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Engineered control of the CAR-T-tumor synapse using customized DNA linkers

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Engineered control of the CAR-T-tumor synapse using customized DNA linkers

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Content 1

Engineered Control of the CAR-T-Tumor Synapse
Using Customized DNA linkers

Shayian Jalilian
May 2019
M.S. Experimental and Molecular Pathology
Keck School of Medicine
University of Southern California

2

Table of Contents
Abstract 3
Introduction 4
Materials and Methods 5
Results 8
Discussion 21
References 23

3

Abstract
Chimeric Antigen Receptor (CAR) T-cell therapy represents a class of powerful cell-
based therapeutics approved for use in patients with advanced lymphomas. Despite the
clinical success of CAR-T, patients can experience a severe, uncontrolled and
potentially life-threatening activation of engrafted CAR-T. One approach to overcome
this effect is to engineer CAR containing an ON-switch, enabling tight regulation of
activation. Here, we leverage recent advances in our understanding of DNA base
modifications and sensor proteins to design and engineer CAR-T cells that establish a
tumor-T cell synapse specifically in the presence of customized tumor antigen-targeting
modules and modified DNA linkers. We establish a minimal system comprised of three
components: a customized DNA linker containing 5-methylcytosine (5mC) and N
6
-
methyladenine (6mA) DNA base modifications, a synthetic fusion of a single-chain
variable fragment (scFv) targeting a cell surface antigen (LaG_16) with a methyl-binding
domain (MBD) that binds to the 5mC modification; and T-cells expressing a CAR
containing an extracellular restriction enzyme adenine methylase associated (RAMA)
domain, which acts as a selective sensor to the 6mA modification. Activation of CAR-T
against tumor cells occurs exclusively in the presence of the LaG_16-MBD antigen-
targeting module, a customized DNA linker containing both 5mC and 6mA
modifications, and the RAMA-CAR. Our results establish proof-of-concept for regulation
of a CAR-T-tumor synapse using customizable targeting modules and modified DNA
linkers.

4

Introduction
Recent reprogramming of T cells or engineered T cell receptors (TCRs) to
express chimeric antigen receptors (CARs) that target specific antigens has resulted in
a major breakthrough in immunotherapy
1-3
(Figure 1A). In one early clinical trial, 27 of
30 (90%) patients experienced a complete remission of Acute Lymphoblastic Leukemia
(ALL), which paved the way for a larger clinical trial and the development of the first
FDA approved CAR-T cell therapy (Kymriah)
4
. Unfortunately, despite successful clinical
outcomes, side effects have also been reported to be associated with CAR-T therapy,
which in some cases can be fatal
5-8
. In fact, the death of several patients prompted the
termination of a trial
9
.
Unregulated CAR T cell activation in patients prompted the idea of designing
receptors that address the issue of controllability and specificity. One example includes
splitting the intracellular CAR components that re-assemble and activates specifically in
the presence of a small molecule
10
(Figure 1B). In a different design, the recognition of
an antigen by a synthetic Notch receptor induces the expression of a second receptor
(CAR) that recognizes a different antigen, allowing for a dual-receptor activated only in
the presence of two antigens
11
(Figure 1D). Another example includes the use of a
SUPRA CAR that splits the extracellular receptor into two parts: one segment attached
to the intracellular signaling domains and a distinguishable scFv that targets tumor
antigen
12
(Figure 1C). In contrast to CAR systems, a novel design for T Cell Receptor
(TCR) has also been synthesized. A scFv targeting a tumor antigen was added to the
extracellular co-receptor domain of T cells, hence bypassing the necessity of T cell
activation through MHC bound antigen and reducing the toxicity of T cell activation
13
.
5

