPLSLRPEACRP
AAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPV
QTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDV
LDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGL
YQGLSTATKDTYDALHMQALPPR

CD19-BB3z
MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQ
KPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTF
GGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYG
VSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAI
YYCAKHYYYGGSYAMDYWGQGTSVTVSSAVPPQQWALSTTTPAPRPPTPAPTIASQ
PLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYI
FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELN
LGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERR
RGKGHDGLYQGLSTATKDTYDALHMQALPPR

huLym-1-B-
BB3z
MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLTCTASGFSLTSYGVHWV
RQPPGKGLEWLAVIWSDGSTTYNSALKSRLTISKDNSKSQVYLQMNSLRAEDTAVYYC
ARHYGSTLAFASWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGD
RVTITCRASVNIYSYLAWYQQKPGKAPNLLIYNAKILAEGVPSRFSGSGSGTDFTLTISSL
QPEDFASYYCQHHYGTFTFGQGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAA
GGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQT
TQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLD
KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ
GLSTATKDTYDALHMQALPPR
huLym-1-B-
DAP
MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLTCTASGFSLTSYGVHWV
RQPPGKGLEWLAVIWSDGSTTYNSALKSRLTISKDNSKSQVYLQMNSLRAEDTAVYYC
ARHYGSTLAFASWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGD
RVTITCRASVNIYSYLAWYQQKPGKAPNLLIYNAKILAEGVPSRFSGSGSGTDFTLTISSL
QPEDFASYYCQHHYGTFTFGQGTKVEIKAVPPQQWALSTTTPAPRPPTPAPTIASQPL
SLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCLCARPRRSPA
QDGKVYINMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDL
NTQRPYYK
94

CAR
construct
Amino Acid Sequences
CD19-DAP
MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQ
KPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTF
GGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYG
VSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAI
YYCAKHYYYGGSYAMDYWGQGTSVTVSSAVPPQQWALSTTTPAPRPPTPAPTIASQ
PLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCLCARPRRSP
AQDGKVYINMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSD
LNTQRPYYK

95
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Abstract (if available)

Abstract The Lym-1 antibody targets a unique, discontinuous epitope (Lym-1 epitope) on HLA-DR proteins preferentially upregulated in human B-cell lymphomas and leukemias, without significant expression in normal tissues. It was developed in the late 1970’s by Dr. Alan Epstein and found to be clinically safe and effective as an I-131 radioimmunoconjugate. Recently, SH7129, a molecule that mimics Lym-1 designed by SHAL Technologies, was demonstrated to stain a subset of solid tumors suggesting the application of Lym-1 epitope targeted immunotherapies can go beyond hematological malignancies. Hence, we leverage the distinct binding profile of Lym-1 antibody with the goal of developing effective chimeric antigen receptors (CARs) modified T cells to treat Lym-1 epitope positive cancers, including solid tumors. ❧ CARs are synthetic molecules that are capable of mediating T cell effector functions upon engaging the target molecules in a major histocompatibility complex (MHC) independent manner. This dissertation describes the development and evaluation of Lym-1 epitope targeted CAR T cells generated using parental Lym-1 and a humanized version (huLym-1-B). Both exhibited impaired expansion and progressive upregulation of exhaustion markers when the signaling domain consisted of 4-1BB-CD3ζ(BB3z) which is the conventional component of 2nd generation CAR framework. Here, we identified the underlying mechanisms of huLym-1-B-BB3z CAR T cells expansion failure and construct an intracellular DAP10-DAP12 signaling domain-based (huLym-1-B DAP) CAR. This novel signaling overcomes impaired expansion seen in BB3z based Lym-1 CAR T cells and mediates significantly better tumor control in a systemic human lymphoma model in NSG mice. Our results demonstrate that huLym-1-B DAP CAR T cells are a promising modality to explore in the clinic.

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Asset Metadata

Creator Zheng, Long (author)

Core Title Lym-1 epitope targeted chimeric antigen receptor (CAR) T cells for the immunotherapy of cancer

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Medical Biology

Publication Date 04/29/2021

Defense Date 03/17/2021

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

Tag B-cell lymphoma,cancer,chimeric antigen receptor,DAP10,DAP12,immunotherapy,Lym-1,OAI-PMH Harvest,T cells

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Neman-Ebrahim, Josh (committee chair), Epstein, Alan (committee member), Kaslow, Harvey (committee member)

Creator Email longzhen@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-456727

Unique identifier UC11668679

Identifier etd-ZhengLong-9564.pdf (filename),usctheses-c89-456727 (legacy record id)

Legacy Identifier etd-ZhengLong-9564.pdf

Dmrecord 456727

Document Type Dissertation

Rights Zheng, Long

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

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