Redirection of genetically engineered CAR-T cells using bifunctional small molecules

Article abstract of DOI:10.1021/jacs.5b00106

Chimeric antigen receptor (CAR)-engineered T cells (CAR-Ts) provide a potent antitumor response and have become a promising treatment option for cancer. However, despite their efficacy, CAR-T cells are associated with significant safety challenges related to the inability to control their activation and expansion and terminate their response. Herein, we demonstrate that a bifunctional small molecule switch consisting of folate conjugated to fluorescein isothiocyanate (folate-FITC) can redirect and regulate FITC-specific CAR-T cell activity toward folate receptor (FR)-overexpressing tumor cells. This system was shown to be highly cytotoxic to FR-positive cells with no activity against FR-negative cells, demonstrating the specificity of redirection by folate-FITC. Anti-FITC-CAR-T cell activation and proliferation was strictly dependent on the presence of both folate-FITC and FR-positive cells and was dose titratable with folate-FITC switch. This novel treatment paradigm may ultimately lead to increased safety for CAR-T cell immunotherapy.

Full text of DOI:10.1021/jacs.5b00106

Communication
pubs.acs.org/JACS
Redirection of Genetically Engineered CAR‑T Cells Using Bifunctional
Small Molecules
Min Soo Kim,† Jennifer S. Y. Ma, Hwayoung Yun,
,§
†,§
‡,§,∥
Yu Cao, Ji Young Kim, Victor Chi,
‡
†
†
†
†
†
†
†
Danling Wang, Ashley Woods, Lance Sherwood, Dawna Caballero, Jose Gonzalez,
,
†,‡
,†
,†
Peter G. Schultz,* Travis S. Young,* and Chan Hyuk Kim*
†
California Institute for Biomedical Research, 11119 North Torrey Pines Road, Suite 100, La Jolla, California 92037, United States
Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines
‡
Road, La Jolla, California 92037, United States
*
S Supporting Information
tumors, where tumor associated antigens (TAAs) are not as
restricted as hematological markers and may be found on normal
tissues.
ABSTRACT: Chimeric antigen receptor (CAR)-engi-
neered T cells (CAR-Ts) provide a potent antitumor
response and have become a promising treatment option
for cancer. However, despite their efficacy, CAR-T cells are
associated with significant safety challenges related to the
inability to control their activation and expansion and
terminate their response. Herein, we demonstrate that a
bifunctional small molecule “switch” consisting of folate
conjugated to fluorescein isothiocyanate (folate-FITC) can
redirect and regulate FITC-specific CAR-T cell activity
toward folate receptor (FR)-overexpressing tumor cells.
This system was shown to be highly cytotoxic to FR-
positive cells with no activity against FR-negative cells,
demonstrating the specificity of redirection by folate-
FITC. Anti-FITC-CAR-T cell activation and proliferation
was strictly dependent on the presence of both folate-
FITC and FR-positive cells and was dose titratable with
folate-FITC switch. This novel treatment paradigm may
ultimately lead to increased safety for CAR-T cell
immunotherapy.
7
−10
One approach to control the activity of CAR-T cells is to use
an intermediate bifunctional molecule as a “switch” to redirect
the specificity of CAR-T cells. Unlike conventional CAR-T cells,
the extracellular domain of such a CAR does not directly bind to
any TAA; thus, these CAR-T cells are essentially inert in the
presence of normal and cancerous cells. However, in the
presence of a bifunctional molecule that is specific for both the
TAA and the CAR, CAR-T cells can be redirected specifically to
target cancer cells. Moreover, the activity of these CAR-T cells
can be titrated based on the dosage of switch molecule. The
feasibility of this concept has been previously demonstrated in in
vitro and in mouse models using FITC- or biotin-tagged
11−15
antibodies.
More recently, Campana and co-workers have
employed a similar approach using CAR-T cells that binds to the
Fc portion of a full-length antibody, which enables the targeting
15
of cancer cells in an antibody-dependent manner. Herein, we
show that the specificity of CAR-T cells can be redirected using a
bifunctional small molecule. Small molecules have excellent
tumor penetration and can be readily modified by medicinal
chemistry to produce candidates with optimized biophysical and
ecently, immunotherapies have emerged as a powerful
16,17
pharmacokinetic properties.
