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Generation and characterization of humanized anti-CD19 chimeric antigen receptor T (CAR-T) cells for the treatment of hematologic malignancies
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Generation and characterization of humanized anti-CD19 chimeric antigen receptor T (CAR-T) cells for the treatment of hematologic malignancies
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Content
Generation and characterization of humanized anti-CD19
chimeric antigen receptor T (CAR-T) cells for the treatment of
hematologic malignancies
By
Xu Han
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In partial Fulfillment of the
Requirement for the Degree
MASTER OF SCIENCE
(Biochemistry and Molecular Biology)
May 2016
Copyright 2016 Xu Han
ACKNOWLEDGEMENTS
I would like to express my special appreciation and thanks to my adviser Dr.
Preet Chaudhary for your constant encouragement and guidance towards
research and writing thesis. You always support me on both research and
career, and give me opportunity to participate and learn so much in the lab.
I would like to thank all members of Dr. Chaudhary’s Lab for their help in
my research and for guidance in writing my thesis. In particular, I will like
to thank Drs. Hittu Matta, Venkatesh Natarajan, Sunju Choi, and
Ramakrishnan Gopalakrishnan. It has been a pleasure for me to work with
them.
I would like to thank the members of my committee for their kind support:
Dr. Michael Lieber and Dr. Robert Maxson. Their guidance has been most
appreciated. I would also like to thank Dr. Zoltan Tokes for guiding me
through this two-year program with kindness.
Thank you every one for your help and support!
ABSTRACT
Chimeric antigen receptor expressing T cells (CAR-T) represent a novel approach to
cancer therapy. Recent studies have shown successful targeting and killing of CD19
expressing malignancies with CAR-T cells targeting the CD19 antigen. Dramatic
responses have been observed with this approach in patients with relapsed and refractory
acute and chronic lymphocytic leukemia and lymphomas. The CD19 CARs in current
clinical trials contain scFv fragments derived from murine monoclonal antibodies. The
development of antibodies against the murine antibody fragments not only contributes to
lack of persistence of T cells expressing such CARs but could also lead to allergic
reactions, including anaphylaxis. To overcome this limitation, we have generated a CD19
CAR containing scFv fragment derived from a humanized CD19 monoclonal antibody.
We demonstrate that this CAR is expressed on the surface of T cells and induce selective
killing of CD19 expressing lymphoma cells.
i
Table of Contents
Introduction .................................................................................................................. 1
Structural construction of CARs .................................................................................... 2
Different generations of CARs ...................................................................................... 5
Components of successful immunotherapy of CARs .................................................... 6
Other immune cells used in CAR .................................................................................. 8
Lymphoma and CAR-T cell therapy .............................................................................. 9
B-cell chronic lymphocytic leukemia .......................................................................... 11
Limitation of CARs ...................................................................................................... 12
Objectives of Project .................................................................................................. 15
Result ........................................................................................................................... 16
Construction of humanized CAR plasmids .................................................................. 16
Detection of Myc expression in CAR T-cells .............................................................. 18
Humanized CD19 CAR expressing T cells bind to soluble CD19 .............................. 22
Cytotoxicity Assay ....................................................................................................... 23
Interferon-γ Enzyme-linked Immunosorbent Assay (ELISA) ..................................... 24
Discussion .................................................................................................................... 26
Materials and methods .............................................................................................. 28
Generation of lentiviral CD19-CAR constructs: .......................................................... 28
Virus generation ........................................................................................................... 29
ii
Viral Transduction ........................................................................................................ 30
Flow cytometry of Myc expression ............................................................................. 30
ELISA .......................................................................................................................... 31
References ................................................................................................................... 32
1
Introduction
Immune system has evolved to protect our body from extrinsic damage caused by
pathogens (virus, bacteria) invasion and intrinsic evolution of tumor cells. Humoral
immunity conferred by B cells and cell mediated immunity conferred by T-cells
collectively work to eradicate infections and tumor cells. The fundamental and foremost
step in this process is the recognition of antigen by antibodies or T cell receptors which
differ in their mode of interaction. In contrast to antibodies which recognize native
antigens, T cell receptors interact with fragment of antigen bound to the major
histocompatibility complex (MHC). T-cells can recognize tumor associated antigens
(TAA) which lead to activation of signaling mechanisms which lead to killing of tumor
cells. However, tumor specific T-cells are rare and effective killing of tumor cells
requires enrichment of this subset to effectively eliminate all tumor cells leaving no
residual tumor cells to prevent recurrence. One approach to isolate this rare tumor
reactive T-cells from patients expand them ex vivo and infuse into the cancer patients.