Although these systems have enabled the improvement of CAR T therapy, the designs
can be static and limit the ability to change the environment of CAR-T-tumor synapse.
Therefore, to increase the efficiency of CAR T therapy, our engineered CAR design
adds a new dimension of flexibility in addition to specificity and controllability.
Here, we design an engineered CAR-T-tumor synapse composed of a synthetic
fusion protein targeting a tumor surface antigen and a DNA linker; and a customized
CAR consisting of a DNA base modification sensor domain targeting the same DNA
customized linker. Hence, the interaction of a synthetic fusion protein with a customized
DNA linker bridges the tumor cell with a CAR T cell. This system creates an ON-switch
for CAR T cell activation by regulating the presence of customized DNA linker.
Moreover, the use of a customized DNA linker allows for a more vigorous immune
response by enabling the ability to recruit additional immune cells to the CAR-T-tumor
synapse microenvironment. For example, DNA modifications containing natural killer
(NK) cell ligands allows the recruitment of NK cells to the CAR-T-tumor synapse. We
show that our design permits a flexible, controllable, and reversible activation of CAR T
cells.
Materials and Methods
Expression Vectors
For bacterial expression of Myc/His-tagged LaG_16-MBD protein, the codon was cloned
into NheI/HindIII sites of pcDNA3.1/Hygro/LacZ vector.

For bacterial expression of RAMA-CAR-4-1BB-CD3ζ, the codon was synthesized
(Genewiz) and cloned into BamHI/XbaI sites of pcDNA3.1/Hygro/LacZ vector.
6

Protein Extraction and Immunoblotting
To purify LaG_16-MBD, HEK293T cells were transfected with Myc/His-LaG16-MBD
plasmid and allowed to proliferate for 72h. The media was extracted and centrifuged at
4,800 rpm for 5 min. An appropriate amount of PIC 300x was added to the media,
followed by 1M PIT 1000x. The media was then incubated after adding Ni-resin at 4°C
for 1h 30min. The media was centrifuged at 4,800rpm for 3min followed by the disposal
of supernatant. The Ni-resin with LaG16-MBD was then washed first with 1x PBS then
twice with a wash buffer (1xPBS, 20mM imidazole, 100mM NaCl, pH 8.0). LaG16-MBD
protein was then eluted in two increments using an elution buffer (1xPBS, 20mM
imidazole, 350mM NaCl, pH 8.0). To isolate LaG_16-MBD in cells, the cells were lysed
and centrifuged at 4,000rpm for 2min. The pellet was then resuspended with 1mL PBS
and centrifuged at 4,000rpm for 2min. Finally, the sample underwent multiple rounds of
sonication.

To confirm expression of RAMA-CAR, HEK293T cells were transfected with HA-tagged
RAMA-CAR plasmid. After 72h, the cells were centrifuged at 4,000rpm for 2min. The
supernatant was then disposed and 1mL of PBS was added to the pellet. The sample
was then centrifuged at 4,000rpm for 2min. The supernatant was disposed and 400μL
of RIPA buffer was resuspended with the pellet. Then, the sample was incubated on a
shaker for 45min at 4°C. After centrifuging at 13,000rpm for 1min, 300μL of sample was
extracted and used for SDS-PAGE.

7

For immunoblot analysis, the extracted LaG_16-MBD or RAMA-CAR was mixed with 6X
SDS. After the sample finished running on a gel, it was transferred to a nitrocellulose
membrane for 2hr. After washing the membrane with ponceau, 5% milk was added to
the membrane and left on a shaker for 20min. The membrane was then incubated
overnight at 4°C with an anti-myc or anti-HA antibody (primary antibody). After washing
the membrane with TBS-T 3x, the secondary antibody was added and incubated on a
shaker for 40min. The membrane was then washed 3x with TBS-T and SuperSignal
West Pico PLUS (Thermo Fisher) was used to detect the immunoblot bands.

DNA Synthesis
To synthesize 5mC and 6mA base modified DNA, oligonucleotides containing 5mC and
6mA base modifications were synthesized with 15bp overlaps. Next, the fragments were
added to NEBuilder HiFi DNA Assembly Master Mix and incubated at 50°C for 20min.

