1
Selective Targeting of Glioblastoma with EGFRvIII/EGFR Bi-targeted Chimeric
Antigen Receptor T Cell
Hua Jiang1, Huiping Gao
1, Juan Kong
1, Bo Song
2, Peng Wang
2, Bizhi Shi
1, Huamao Wang
2,
Zonghai Li1, 2*
1State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji
Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
2CARsgen Therapeutics, Shanghai, China
Running title: EGFR/EGFRvIII bi-targeted CART cells for cancer treatment
Corresponding Author: Zonghai Li, MD, State Key Laboratory of Oncogenes and Related
Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiaotong University School of
Medicine, No.25/Ln2200, XieTu Road, Shanghai 200032, China. E-mail:
Disclosure of Potential Conflicts of Interest
Dr. Zonghai Li has ownership interests in anti-EGFR/EGFRvIII CAR-T. No other potential
conflicts of interest were disclosed by the other authors.
Word count: 5329 words (including Materials and Methods)
Total number of figures: 6 Figures, 7 Supplementary Figures, 1 Supplementary Tables
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Abstract
The heterogeneous expression of EGFRvIII [variant III mutant of epidermal growth
factor receptor (EGFR)] in glioblastoma has significant impact on the clinical response to the
treatment of EGFRvIII-specific chimeric antigen receptor–engineered T (CART) cells. We
hypothesized that CAR T cells that could target both EGFRvIII and the form of EGFR
expressed on tumor cells, but not EGFR on normal cells, would greatly improve efficacy
without inducing on-target, off-tumor toxicity. Therefore, we developed a humanized
single-chain antibody, M27, with a single specificity that binds to an epitope found both on
wild-type EGFR- and EGFRvIII-overexpressing tumor cells, but not EGFR-expressing
normal cells, including primary keratinocytes, on which wild-type EGFR is highly expressed.
M27-derived CAR T cells effectively lysed EGFRvIII- or EGFR-overexpressing tumor cells,
but showed no observable toxicity on normal cells. Inclusion of the CD137 (4-1BB)
costimulatory intracellular domain in the M27-28BBZ CAR provided CAR T cells with
higher tumor lysis activity than when not included (as in the M27-28Z CAR). The growth of
established EGFR- or EGFRvIII-overexpressing glioma xenografts was suppressed by
M27-28BBZ CAR T cells as well. The growth of heterogeneic xenograft tumors, created by
mixing EGFR and EGFR-overexpressing glioblastoma cells, was also effectively inhibited by
M27-28BBZ CAR T cells. The survival of mice in the orthotopic models was significantly
prolonged after M27-28BBZ CAR T-cell infusion. These results suggested that
tumor-selective, bi-targeted anti-EGFR/EGFRvIII CART cells may be a promising modality
for the treatment of patients with EGFR/EGFRvIII-overexpressing glioblastoma.
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Introduction
Glioblastoma is the most common and aggressive brain malignancy without effective
treatment options (1, 2). In the US, approximately 10,000 new cases are diagnosed annually
(3). Standard of care procedures include surgical resection followed by radiation therapy
and/or chemotherapy with the alkylating agent temozolomide (4). Despite therapeutic
advances, the prognosis of glioblastoma remains very poor, with a median survival of less
than two years (5). The resistance of glioblastoma to standard therapies results in a high rate
of tumor recurrence, and the lack of tumor specificity of the treatment may result in a
significant damage to healthy brain tissues (6). There is consequently a high unmet medical
need for a safe and efficacious tumor-selective therapy against glioblastoma.
Chimeric antigen receptor (CAR)–engineered T (CART) cells are a promising strategy for
cancer treatment (7). A classic CART structure includes an extracellular tumor-targeting
single-chain variable fragment from an antibody (scFv domain), a short transmembrane
domain, and a tandemly assembled intracellular costimulatory domain with intracellular
T-cell signaling moieties. Preclinical and clinical studies have shown CAR T cells redirected
to IL13Rα2 and HER2 could induce tumor regression in glioblastoma (8-11), suggesting that
CAR T cells could be developed as next-generation therapeutics for glioblastoma treatment.