This procedure referred as “Adoptive cell transfer (ACT)” was first demonstrated by
isolating tumor infiltrating T-cells (TILs) from surgically removed metastatic melanoma,
but technical limitations restricted its wide spread application in clinic. Another approach
would be to engineer T-cells with receptors with specificity to known tumor antigens.
This needs cloning of the TAA-specific TCR, expressing them in T-cells, and infusing
2
them into patients.
Chimeric antigen receptors offer an alternative approach to artificially enforce the tumor
recognition to specific TAA using an antibody binding domain instead of extracellular
region of TCR to mediate antigen recognition. CARs are synthetic receptors that
combine the binding of an antibody and signaling functions of T cell receptor. As target
recognition is dictated by antibodies, CARs can be designed to recognize an expanded
range of potential proteins targets and, even carbohydrate and glycolipids. However,
application of CAR is limited to cell surface molecules as intracellular targets are not
accessible (Saar G, 2015).
Unlike T-cell receptor, CARs confer HLA-independent recognition and eliminate
additional steps of antigen processing and presentation (Shivani S, 2015). This feature of
CARs (Shivani S, 2015) also enables them to bypass tumor escape and tolerance
induction mechanism from TCR-mediated immunity (Zhou G, 2012). Furthermore,
CARs can work under any HLA backgrounds which overcome the barrier that TCRs
need to match haplotype of patients.
Structural construction of CARs
The CAR construct consists of an extracellular antigen-recognition region that includes
antibodies in the form of single-chain variable fragments (scFv), a hinge region and a
transmembrane domain that anchors CAR to the cell membrane and an intracellular
3
signal domain for transducing signals from TCR complex and co-stimulatory molecules
(Fig.1). Upon interaction with antigen on tumor surface, these signaling domains mediate
the effector functions including cytokines expression, secretion and lysis of the target
cells.
Antigen specificity of CAR is based on antibodies, which can be derived from mouse
monoclonal, humanized or fully human Abs (Claudia G, 2015). For the purpose of
clinical applications, an ideal CAR should be designed to maximize interaction with
tumor and minimize toxicities to normal tissues. The scFv plays important role in
determining the antigen specificity of CAR and there is considerable effort underway by
various research teams to identify different scFvs capable of recognizing new tumor
antigens or to improve their interaction with known target antigens.
Hinge region of CARs provide flexibility to scFv and has significant impact on CAR-T
cell properties (Claudia G, 2015). The length of hinge relates to the location of epitope
targeted by scFv (Ryan G, 2005).
Transmembrane (TM) domain helps CARs to anchor to cell surface. CD28 TM portion is
generally used in CARs along with 4-1BB which is co-stimulatory endodomain.
4
Fig. 1. Structure of CAR molecules. Optimization of scFv affinity with target antigen
may reduce potential toxicities. scFv can be inclined to bind antigen in membrane bound
form rather than a soluble form by selection. scFv may cause spontaneous signaling into
T-cells or T cell rejection because of its immunogenicity. Hinge length affects antigen
binding. Fc receptor positive cells can be activated by hinge and long fragment of the Fc
part in immunoglobulin (Ig). Combination of signaling domains or CD28 and 4-1BB
co-stimulation can affect CAR persistence, proliferation and effector functions. TM
affect CAR anchor and may affect CAR expression.
5
Different generations of CARs
First generation CARs are composed of antigen specific scFv linked to cytoplasmic
signaling domain (e.g., CD3-ζ or Fc receptor [FCR]-γ chains) through a hinge and a TM
domain (Jae P and Renier B, 2015). Due to the lack of costimulation, T cell proliferation
is limited in first generation CARs. Second generation of CARs overcomes the lack of
T-cell costimulation based on modified first generation CARs by incorporating CD3-ζ
signaling domain of T-cell costimulatory molecules (e.g., CD28, OX40, 4-1BB), which
can enhance T-cell proliferation and persistence. This was demonstrated with
CD19-specific CAR T-cells containing 4-1BB which showed longer persistence and
biological functions as compared to first generation CARs. Third-generation CARs
contain tandem cytoplasmic signaling domains from more than one costimulatory
receptors, CD28/OX40 or CD28/4-1BB (Carpenito C, 2009). These signal moieties
activate downstream kinase pathways that support gene transcription and functional
cellular response (Claudia G, 2015). The benefits of additional costimulatory receptors in
the 3
rd
generation over the 2
nd
generation are not very clear. Optimal design a CAR
remains as a challenge and needs to be empirically evaluated for the treatment of
different tumors (Fig. 3).
Recently, cytokine-secreting and ligand modified CAR T-cells have been designed which
are able to reprogram immunosuppressive tumor microenvironment (Hanren D, 2016).
Fourth-generation CAR construct have a cytokine expression cassette to attract innate
6
immune response.