T-cell Activation Assay
To test the activation of T cells, Jurkat T cells were transfected with RAMA-
CAR+pGL3_NFAT plasmid and empty vector (control)+pGL3_NFAT plasmid.
Approximately 72h after transfection, the Jurkat T cells were incubated with HEK293T
cells transfected with and without sGFP. Jurkat T cells and HEK293T cells were then
incubated in the presence and absence of DNA linker and LaG_16-MBD depending on
the experimental condition (Figure 7B). After 24h, the protocol for Pierce Firefly
Luciferase Glow Assay Kit (Thermo Scientific) was followed to determine the level of T
cell activation.
8

Fluorescence Assay
To measure the recruitment of DNA to sGFP antigens, HEK293T cells were transfected
with sGFP plasmid and oligonucleotides were labeled with 5’FAM. Modified and non-
modified oligonucleotides were then incubated with and without sGFP expressing
HEK293T cells at different experimental conditions in a 96-well plate. The fluorescence
level was measured using BioTek Synergy H1.

Cell Culture
HEK293T and Jurkat T cells were cultured with Dulbecco’s Modified Eagle’s Medium –
high glucose (Sigma).

Cell Transfections
For 10cm petri dishes and 12-well plates, the cells were transfected with 8μg and 1μg
plasmid DNA, respectively, using Bio-T (Bioland Scientific) according to the
manufacturer’s protocol.
Results
Design of the CAR-T-tumor synapse
Our CAR T cell system consists of three components (Figure 2): a customized
DNA linker containing 5mC and 6mA DNA base modifications, a synthetic fusion protein
consisting of a scFv targeting tumor surface antigen (LaG_16) connected to a MBD
through a linker; and a novel CAR consisting of a RAMA domain linked to intracellular
T-cell CD28/4-1BB costimulatory domains and a CD3ζ signaling domain that interacts
9

with the same DNA linker. Thus, the customized DNA linker interacts with two sensor
domain proteins: the MBD that interacts with 5mC and a newly identified RAMA sensor
domain that targets 6mA. This design offers several advantages for CAR T therapy, for
example it allows the 1) changing of LaG_16 protein to target a different tumor surface
antigen, 2) addition of DNA base modifications enabling the recruitment of further
immune cells, and 3) controllability of T cell activation by manipulating the presence of
the customized DNA linker.
10

C
A
B
D
Figure 1. Structural overview of conventional TCR and CAR with modern approaches of re-designing
receptors to provide regulated control over T cell activation
(A) TCR versus CAR receptor design over three generations. First generation CAR includes a scFv linked to an
intracellular CD3ζ domain. Second and third generation CAR includes the addition of costimulatory domains:
CD28 or 4-1BB. (June et al., 2018)
(B) Small molecule-gated CAR. Segregated intracellular receptor domains unite and activate T cell exclusively
in the presence of the small molecule. (Wu et al., 2015)
(C) Split, universal, and programmable (SUPRA) CAR system. An AZip connected to a scFv attaches to BZip
connected to intracellular signaling domains. T cell gets activated upon interaction of AZip with BZip. (Cho et
al, 2018)
(D) Dual receptor CAR. SynNotch receptor activates TF upon interacting with antigen A inducing the
expression of CAR on cell. T cell then becomes activated upon interacting with antigen B. (Roybal et al., 2016)

11

--
6mA
6mA
5mC
6mA
5mC
5mC
LaG_16
--
--
Tumor Cell
sGFP
--
---
----
MBD RAMA
T-Cell
Activation
CD28/4-1BB
CD3ζ
CAR-T Cell
Figure 2. Strategy for engineered control of CAR-T-tumor synapse
A targeting module (LaG_16-MBD) containing a single chain variable fragment (scFv) binds to a test antigen
(GFP) on the tumor cell and to an oligonucleotide containing 5-methylcytosine (5mC) via the MBD domain.
N
6
-methyladenine (6mA) modifications on the same DNA linker bind to the RAMA domain of the RAMA-CAR
expressed on a T cell allowing for a regulated control of T cell activation.