R
alternative to conventional chemotherapy for the treatment
As a proof-of-concept, we designed a heterobifunctional folate-
FITC conjugate (Figure 1A) that can simultaneously bind to
folate receptor (FR) overexpressed on malignant cells and to a
CAR-T cell with engineered specificity for FITC. The alpha
isoform of FR is expressed on nearly 50% of cancers with a
particularly high incidence in breast, lung, uterus, and ovarian
cancers, whereas very low levels are detectable on normal
1
of cancer. One exciting approach is the use of engineered
2
chimeric antigen receptor (CAR) T cells (CAR-Ts). In this
approach, T cells are genetically modified to express a chimeric
receptor that consists of an extracellular binding domain (e.g., a
single-chain antibody variable region, scFv) and a combination of
intracellular signaling domains (e.g., CD3ζ and CD137). After
transfer back into patients, these cells robustly proliferate to
mount a potent response against malignant cells. Indeed, CAR-
Ts engineered to target B-cell antigen CD19 (CART-19) have
demonstrated unprecedented responses in recent clinical trials in3
the treatment of relapsed or refractory leukemia and lymphoma.
Despite these successes, there are significant safety challenges
associated with the use of CAR-T cells. For example, robust T
1
8−20
tissues.
In addition, the endogenous ligand, folate, has a high
21
affinity (K ≈ 1 nM) to FR, making it an excellent cancer-
d
targeting small molecule for use as a switch. The use of the
synthetic molecule FITC as a ligand for the CAR reduces the
potential for off-target cross-reactivity of CAR and eliminates
competition by endogenous ligands.
A folate-FITC conjugate has been previously reported by
2
2
Endocyte (clinically as EC-17) for use in image-guided surgery
and hapten conjugate-targeted immunotherapy
cell proliferation has caused cytokine release syndrome and CD-
23−25
4
in FR-
1
9 targeting has resulted in long-term B-cell aplasia. These
adverse effects are due to the inability to regulate persistent CAR-
2
,5,6
T activity.
More importantly, these safety issues pose a
Received: January 5, 2015
Published: February 18, 2015
greater problem when this approach is used to target solid
©
2015 American Chemical Society
2832
DOI: 10.1021/jacs.5b00106
J. Am. Chem. Soc. 2015, 137, 2832−2835

Journal of the American Chemical Society
Communication
effector to target cell ratio of 10 to 1 in folate deficient media,
folate-FITC demonstrated highly potent (EC of ∼9 pM, Figure
50
2
A) redirection of anti-FITC CAR-T cells to KB cells over 24 h.
Figure 1. (A) Structure of folate-FITC. (B) Assessment of folate-FITC
binding to KB (FR+) and A549 (FR-) cell lines by flow cytometry. The
cells were labeled with (red line) or without (black line) 50 nM folate-
FITC and analyzed by flow cytometry in the FITC channel. (C) Anti-
FITC CAR lentiviral construct. (D) Anti-FITC CAR expression on
human T cells was assessed by direct staining with antimouse IgG
F(ab′)2 antibody (Jackson Immuno Research) or a FITC-conjugated
isotype antibody (eBiosciences). Expression was also detected by
primary staining with folate-FITC and secondary staining with an
antifolate antibody (Novus Biologicals) conjugated with Alexa Fluor
6
47 dye. Results shown represent findings observed in seven (B) and
three (D) experiments.
overexpressing cancer patients. EC-17 has been thoroughly
characterized in preclinical and clinical studies. In addition, a
recently published crystal structure of human FR in complex with
folate suggests that the FITC conjugation site in folate-FITC is
solvent-exposed and will not likely interfere with FITC binding
Figure 2. (A) Twenty-four hours cytotoxicity assay of anti-FITC CAR-
T cells targeting KB (FR+) and A549 (FR-) cancer cells cultured with
serial dilutions of folate-FITC at an effector to target cell ratio of 10:1 (1
× 10 to 1 × 10 cells per well). Cytolytic activity was determined by
measuring the amount of LDH released into cultured media using
CytoTox 96 Nonradioactive cytotoxicity assay kit (Promega). Each
concentration was carried out in triplicate and error bars represent SEM.
5
4
2
1
to CAR-T cells. To synthesize the folate-FITC switch, we
25
followed established synthetic schemes (Supplementary Figure
) and confirmed the purity (>95%) using high-resolution mass
1
spectrometry and NMR. The selectivity of folate-FITC was
(B) Quantification of cytokine (IFN-γ and IL-2) levels in cultures
verified by flow cytometry using FR-positive cervical carcinoma
described in panel A by ELISA. Error bars represent SEM derived from
triplicate samples. (C) Microscopic images of cytotoxicity assay
described in panel A obtained on Zeiss Axio Observer A1.