Fig. 2. Three generations of CAR-s. First-generation contain one single cytoplasmic
domain for signaling. Second- and third-generation have combinations of signaling
domains with different costimulatory endodomain.
Components of successful immunotherapy of CARs
For successful clinical application, CAR T-cells needs to traffic to the tumor site,
recognize desired antigen, proliferate, evade suppression form tumor microenvironment,
kill tumor cells and persist long enough to monitor and eliminate residual tumor cell
growth (Fig. 3).
CAR-T-cells persistence can be fulfilled by using gamma-retrovirus and lentivirus to
result in stable integration into genome. Chemotherapy and radiation given prior to
7
CAR-T cell infusion also help in T cell homeostatic proliferation and persistence (Mark
D, 2004).
For CAR-T-cells migration throughout the body and homing to antigen expressed sites,
expression of proper chemokine receptors is needed. It was shown that Hodgkin’s
lymphoma marker CD30 help T-cells localization to tumor sites which is modified to
express CCR3 on the surface (Christian C, 2009).
Fig. 3. Components of successful adoptive cellular immunotherapy (Saar G, 2015).
Processes of CAR T-cells in clinical treatment includes preconditioning with
chemotherapy, then CAR T-cells circulation after intravenous infusion, migration to
tumor sites, accumulation at tumor sites, further execution of their effector functions.
CAR T-cells need to have the ability to overcome immunosuppression and survive under
tumor microenvironment, their persistence is essential for eliminating all malignant-cells.
8
Other immune cells used in CAR
NK cells: Natural killer (NK) cells are important innate lymphoid cells because they can
lyse malignant or infected cells in human leukocyte antigens (HLA)-independent manner
and have a crucial role in defense of cancer development and viral infection (Cristina E,
2014). NK cells can be derived from peripheral blood (PB), bone marrow (BM) and
human embryonic stem cells (hESCs). NK cells are CD56
+
/CD3
-
and are subdivided into
cytotoxic CD56
dim
CD16
+
and immune-regulatory CD56
bright
CD16
−
cells (Wolfgang G,
2015). Similar to CAR T-cells, NK cells becomes another potential candidate for
adoptive immunotherapy.
Jurkat-NFAT-eGFP T-cells for experiment: Jurkat cell are an immortalized line of human
T lymphocyte cells. Unlike primary T-cells, Jurkat cells do not have the ability of killing
tumor cells, but share other feature similar with primary T cells and can be used for
detecting CAR T-cells interaction with target tumor cells. Compared to primary T-cells,
Jurkat cells have rapid growth and stable characteristics during culture. In some
experiment, CAR modified Jurkat cells are used to detect interaction between CAR
Jurkat cells with target cells (Fig. 4).
9
Fig. 4. One example of Jurkat cell activation. When receptor on the surface of Jurkat cell
interact with antigen on the tumor target-cells, cytoplasmic signaling pathway is
activated, NFAT promoter is turned on and its downstream gene (GFP reporter gene) is
expressed.
Lymphoma and CAR-T cell therapy
Lymphoma is a kind of blood cell tumors developed from clonal expansion of lymphatic
cells at different stages of differentiation; with symptoms such has enlarged lymph nodes,
fever, weight loss and drenching sweats. There are two main categories: Hodgkin
lymphomas (HL) and the non-Hodgkin lymphomas (NHL). HL is a rare cancer with high
cure rate (Roberta F, 2015).
Two grades of NHL are known. Indolent (low grade) NHL grow slowly and aggressive
(high-grade) NHL grow more quickly and are rapidly fatal without treatment (Elroy W,
10
2015). NHL incidence is increased in patients with AIDS (Fig. 5).
Strategies of immunotherapies in lymphoma are divided into drug-base or cell-base.
Drug-base immunotherapy includes general stimulation of immune response or
modification of the crosstalk between cytotoxic T-cells and malignant cells with agents
(Benjamin K, 2016). Cell-based immunotherapy includes allogeneic hematopoietic stem
cell transplantation (allo-HCT), chimeric antigen receptor (CAR), ex vivo stimulation
and reinfusion of autologous T-cells.
Fig. 5. Comparison of characteristics of different histological types of AIDS-related
non-Hodgkin's lymphomas (ARLs) (Yibin C, 2007)
Efficiency of CARs depend on affinities of CAR itself, on the properties of antigen
epitope function to T-cells and even location of the recognized epitope on the antigen.
T cell activation relies on the phosphorylation of immune-receptor tyrosine-based
activation motifs (ITAMs) present in the cytoplasmic CD3-ζ domain of the TCR complex
11
(Carlos R, 2016). CD19 is used for target of CAR-T-cells to eliminate malignant B cells.