Epidermal growth factor receptor (EGFR) is amplified in approximately 40-60% of
glioblastomas (12, 13). EGFRvIII, a cancer-specific variant of EGFR, is found in
approximately 31% of glioblastoma patients (12). Antibodies to EGFR and small molecule
inhibitors targeting EGFR have been used to treat patients with glioblastoma, but were not
clinically efficacious (14, 15). CAR T cells targeting EGFRvIII can selectively eliminate
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EGFRvIII-expressing glioblastoma cells (14, 16, 17). Although CAR T cells efficiently
infiltrated and eliminated EGFRvIII tumor cells in some patients, neither partial nor complete
responses were achieved in the clinical trials (18).
An increasing body of data suggests that intratumoral heterogeneity is one of the principal
causes of tumor relapse (19, 20). The poor clinical outcome of glioblastoma patients treated
with EGFRvIII-targeted CAR T cells might be attributed, in part, to the fact that EGFRvIII
was only expressed in a subpopulation of tumor cells (21, 22). We hypothesized that
bi-targeted CAR T cells that recognize both wild-type EGFR and EGFRvIII overexpressed by
glioma cells would show better efficacy than CAR T cells targeting EGFRvIII alone.
Monoclonal antibody 806 (mAb806) targets a conformationally-exposed epitope, EGFR287-302
,
that is found in both wild-type EGFR that is expressed on tumor cells and the mutated
oncogenic form of EGFR,EGFRvIII (23, 24). ABT-806, a humanized variant of mAb806
shows minimal skin toxicity in treated patients (25). ABT-414, an antibody-drug conjugate
composed of ABT-806 and a potent anti-microtubule agent, monomethyl auristatin F
(MMAF) had no skin toxicity in human studies with an objective response rate of
approximately 6.8% (26). This evidence suggests that the immune-oncology therapeutics
targeting EGFR287-302
have potential for improved safety and efficacy. Therefore, here we
generated CAR T cells recognizing EGFR287-302
and characterized their safety and efficacy
for the treatment of glioblastoma.
Materials and Methods
Cell lines
The human glioblastoma cell lines U87MG, U251, human epidermoid
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carcinoma cell line A431, human oral adenosquamous carcinoma cell line CAL27 and the
human embryonic kidney cell line 293T were obtained from American Type Culture
Collection (ATCC, Manassas, VA). Human hepatocyte L02 cell and human prostate
epithelial RWPE-1 cell were purchased from the Cell Bank of the Chinese Academy of
Sciences (Shanghai, China). U87MG-EGFR and U251-EGFR cells stably expressing human
EGFR protein were generated in our laboratory by transducing the U87MG and U251 cells
with a VSV-G pseudotyped lentiviral vector encoding wild-type EGFR. U87MG-EGFRvIII
and U251-EGFRvIII cells stably expressing EGFRvIII protein were generated in our
laboratory by transducing the U87MG and U251 cells with a VSV-G pseudotyped lentiviral
vector encoding exon 2–7-deleted EGFR. U251-luci-EGFR and U251-luci-EGFRvIII cells
expressing firefly luciferase were also established by lentiviral transduction. All cells were
cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% FCS (Life Technologies,
Mountain View, CA) with 100 μg/ml penicillin and 100 U/ml streptomycin (Life
Technologies). Human primary keratinocytes were isolated from healthy human skin and
cultured in EpiLife Medium with 60 µM calcium (Life Technologies, Mountain View, CA)
with Human Keratinocyte Growth Supplement (HKGS) (Life Technologies,
Mountain View, CA). The cell lines were authenticated by using short tandem repeat (STR)
analysis. Mycoplasma contamination testing was routinely performed by using PCR every 3
months in our laboratory. These cell lines had been cultured for 3-4 years in our lab and were
cryopreserved to create a working cell bank in which each vial of cells was subject to
subculture for up to 4 weeks after recovery. All cells were maintained at 37°C in humidified
air with 5% CO2.