12

Structure and Immunoblot Analysis of RAMA-CAR
Our RAMA-CAR structure consists of a 6mA sensor domain protein attached to
intracellular T cell costimulatory domains (Figure 3A). Therefore, the activation of T cell
occurs solely in the presence of 6mA DNA base modifications. To confirm the
expression of RAMA-CAR in Jurkat T cells, we first transfected HEK293T cells with
RAMA-CAR plasmid tagged with an HA sequence (YPYDVPDYA). After the cells were
grown for 72h, the cells were lysed and run on SDS-PAGE. An anti-HA antibody was
used to target the RAMA-CAR and visualize the protein band on the membrane. The
original protein weight is 42.48kDa, however due to post-translational cleavage, the
weight of the protein observed is ~38kDa (Figure 3B).

13

Figure 3. Structure and immunoblot analysis of RAMA-CAR expressed in HEK293T cells
(A) Structure of RAMA-CAR expressed in T cells.
(B) To confirm expression of RAMA-CAR, HEK293T cells were transfected with HA-tagged RAMA-
CAR plasmid. 72h after transfection, the cells were lysed and anti-HA antibodies were used for
immunoblot analysis of RAMA-CAR expression.
B
50 kDa
37 kDa
25 kDa
Empty Vector
RAMA-CAR
CD28/4-1BB
CD3ζ
Transmembrane
domain
RAMA
A
14

Immunoblot analysis of LaG_16-MBD
To confirm the synthesis and presence of LaG_16-MBD protein (Figure 4A), we
performed an immunoblot analysis. We transfected HEK293T cells with a LaG_16-MBD
plasmid tagged with His and allowed the cells to grow for approximately 72h. Using the
His-tag on LaG_16-MBD, the protein was eluted through nickel affinity chromatography.
In addition, the cells were also lysed for confirmation of protein secretion. Finally, an
anti-myc antibody bound to the myc-tag on LaG_16-MBD was used for visualizing the
presence of the protein at ~44kDa (Figure 4B).
15

75 kDa
50 kDa
Cell lysate
Elution #1
Elution #2
B
LaG_16 MBD
Linker Myc-His
A
Figure 4. Domain diagram and immunoblot analysis of purified LaG16-MBD protein extracted
from HEK293T cells
(A) Domain structure of LaG_16-MBD protein.
(B) To purify LaG_16-MBD, HEK293T cells were transfected with Myc-LaG_16-MBD plasmid. 72h
after transfection, soluble LaG_16-MBD was recovered using nickel affinity chromatography. Anti-
myc antibodies were used for immunoblot analysis of LaG_16-MBD in cell lysates and protein eluted
from the nickel resin.
16

Recruitment of DNA to target cells
To confirm the recruitment of customized DNA oligonucleotides to tumor cells,
we next measured the level of DNA associated with surface GFP (sGFP) present on
tumor cells. Oligonucleotides were labeled with 5’-FAM to quantify the level of
fluorescence upon interaction with target sGFP. In addition, HEK293T cells were
transfected with and without sGFP plasmid and incubated at different conditions (Figure
5B): no treatment, DNA not modified with 5mC and 6mA, 5mC modified DNA,
unmodified DNA with LaG_16, and 5mC modified DNA with LaG_16. Since LaG_16-
MBD links the tumor cell with DNA, we expect to observe the highest amount of
fluorescence exclusively in the presence of LaG_16-MBD and modified DNA. The
results revealed that relative to control, a significant amount of DNA was bound to sGFP
solely in the presence of LaG_16 and 5mC modified DNA (Figure 5B). Thus, these
results confirm the capability of LaG_16-MBD to recruit 5mC modified DNA to target
cells.
17