One report of a second-generation CAR with CD-19 and CD28 usage in clinical, shows
tumor underwent partial regression and B cells were absent from circulation after T cell
infusion in patient with advanced follicular lymphoma (Carlos R, 2016). 4-1BB (CD137)
as an alternative costimuatory domain in CAR-T-cells help improves expansion and
persistence. CD28 represents an ”early” costimulatory signal and tumor necrosis factor
receptor (TNFR) family play crucial role as “late” costimulatory molecules, such as
4-1BB and OX40.
B-cell chronic lymphocytic leukemia
B-cell chronic lymphocytic leukemia (CLL), a type of leukemia, starts in bone marrow
then goes to the blood and spread other part of body. It is a clonal expansion of
CD5
+
CD19
+
B lymphocytes and can affect B cell lymphocytes which normally fight
infection by producing antibodies (Robbert H, 2013). In CLL, the cells can mature partly
but not completely, so the ability of fight infection is diminished.
CLL can be distinguished as carrying either unmutated (U-CLL) which express
low-affinity or self-reactive B cell receptors (BCRs) or mutated Igs (M-CLL) of
stereotypic BCRs. U-CLL and M-CLL share a highly similar gene expression profile (Ulf
K, 2001). Although CLL takes long time to form cancers, whose patients are usually over
50 years old, it generally harder to cure than acute leukemia.
In CLL clinical treatment on patients, CD19 CAR-T-cells with 41BB constimulatory
12
domain and CD3 ζ activation domain are shown elimination of high tumor burdens and
active persistence last for three years (Michael K, 2011 and David P, 2011). Ten of forty
patients use CD19-specific CAR-T-cells along with chemotherapy achieved completes
remission, and ten achieved partial remission.
Limitation of CARs
“Cytokine release syndrome’ (CRS) is a common side effect of CAR in clinical use. It
is caused by over expression and excessive release of proinflammatory cytokines.
Symptoms include cardiac dysfunction, fever, hypotension, kidney failure, hypoxemia,
and electrolyte abnormalities. Patients under CAR treatment need close monitoring. In a
significant number of cases, CAR T-cells therapy can also lead to neurologic symptoms
including tremor, seizures and death.
The CD19 CARs in current clinical use contain scFV fragments derived from murine
monoclonal antibodies. The development of antibodies against the murine antibody
fragments lead to lack of persistence of T cells expressing such CARs. Such antibodies
can also lead to allergic reactions, including anaphylaxis.
Mechanism of gene therapy with lentivirus vector
Lentivirus is a genus of retroviridae family, which can integrate genetic information into
host cell genome. Lentiviral vectors are frequently used to express CAR in T-cells.
13
Lentiviral infection has high-efficiency of infection of both dividing and non-dividing
cells with low immunogenicity and lead to long-term stable expression of transgenes.
Lentiviral vectors used for gene therapy are replication-defective or self-inactivating;
meaning that delivery of desired sequence is allowed but viral replication does not occur
in host cells. When transfected into host cell, we called packaging cells, lentiviral gene is
disrupted by three parts and packaged into three vectors: one has 5’ LTR to drive
expression of package genomic DNA/RNA, two helper plasmids that encode the
structural and envelope proteins. After transfection into packaging cells, intact virus is
generated with the ability of introducing desired gene into host cell genome (Fig. 6).
In clinical, first report is utilizing lentivirus which is HIV-based, and shows advantage of
both safety and efficacy. Gene therapy of using virus to carry target gene into host aims
to cure some genetic disease, like gene deletion of malfunction. Through the ability of
virus can insert its gene into host genome, lentivirus is used as a kind of therapy. In CAR
modified T-cells, same mechanism is applied.
14
Fig. 6. Production of recombinant lentiviral vectors. Gag and Pol are needed for the
maturation of virion. Vesicular stomatitis virus (VSV-G) is for coding fusogenic envelope
G glycoprotein. The packaging cells produce infectious particles, whose genome only
encodes sequences from the transfer plasmid, which can be used to transduce the
targeT-cells.
15
Objectives of Project
CAR engineered T-cells offers a promising approach for cancer immune therapy.
Although several clinical trials were conducted in past two decades after the concept of
CAR was initially proposed, several of them failed due immunogenicity and short
persistence of engineered CAR-T-cells in vivo. Recently, T-cells engineered with CD19
specific CAR containing costimulatory domains have shown promising clinical benefits
in B cell malignancies, reviving enthusiasm in this field. However, host anti-CAR
immune response can limit the life span of CAR-T-cells. The target binding domain
(scFv) of CARs is generally derived from murine monoclonal antibodies which elicits
anti-mouse response in humans leading to clearance of CAR T-cells after infusion. The
objective of this thesis is to generate a humanized version of anti-CD19-CAR, express
them in T-cells and functionally evaluate its ability to kill tumor cells.