(
1
KB) and FR-negative lung carcinoma (A549) cell lines (Figure
B).
To create a FITC-specific CAR, the anti-FITC scFv clone
2
6
4
M5.3 was subcloned into a lentiviral vector containing a
These results were reproducible across multiple T cell donors
with similar efficacies (average EC50 = 9.7 ± 3.2 pM,
Supplementary Figure 2). Folate-FITC failed to induce lysis of
KB cells in the presence of nontransduced T cells. Furthermore,
no significant cytotoxic activity was observed with A549 cells at
concentrations of folate-FITC up to 10 nM, demonstrating the
high selectivity of folate-FITC and anti-FITC CAR-T cells. The
amount of IFN-γ and IL-2 cytokines released during the 24 h
assay positively correlated with the concentration of FITC-folate
and was dependent on the presence of FR-positive cells (Figure
2B). Lastly, selective formation of aggregates, indicative of cross-
linking between T cells and target cells, was only visible in
cocultures of anti-FITC CAR and KB cells with folate-FTIC at
100 pM, but not in any of the controls (Figure 2C). Overall, our
findings demonstrate that folate-FITC, as a small molecule
“switch”, can specifically and dose-dependently redirect anti-
FITC CAR-T cells to target FR-positive cells.
second generation CAR (from C-terminus: CD3ζ, 4-1BB, CD8
27
transmembrane domain, and anti-FITC scFv; Figure 1C).
Lentiviral particles were generated in HEK293FT cells and used
directly to transduce human T cells (Supporting Information).
Transduction efficiencies of 40−50% were measured by flow
cytometry by staining for scFv surface expression using
antimouse IgG antibody (Figure 1D and Supplementary Figure
2
). To confirm that the scFv was properly folded on the cell
surface and retained its affinity for FITC, flow cytometry was
performed using a commercially available FITC-conjugate
antibody. We observed binding of FITC-conjugated antibody
(
44.4% positive, Figure 1D) that was similar to the transduction
efficiency measured by the antimouse IgG antibody described
above. In addition, the binding of an antifolate antibody to folate-
FITC labeled CAR-T cells (52.1% positive, Figure 1D) indicates
that the folate group of the folate-FITC is solvent-exposed and is
accessible for the simultaneous binding to FR-positive cells.
Next, we evaluated the ability of folate-FITC to redirect anti-
We next confirmed the specificity of anti-FITC CAR-T cell
activation by monitoring expression of cell surface activation
markers, CD25 and CD69, on CAR-T cells in coculture with KB
cells. After a 24 h incubation in the presence (2.5 nM) or absence
6
FITC CAR-T cells to FR-positive KB cell lines (∼1.5 × 10 FR/
cell), using FR-negative A549 cell lines as a control. At an
2
8
2
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DOI: 10.1021/jacs.5b00106
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Journal of the American Chemical Society
Communication
of folate-FITC, the anti-FITC CAR-T cells were labeled with
anti-CD25 and anti-CD69 antibodies and analyzed by flow
cytometry. As anticipated, folate-FITC increased CD25 and
CD69 expression levels on anti-FITC CAR-T cells compared to
nontransduced T cells in the presence of FR-positive cells
(
Figure 3A and Supplementary Figure 3). In addition to T cell
Figure 4. (A) Twenty-four hours of cytotoxicity assay of anti-FITC
5
4
CAR-T cells (1 × 10 ) targeting KB cells (1 × 10 ) in the presence of
200 pM folate-FITC and varied concentrations of free fluorescein (0−
1000-fold of folate-FITC). Each data point represents a mean of
triplicates, and error bars represent SEM. (B) Twenty-four hours of
5
cytotoxicity assay of anti-FITC CAR-T cells (1 × 10 ) targeting KB (1 ×
4
1
0 ) cells in the presence of different concentrations of folate-FITC in
folate-free media supplemented with the indicated concentrations of
free folate. Each data point represents a mean of triplicate samples, and
error bars represent SEM.