0
100
200
300
400
500
600
700
800
Background Unmodified DNA 5mC DNA Unmodified DNA
+ Lag_16
5mC + LaG_16
Cell Surface Fluorescence
Recruitment of DNA to Target Cells
ΔsGFP Cells sGFP Cells
293T
Cells
293T-
GFP
Cells
B
A
Figure 5. Measuring the recruitment of DNA to target cells
(A) HEK293T cells were transfected with surface-GFP (sGFP) plasmid then incubated with the
indicated combinations of LaG_16-MBD and fluorescein (5’FAM) labeled DNA oligonucleotide.
Fluorescence of DNA stably bound to the cell surface was measured to quantify the interaction of
each DNA type with sGFP target antigen.
(B) Chart specifying the components of each experimental setup.
18

Combining 5mC and 6mA oligonucleotides
To obtain the customized 5mC and 6mA DNA linker, we combined distinct 5mC
and 6mA oligonucleotides. After synthesizing 15bp overlaps in 5mC and 6mA
oligonucleotides, the DNA was incubated with a DNA Assembly Master Mix at 50°C for
20min (Figure 6).

5mC
6mA
5mC
6mA
15 bp
https://www.neb.com/-/media/catalog/datacards-or-manuals/manuale2621.pdf
Figure 6. Strategy for combining 5mC and 6mA oligonucleotides
5mC and 6mA oligonucleotides were designed with 15bp overlaps, then added to NEBuilder HiFi
DNA Assembly Master Mix and incubated at 50°C for 20min.
19

CAR-T activation levels
Finally, we tested the activation of Jurkat T cells at different experimental settings
(Figure 7B) to confirm the functionality of our CAR T design. Jurkat T cells were
transfected with RAMA-CAR+pGL3_NFAT or empty vector+pGL3_NFAT vectors. After
transfection, the Jurkat T cells were added to HEK293T cells transfected with and
without sGFP plasmid. Next, depending on the experimental setup, the cells were
incubated in the presence and absence of DNA linker and LaG_16-MBD. To measure
Jurkat T cell activation, transcriptional activation of the pGL3_NFAT reporter was
measured using luminescence assay (Figure 7A). The formation of a CAR-T-tumor
synapse is expected to occur exclusively in the presence of Jurkat T cells, HEK293T-
sGFP cells, DNA linker, and LaG_16-MBD proteins, which is indicated by “complete” in
results (Figure 7A). Moreover, we observed the highest level of T cell activation
targeting tumor cells in the presence of DNA linkers (Figure 7A).

20

A
Figure 7. CAR-T activation levels at distinct experimental setups
(A) Jurkat T cells were transfected with RAMA-CAR+pGL3_NFAT or empty vector+pGL3_NFAT
plasmid. 72h after transfection Jurkat T cells were added to wells containing HEK293T cells
transfected with empty vector or sGFP plasmid, in the absence or presence of the indicated DNA
linkers and LaG_16-MBD proteins. Transcriptional activation of the pGL3-NFAT reporter was
measured by luminescence assay.
(B) Chart specifying the components for each trial.
B
-2
0
2
4
6
8
10
Complete
(+DNA)
Complete
(++DNA)
ΔRAMA-CAR ΔDNA Linker ΔsGFP ΔDNA Linker
& LaG16
293T-GFP
Cells
Relative CAR T-Cell Activity
T Cell Activity in Engineered CAR-T-Tumor Synapse
21