16
Result
Construction of humanized CAR plasmids
We generated CAR constructs targeting murine CD19 (FMC63), and a humanized CD19,
both targeting CD19 and a control CAR (4C3). The control CAR only recognize viral
antigen and does not interact with mouse or human CD19. The final constructs were
designated as
pLenti-EF1∆Xho-Nhe-mCD19 (FMC63)-Mlu-MYC-CD8TM-BBZ-P2A-T2A-Pac,
pLenti-EF1∆Xho-Nhe-hCD19-Mlu-MYC-CD8TM-BBZ-P2A-T2A-Pac,
pLenti-EF1∆Xho-Nhe- 4C3-Mlu-MYC-CD8TM-BBZ-P2A-T2A-Pac.
In each case, cDNA encoding corresponding scFv fused to CD8 signal peptide was PCR
amplified from synthetic gene fragments and cloned in frame with a human CD8 signal
peptide, a Myc epitope tag, the hinge and transmembrane domain of human CD8, the
cytosolic domain of human 41BB (CD137) receptor, the cytosolic domain of human
CD3z, a 2A ribosomal skip sequence and a cDNA encoding a puromycin resistance gene
(Fig.7).
17
Fig.7. A schematic representation of the hCD19-CAR construct:
pLenti-EF1∆Xho-Nhe-hCD19-Mlu-MYC-CD8TM-BBZ-P2A-T2A-Pac
Recombinant plasmids were transformed into competent cells and plated on carbenicillin
plates. Colony PCR was used as first step to detect the recombinant clones (Fig. 8). The
positive clones were used for plasmid isolation and their sequence confirmed by
automated sequencing.
Fig.8 For colony PCR screening, we
picked 8 colonies. For PCR, we used one
primer specific for insert and the second
primer specific for vector. The expected
size for positive clones was approximately
300-400bps.
18
Detection of Myc expression in CAR T-cells
To generate virus, we transfected our CAR plasmids along with packaging plasmids
(pLP/VSVG and psPAX2) into HEK-293 cells through calcium phosphate transfection
method.
The viral supernatants were collected 72 hours post transfection and concentrated with
ultracentrifugation at 18,000 rpm for 2 hours at 4° C. Primary T cells were isolated from
de-identified donors with CD3-microbeads (Miltenyi Biotech) and infected with the
lentiviruses encoding the different CAR constructs in the presence of polybrene. To
select CAR-positive T-cells, we used puromycin at a dose of 300ng/ml. The uninfected
T-cells were dead after approximately 10 days of puromycin selection. We performed
flow cytometery experiment with Myc-APC antibody to confirm the expression of CAR
in T-cells. Uninfected T-cells were used as negative control.
A
19
B
C
20
Fig. 9. Flow cytometric analysis to confirm CAR expression on cell surface of T-cells.
Uninfected or T-cells infected with different lentiviral CARs were stained with
APC-conjugated Myc antibody to detect Myc tag fused to CARs. (A) Uninfected control
T-cells. (B) T-cells transduced with lentiviral hCD19 construct before (upper panel) and
after puromycin selection (lower panel). (C) T-cells transduced with a control lentiviral
(4C3) construct before (upper panel) and after (lower panel) puromycin selection. (D)
T-cells transduced with lentiviral mCD19 (FMC63) construct before (upper panel) and
after (lower panel) puromycin selection.
As shown in Fig 9A, uninfected T-cells did not show any expression of Myc. In contrast,
T cells infected with the humanized hCD19 CAR construct showed significant
D
21
expression of Myc on the cell surface, which was increased further in T cells that had
been selected with puromycin (Fig 9B).
Myc expression was seen in all other CAR-engineered T cells including negative control
(4C3) and positive control (FMC63), showing an efficient transduction of T- cells with
CAR viral supernatant. Again, selection with puromycin further increased the proportion
of Myc-expressing T cells in the above cases as well as the mean fluorescence intensity
of Myc expressing cells.