decrease in potency likely results from multivalent binding of
folate-FITC to anti-FITC CAR-T cells in the presence of KB
cells, which provides sufficient avidity to overcome competition
from physiological levels of folate. At supra-physiological
concentrations of folate (2 uM), the EC50 of folate-FITC is
reduced 600-fold, further confirming that the activity of anti-
FITC CAR-T is mediated by the binding of folate-FITC. Further
evaluation by flow cytometry revealed that excess folate and
fluorescein inhibited folate-FITC binding to KB and ant-FITC
CAR-T cells, respectively, in a concentration-dependent manner
Figure 3. (A) Folate-FITC dependent activation of anti-FITC CAR-T
5
cells. Anti-FITC CAR-T cells (1 × 10 cells) were cocultured with KB
5
cells (1 × 10 cells) for 24 h in the presence (2.5 nM) or absence of
folate-FITC, and subsequently stained with APC-conjugated antimouse
IgG F(ab′)2 antibody followed by PE-conjugated anti-CD25 and
PerCP-Cy5.5-conjugated anti-CD69 antibodies (eBioscience). (B) T
cell proliferation assay. Folate-FITC (10 nM) and beads (Dynabeads,
Invitrogen) conjugated with the indicated antibodies were added to anti-
5
FITC CAR-T or nontransduced T cells (1 × 10 cells) and cultured for
72 h. T cell proliferation was measured using the CellTiter 96 AQueous
One Solution Assay kit (Promega). Results are shown as an average
absorbance at 490 nm. Error bars represent SEM derived from triplicate
samples. *P ≤ 0.05 was determined by 2-paired Student t test. Results
represent findings from three independent experiments.
(
Supplementary Figure 4), suggesting that the attenuated activity
of anti-FITC CAR-T shown in Figure 4 is a result of reduced
binding of folate-FITC.
In conclusion, we have demonstrated a novel therapeutic
approach that combines the dose titratability of small molecules
with the potency of CAR-T cell therapy. Future studies will
evaluate the in vivo efficacy of folate-FITC-controlled anti-FITC
CAR-T cells in FR-positive xenograft tumor models. This
switchable platform may overcome some of the safety hurdles
associated with current CAR-T cell approaches and provide a
promising immunotherapeutic for FR-positive cancer patients.
activation, we also assessed the proliferative response of the anti-
FITC CAR-T cells in the presence of folate-FITC using
antifolate antibody-conjugated beads to mimic FR-overexpress-
ing cells. Anti-FITC CAR-T cells cultured in the presence of
folate-FITC (10 nM), and these beads induced significant CAR-
T cell proliferation over 72 h (Figure 3B, p < 0.01).
Lastly, to determine whether the mechanism of in vitro
cytotoxic activity of CAR-T cells is strictly dependent upon the
bispecific binding of folate-FITC to both FR and anti-FITC scFv,
we carried out competition assays using excess amount of
nonconjugated folate or fluorescein. As shown in Figure 4A, the
presence of a 1000-fold molar excess of fluorescein reduced the
lysis of KB cells from 64 to 24%. Furthermore, these results
suggest that administration of free fluorescein molecule may be
used to attenuate anti-FITC CAR-T cell activity in the case of an
adverse event, thereby providing an additional method of
regulating CAR-T cell activity. Unlike fluorescein, folate is
normally present in blood and thus may compete with folate-
FITC for FR binding, which may reduce the therapeutic efficacy
of CAR-T cells. To assess this possibility, we examined the
cytotoxic activity of CAR-T cells in the presence of folate
concentrations relevant to physiologically relevant folate levels in
human (20 nM) and rodent (200 nM) serum. Notably, certain
cell culture media, such as RPMI-1640, contains up to 2 μM
folate. Indeed, as shown in Figure 4B, the presence of folate in
media reduced the potency of folate-FITC; however, folate-
FITC still remained highly potent at concentrations of folate,
which are physiologically relevant in human (EC50 = 9.3 ± 1.5
pM) and rodents (EC50 = 66.9 ± 11.6 pM). This minimal
ASSOCIATED CONTENT
Supporting Information
Supplementary Figures S1−S4 and detailed materials and
methods. This material is available free of charge via the Internet
at http://pubs.acs.org.
■
*
S
AUTHOR INFORMATION
Corresponding Authors
schultz@calibr.org
tyoung@calibr.org
chkim@calibr.org
Present Address
College of Pharmacy, Pusan National University, Geumjeong-
gu, Busan 609−735, Korea
Author Contributions
■
*
*
*
∥
29
§
These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
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