Discussion

The ability to regulate the microenvironment of a CAR-T-tumor synapse allows
tight regulation over T cell activation in addition to providing a robust and specific
immune response against tumor cells. Here, we show that integrating DNA sensor
proteins to modify components of the CAR T-tumor synapse potentially increases the
specificity, controllability, and flexibility of CAR T therapy. A synthetic fusion protein,
LaG_16-MBD, binds to a tumor cell and a customized DNA linker; while a novel CAR,
RAMA-CAR, interacts with the same customized DNA, (RAMA-CAR of T
cellàDNAàLaG_16-MBDàtumor cell) generating an anti-tumor immune response to
kill tumor cells. Our approach of using a customized DNA linker as the limiting factor to
activate CAR T cells provides a reversible approach for CAR T cell activation by
regulating the presence of DNA. Moreover, the scFv connected to MBD, LaG_16, can
be swapped with a different antibody to target additional tumor specific antigens,
increasing the compatibility of our system in different settings.
Moreover, our novel design of using a customized DNA linker and an isolated
scFv provides a new platform in CAR T therapy by facilitating the recruitment of
additional cellular components to tumor cells and increasing the array of target antigens.
For example, we can recruit CAR T cells to more than one type of tumor cell by adding
additional scFv, linked to MBD, targeting different tumor specific antigens. Although
targeting multiple antigens might appear to increase cytotoxicity, our design of CAR T
therapy prevents T cell activation against non-tumorigenic antigens by regulating the
presence of DNA. In addition to targeting multiple antigens, we can also add an
22

additional scFv linked to MBD that targets NK cell ligands to recruit NK cells to the CAR-
T-tumor synapse, providing a more vigorous immune response against tumor cells.
Future studies include swapping the LaG_16 targeting module with scFv specific
for tumor antigens and observing the effect in vivo. For this study, we can change the
LaG_16 scFv to target CD19 and test the efficacy of our system on an Acute
Lymphoblastic Leukemia (ALL) tumor model in mice. Moreover, the effect of our
engineered CAR design can also be compared to conventional CAR in vivo to
determine the safety and efficacy for potential therapeutic use of our CAR design.
Because nucleases will rapidly breakdown DNA in vivo, this allows us to have tight
regulation of CAR T cell activation by having more control of DNA present in peripheral
blood. Moreover, as oligonucleotides have been approved for therapeutic treatment by
the FDA, we suspect a positive outcome for T cell activation using DNA. Nonetheless, a
dose titration study will be necessary to prove our hypothesis.