Name Events %Parent
T-UI:P1 9,618 45.20
T-UI:P2 94 0.98
T-FMC63-BBZ-PAC-A13-NO PURO:P1 9,776 69.19
T-FMC63-BBZ-PAC-A13-NO PURO:P2 2,528 25.86
T-FMC63-BBZ-PAC-A13-WITH PURO:P1 10,000 68.01
T-FMC63-BBZ-PAC-A13-WITH PURO:P2 9,004 90.04
T-hCD19-BBZ-PAC-P08-NO PURO:P1 10,000 62.73
T-hCD19-BBZ-PAC-P08-NO PURO:P2 6,884 68.84
T-hCD19-BBZ-PAC-P08-WITH PURO:P1 10,000 78.80
T-hCD19-BBZ-PAC-P08-WITH PURO:P2 9,371 93.71
T-4C3-BBZ-PAC-N01-NO PURO:P1 10,000 74.99
T-4C3-BBZ-PAC-N01-NO PURO:P2 1,808 18.08
T-4C3-BBZ-PAC-N01-WITH PURO:P1 10,000 65.11
T-4C3-BBZ-PAC-N01-WITH PURO:P2 5,063 50.63
Table. 1 Statistic data of flow cytometery with Myc-APC. P1 gate represents live cell
population and P2 gate is Myc positive cells.
22
Humanized CD19 CAR expressing T cells bind to soluble CD19
We next tested whether the CAR can actually bind to CD19. Jurkat-NFAT-Luc cells were
stably transduced with the different CD19 targeted CAR constructs and selected in
puromycin. Cells were incubated with soluble CD19 (sCD19) supernatants and after
extensive washes assayed for sCD19 binding by an enzyme linked reporter assay. Figure
10 shows strong binding of Jurkats expressing FMC63-BBz-PAC-A13 and hCD19-
-BBz-PAC-P08 CAR constructs to sCD19, while no significant binding was observed on
uninfected cells or those expressing 4C3-BBz-PAC-N01 control CAR. Essentially similar
results were obtained when the experiment was performed on T cells expressing the
different CAR constructs.
Fig. 10 Strong binding of sCD19 to Jurkat cells expressing the FMC63 and hCD19 CAR
constructs as compared to uninfected (UI) Jurkat cells and those expressing the 4C3
control CAR.
23
Cytotoxicity Assay
We conducted these cytotoxicity experiments at two different time points. The effecter
CAR T-cells were incubated with RAJI target cells either for 4 hours (effector: target
ratio of 10:1) or for 96 hours (effector: target ratio of 1:1) following which the extent of
cell death was assessed by an enzymatic assay. Culture medium was used as a blank in
cell death assay. To get rid of any background interference in cell death assay. These
results show effective lysis of of target RAJI cells by CAR-T cells(Fig. 11).
Fig.11 Cell death assay of CAR T cell treated cancer cells. Different CAR T-cells
co-cultured with CD19 positive RAJI cells for (A) 4 hours and (B) For 96 hours.
No cell death of RAJI cells was observed when incubated with uninfected T-cells (UI).
Similarly control (4C3) CAR T-cells did not kill target cells because it was not able to
recognize specific antigen on tumor cells. The positive control mCD19 (FMC63) CAR T
cells killed RAJI cells. The puromycin-selected CAR-T cells were more efficient in
24
killing target cells than those cells that had not been selected with puromycin.
The humanized CD19 CAR T-cells showed more cytotoxicity towards RAJI cells as
compared to mCD19 (FMC63) CAR T-cells. Thus, humanized anti-CD19 CAR not only
retained but demonstrated improved cytotoxicity against target cells.
We observed essentially similar results when target cells were co-cultured with effector
cells for 96 hours as those observed in 4 hours of co-culture experiment. This clearly
shows that the cytotoxic effect of CAR cells was persistent when the effector and target
cells were incubated over a longer period of time.
Interferon-γ Enzyme-linked Immunosorbent Assay (ELISA)
To detect whether increased cytokine secretion could contribute to the cytotoxic effect of
CD19-CAR, we measured the secretion of interferon-γ (IFN-γ) via ELISA. The
supernatant from the CAR T-cells was collected after they had been co-cultured with
target cells (CD19
+
RAJI cells) for 96 hours at an effector: target cells ratio of 1:1. As
IFN-γ is known to be involved in cytotoxicity of CAR-T cells, we specifically examined
its secretion by ELISA. As controls, we also checked IFN-γ secretion in uninfected T cell
to control for background interference and in RAJI cells alone to see the basal level of
IFN-γ produced by RAJI cells.
When RAJI cells are co-cultured with T-cells expressing mCD19 (FMC63)-CAR or
humanized CD19-CAR, IFN-γ level were significantly higher (Figure 12). Humanized
CD19-CAR-T cells showed higher IFN-γ expression than mCD19 (FMC63) CAR-T cells.
25
Therefore, it is possible that IFN-γ might contribute to the cytotoxic effect of CAR-T
cells. In comparison there was a very low level of IFN-γ secretion by the control (4C3)
CAR-T cells.
Fig. 12 Cytokine secretion by murine and humanized CAR-T cells upon activation with
RAJI cells. FMC63 CAR T-cells showed increased cytokine expression when CAR
T-cells are activated by interaction with RAJI. T-cells modified with expressing
humanized CD19 receptor showed nearly 2-fold higher cytokine secretion than those
expressing FMC63. 4C3 CAR T-cells are negative control, which is not activated by
RAJI. RAJI cells alone do not show IFN-γ expression. CAR-T cells with and without
puromycin selection were tested.
0.0000
1.0000
2.0000
3.0000
4.0000
5.0000
6.0000
7.0000
8.0000
T-UI
T-FMC63-BBZ-
pac-A13-no puro
T-FMC63-BBZ-
pac-A13-with
puro
T-hCD19-BBZ-
pac-PO8-no
puro
T-hCD19-BBZ-
pac-PO8-with
puro
T-4C3-BBZ-pac-
NO1-no puro
T-4C3-BBZ-pac-
NO1-with puro
OD 650nm
hIFN-gamma-T-CAR-cells with RAJI-cells
Alone
RAJI
26
Discussion
Chimeric antigen receptor expressing T cells have risen to the forefront of
immunotherapy for cancer. CAR-T cells directed against CD19 receptor have shown
extremely promising activity against a number of B cell malignancies, including Acute
Lymphocytic Leukemia, Chronic Lymphocytic Leukemia and Lymphomas in patient
with chemotherapy refractory diseases. The response in these clinical trials correlates
with expansion and long term persistence of CAR modified T cells. All the CAR-T cells
in current clinical trials incorporate a scFv derived from a mouse monoclonal antibody,
which could limit their persistence due to immune response against the murine protein
segments. In addition, murine protein segments could also contribute to allergic
reactions.
In this project, we have generated a CD19 CAR using scFv derived from a humanized
monoclonal antibody against CD19. Our results demonstrate that humanized CD19 CAR
construct is robustly expressed on the surface of T cells as determined by staining with a
Myc antibody. The humanized CAR could also bind to soluble CD19 receptor, thereby
demonstrating that the scFv fragment was functionally active when linked to the rest of
the CAR backbone. The expression of humanized CAR construct was actually better than
that of the FMC63 CAR construct, which is based on a murine CD19 antibody and is
being used in a number of clinical trials.
We also tested the activity of the humanized CAR against CD19 expressing RAJI target
27
cells. The humanized CAR-T cells effectively lysed RAJI cells and were, in fact, better
than the FMC63 CAR-T cells. The humanized CAR-T cells also upregulated the
expression of IFN-γ upon co-culture with the RAJI target cells. Collectively, our results
demonstrate that humanized anti-CD19 CAR-T cells are functionally active and can exert
effective cytotoxicity against CD19 positive target cells.
We are planning to follow these promising in vitro results with in vivo studies in
immunodeficient mice. The ultimate test of humanized anti-CD19 CAR construct
generated in this project will require human clinical trials. Such trials are in the planning
stages.
28
Materials and methods
Generation of lentiviral CD19-CAR constructs:
The pLENTI-Blast vector was derived from pLenti6v5gw_lacz vector (Invitrogen;
ThermoFisher Scientific) by removal of the LacZ gene. pLenti-MP2 was a gift from
Pantelis Tsoulfas (Addgene plasmid # 36097) and was used to generate the
pLENTI-EF1α lentiviral vector by replacing CMV promoter with human EF1α promoter
using standard molecular biology techniques. psPAX2 was a gift from Didier Trono
(Addgene plasmid # 12260). The pLP/VSVG envelope plasmid and 293FT-cells were
obtained from Invitrogen (ThermoFisher Scientific). The retroviral vector MSCVneo,
MSCVhygro, and MSCVpac and the packaging vector pKAT were obtained from Dr.
Robert Illaria’s laboratory. The sequence of the scFV fragment encoding a mouse
monoclonal antibody against human CD19 (FMC63) and a humanized CD19 antibody
(hCD19) were codon optimized using GeneArt
TM
software (Thermo Fisher Scientific)
and gene-fragments encoding the optimized sequences synthesized by GeneArt
TM
or
Integrated DNA Technologies (IDT). The gene fragments were used as templates in PCR
reaction using custom primers digested and then ligated to modified pLENTI-EF1α
vector containing the hinge, transmembrane and cytosolic domains of the CAR cassette
by standard molecular biology techniques. Ligated products were transformed into Stbl3
competent cells (Invitorgen) and colonies were screened using colony-PCR. Plasmids
purification from positive clones was done using standard procedures. The final CAR
29
constructs consisted of human CD8 signal peptide, fused in frame to the scFv
fragments, a Myc epitope tag, the hinge and transmembrane domain of human CD8, the
cytosolic domain of human 41BB (CD137) receptor, the cytosolic domain of human
CD3z, a 2A ribosomal skip sequence and a cDNA encoding a puromycin resistance gene.