23

References
[1] Porter DL, Levine BL, Kalos M, Bagg A, June CH: Chimeric antigen receptor-
modified T cells in chronic lymphoid leukemia. N Engl J Med 2011, 365:725-33.
[2] Urba WJ, Longo DL: Redirecting T cells. N Engl J Med 2011, 365:754-7.
[3] Maude SL, Teachey DT, Porter DL, Grupp SA: CD19-targeted chimeric antigen
receptor T-cell therapy for acute lymphoblastic leukemia. Blood 2015, 125:4017-23.
[4] Maude SL, Frey N, Shaw PA, Aplenc R, Barrett DM, Bunin NJ, Chew A, Gonzalez
VE, Zheng Z, Lacey SF, Mahnke YD, Melenhorst JJ, Rheingold SR, Shen A, Teachey
DT, Levine BL, June CH, Porter DL, Grupp SA: Chimeric antigen receptor T cells for
sustained remissions in leukemia. N Engl J Med 2014, 371:1507-17.
[5] Gust J, Hay KA, Hanafi LA, Li D, Myerson D, Gonzalez-Cuyar LF, Yeung C, Liles
WC, Wurfel M, Lopez JA, Chen J, Chung D, Harju-Baker S, Ozpolat T, Fink KR, Riddell
SR, Maloney DG, Turtle CJ: Endothelial Activation and Blood-Brain Barrier Disruption in
Neurotoxicity after Adoptive Immunotherapy with CD19 CAR-T Cells. Cancer Discov
2017, 7:1404-19.
[6] Davila ML, Riviere I, Wang X, Bartido S, Park J, Curran K, Chung SS, Stefanski J,
Borquez-Ojeda O, Olszewska M, Qu J, Wasielewska T, He Q, Fink M, Shinglot H,
Youssif M, Satter M, Wang Y, Hosey J, Quintanilla H, Halton E, Bernal Y, Bouhassira
DC, Arcila ME, Gonen M, Roboz GJ, Maslak P, Douer D, Frattini MG, Giralt S, Sadelain
M, Brentjens R: Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell
acute lymphoblastic leukemia. Sci Transl Med 2014, 6:224ra25.
[7] Lee DW, Gardner R, Porter DL, Louis CU, Ahmed N, Jensen M, Grupp SA, Mackall
CL: Current concepts in the diagnosis and management of cytokine release syndrome.
Blood 2014, 124:188-95.
[8] Teachey DT, Lacey SF, Shaw PA, Melenhorst JJ, Maude SL, Frey N, Pequignot E,
Gonzalez VE, Chen F, Finklestein J, Barrett DM, Weiss SL, Fitzgerald JC, Berg RA,
Aplenc R, Callahan C, Rheingold SR, Zheng Z, Rose-John S, White JC, Nazimuddin F,
Wertheim G, Levine BL, June CH, Porter DL, Grupp SA: Identification of Predictive
Biomarkers for Cytokine Release Syndrome after Chimeric Antigen Receptor T-cell
Therapy for Acute Lymphoblastic Leukemia. Cancer Discov 2016, 6:664-79.
[9] June CH, O'Connor RS, Kawalekar OU, Ghassemi S, Milone MC: CAR T cell
immunotherapy for human cancer. Science 2018, 359:1361-5.
[10] Wu CY, Roybal KT, Puchner EM, Onuffer J, Lim WA: Remote control of therapeutic
T cells through a small molecule-gated chimeric receptor. Science 2015, 350:aab4077.
[11] Roybal KT, Rupp LJ, Morsut L, Walker WJ, McNally KA, Park JS, Lim WA:
Precision Tumor Recognition by T Cells With Combinatorial Antigen-Sensing Circuits.
Cell 2016, 164:770-9.
[12] Cho JH, Collins JJ, Wong WW: Universal Chimeric Antigen Receptors for
Multiplexed and Logical Control of T Cell Responses. Cell 2018, 173:1426-38 e11.
[13] Helsen CW, Hammill JA, Lau VWC, Mwawasi KA, Afsahi A, Bezverbnaya K,
Newhook L, Hayes DL, Aarts C, Bojovic B, Denisova GF, Kwiecien JM, Brain I,
Derocher H, Milne K, Nelson BH, Bramson JL: The chimeric TAC receptor co-opts the T
cell receptor yielding robust anti-tumor activity without toxicity. Nat Commun 2018,
9:3049.

Abstract (if available)

Abstract Chimeric Antigen Receptor (CAR) T-cell therapy represents a class of powerful cell-based therapeutics approved for use in patients with advanced lymphomas. Despite the clinical success of CAR-T, patients can experience a severe, uncontrolled and potentially life-threatening activation of engrafted CAR-T. One approach to overcome this effect is to engineer CAR containing an ON-switch, enabling tight regulation of activation. Here, we leverage recent advances in our understanding of DNA base modifications and sensor proteins to design and engineer CAR-T cells that establish a tumor-T cell synapse specifically in the presence of customized tumor antigen-targeting modules and modified DNA linkers. We establish a minimal system comprised of three components: a customized DNA linker containing 5-methylcytosine (5mC) and N⁶-methyladenine (6mA) DNA base modifications, a synthetic fusion of a single-chain variable fragment (scFv) targeting a cell surface antigen (LaG_16) with a methyl-binding domain (MBD) that binds to the 5mC modification

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Asset Metadata

Creator Jalilian, Shayian Nicholas (author)

Core Title Engineered control of the CAR-T-tumor synapse using customized DNA linkers

School Keck School of Medicine

Degree Master of Science

Degree Program Experimental and Molecular Pathology

Publication Date 04/23/2019

Defense Date 03/19/2019

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

Tag adoptive cell therapy (ACT),cancer immunotherapy,CAR-T regulation,CAR-T side effects,CAR-T therapy,chimeric antigen receptor (CAR) T cell,OAI-PMH Harvest

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Feldman, Douglas (committee chair)

Creator Email jalilian@usc.edu,snjalilian@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-140637

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