We generated three CAR constructs namely, pLenti-EF1∆Xho-mCD19
(FMC63)-Mlu-MYC-CD8TM-BBZ-P2A-T2A-Pac,
pLenti-EF1∆Xho-hCD19-Mlu-MYC-CD8TM-BBZ-P2A-T2A-Pac and
pLenti-EF1∆Xho-control (4C3)-Mlu-MYC-CD8TM-BBZ-P2A-T2A-Pac (control-CAR)
for this project and sequenced them to confirm their DNA sequence..
Virus generation
To generate CAR-encoding lentivirus, HEK-293 FT-cells were transfected with a mixture
of 10 μg of CAR lentiviral plasmid, 7.5μg psPAX2 containing Gag, Pol, Rev, and Tat
genes for virus packaging; and 2μg pLP/VSVG for expression of the virus G
glycoprotein. Use standard calcium phosphate method. Supernatants were collected at 48
h and 72 h post transfection, filtered through a 0.45µ m filter, concentrated by
ultracentrifugation at 18,500 rpm at 4 C for 2 hours and re-suspended in T-cell medium.
Viruses were stored at -80° C until needed.
30
Viral Transduction
Preparation of T cells: Primary T cells were isolated from PBMCs from a heathy donor
using CD3 microbeads from Miltenyi Biotech. The isolated T cells were cultured in
XVIVO medium from Lonza supplemented with CD3/CD28 soluble antibodies and
purified recombinant human IL2.
Transduction: T-cells (4 million cells/well/2mL) were plated in to a 6-well plate. These
cells were either left un-infected or infected with 300μl of concentrated virus
supernatant/well with 8 µ g/mL polybrene (PB). Cells were centrifuged at 2,800 rpm at
32° C for 90 min andwere left undisturbed at 37 C, 5% CO
2
for about 3 hours. After
incubation, the medium containing viral supernatant and polybrene was replaced with
fresh T-cell medium; plates were incubated overnight at 37° C. Transduction was repeated
two more times for next two days. T-cells infected with CAR expressing lentiviral
supernatants were selected with 400ng/mL of puromycin for CAR-positive T-cells.
Flow cytometry of Myc expression
CAR-expressing primary T cells and un-infected T cells were counted and washed with
Phosphate-buffered saline (PBS) containing 4% Bovine serum albumin (BSA).
Approximately 1x10
6
/mL were stained with Myc-APC antibody and incubated in dark at
4C for 1 hour followed by three washes with cold PBS-BSA wash buffer. The cells were
re-suspended in 500 μl of PBS and acquired using FACSverse flow machine for BD
biosciences to check Myc expression in CAR-positive cells.
31
ELISA
ELISA plate (384-well, nunc) was coated with capture antibody (50 μL per well) diluted
to a working concentration in PBS for overnight at room temperature. Next day, the plate
was washed two times with wash buffer and incubated with blocking buffer (1% BSA in
PBS) for 2 hrs at room temperature. Wells were washed three times prior to adding the
cell supernatants to be assayed for IFN-γ. After, 2 hrs incubation at room temperature, the
plates were washed three times and incubated for additional 2 hrs with detection antibody
diluted in reagent diluent.. The plates were washed again and incubated with
streptavidin-HRP conjugate in dark for 20 minutes. Finally substrate solution (1:1
mixture of color reagent A, H
2
O
2
and color reagent B Tetramethylbenzidine) was added
to each well after washing the plate three times with wash buffer. The plate was
incubated for another 20 minutes and absorbance was read at 655nm.
32
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Abstract (if available)
Abstract
Chimeric antigen receptor expressing T cells (CAR-T) represent a novel approach to cancer therapy. Recent studies have shown successful targeting and killing of CD19 expressing malignancies with CAR-T cells targeting the CD19 antigen. Dramatic responses have been observed with this approach in patients with relapsed and refractory acute and chronic lymphocytic leukemia and lymphomas. The CD19 CARs in current clinical trials contain scFv fragments derived from murine monoclonal antibodies. The development of antibodies against the murine antibody fragments not only contributes to lack of persistence of T cells expressing such CARs but could also lead to allergic reactions, including anaphylaxis. To overcome this limitation, we have generated a CD19 CAR containing scFv fragment derived from a humanized CD19 monoclonal antibody. We demonstrate that this CAR is expressed on the surface of T cells and induce selective killing of CD19 expressing lymphoma cells.
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Creator
Han, Xu
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Generation and characterization of humanized anti-CD19 chimeric antigen receptor T (CAR-T) cells for the treatment of hematologic malignancies
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Biochemistry and Molecular Biology
Publication Date
04/22/2016
Defense Date
03/09/2016
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