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EGFR CAR construction
M27-scFv, an anti-EGFR-specific single-chain variable fragment (scFv), was derived from
the humanized EGFR antibody M27 that recognizes the residues 287-302 of EGFR. The
sequence encoding the M27-scFv antibody in the VL-VH orientation was cloned by PCR
amplification from a plasmid encoding scFv-M27-Fc. Another EGFR-specific scFv, 806-scFv
was derived from the chimeric antibody ch806 (24) that binds amino acids 287-302 of EGFR
as well. The sequence encoding the 806 scFv antibody in the VL-VH orientation was
obtained by PCR amplification from a plasmid encoding scFv-806-Fc. M27-28BBZ and
806-28BBZ CARs contained extracellular domains of the human CD8α signal peptide
(nucleotides 1–63, GenBank NM 001768.6), M27-scFv or 806-scFv, CD8α hinge
(nucleotides 412–546, GenBank NM 001768.6) and CD28 transmembrane domain
(nucleotides 457–537, GenBank NM 006139.3). The intracellular domains consisted of CD28
(“28”; nucleotides 538–660, GenBank NM 006139.3), CD137 (4-1BB, “BB”; nucleotides
640–765, GenBank NM 001561.5) costimulatory domain and CD3ζ (“Z”; nucleotides 154–
492, GenBank NM 198253.2) costimulatory polypeptide. M27-28Z CAR had an extracellular
domain that consisted of human CD8α signal peptide (nucleotides 1–63, GenBank NM
001768.6), M27-scFv and a CD8α hinge (nucleotides 412–546, GenBank NM 001768.6).
The intracellular signaling domain of M27-28Z CAR consisted of CD28 (nucleotides 538–
660, GenBank NM 006139.3) and CD3ζ (nucleotides 154–492, GenBank NM 198253.2)
molecules. The 28BBZ and 28Z fragments were produced by PCR amplification with a
plasmid template encoding the corresponding fragments (27). The DNA sequences of
scFv-28BBZ or scFv-28Z were further cloned by PCR amplification. Both of these fragments
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were designed to have a MluI site at the 5’ end and a SalI site at the 3’ end. The synthesized
fragments were digested with MluI and SalI restriction enzymes (New England Biolabs,
USA), and then inserted into a similarly digested pRRLSIN.cPPT-GFP.WPRE vector plasmid.
The sequence integrity of all the vectors described in this paper was confirmed by DNA
sequencing. The mock construct was transduced by using the pRRLSIN.cPPT-GFP.WPRE
lentiviral vector.
Lentivirus production
Human embryonic kidney 293T cells were seeded at 1.5 × 107 per 15-cm dish for 24 hours
prior to transfection. Expression vector pRRLSIN.cPPT-GFP.WPRE (Mock), or
pRRLSIN-M27-28Z, or pRRLSIN-M27-28BBZ, or pRRLSIN-806-28BBZ was mixed with
two lentiviral packaging plasmids pMDLg/pRRE and pRSV-Rev plus an envelope expressing
plasmid pCMV-VSV-G (from Addgene) to reconstitute a transfection DNA mixture in the
polyethylenimine-based DNA transfection reagent. 293T cells were transfected with the
reconstituted DNA mixture as mentioned above. The viral supernatants were harvested and
filtered (0.45-μm filter) at 72 hours after transfection. The lentiviral particles were
subsequently concentrated 30-fold by ultracentrifugation (Beckman Optima™ XL-100 K,
Carlsbad, CA) for 2 h at 28,000 rpm.
Transduction and culture of primary T-cells
Peripheral blood mononuclear cells (PBMCs) were cultured in AIM-V medium (Life
Technologies) in presence of 2% human AB serum (Huayueyang Biotechnology, Beijing,
China) and recombinant human IL2 (Huaxin High Biotech, Shanghai, China). For the
transduction of primary T cells, PBMCs were activated for 48 hours by Dynabeads Human
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T-Activator CD3/CD28 (Life Technologies, Mountain View, CA) at a bead:cell ratio of 2:1
before infection. The activated T cells were transduced with lentiviral vectors at a multiplicity
of infection (MOI) of 15 in a 24-well plate coated with RetroNectin (Takara, Japan). The
transduced T cells were cultured at a density of 5 × 105 cells/ml in presence of rhIL2 (500
IU/ml).
Flow cytometric analysis
To determine the binding of scFv to target cells, 1×106 cells were incubated with 5μg/ml
scFv-M27-Fc, scFv-806-Fc, or scFv-C225-Fc antibody for 45 minutes at 4°C. PBS was used
as a negative control. After washing with FACS buffer (cold phosphate-buffered saline
containing 1% newborn calf serum), the cells were incubated with FITC-conjugated goat
antihuman IgG (H+L) (Catalog Number: SA00003-12, -Proteintech, Rosemont, IL) for 45
minutes at 4°C. To quantify CAR expression, CAR T cells were incubated with 20 μg/ml
biotinylated anti-human-EGFR-F(ab’)2 fragments at 4°C for 45 minutes. PBS was used as a
negative control. After FACS buffer wash, the cells were incubated with PE-conjugated
streptavidin (eBioscience, San Diego, CA) for 45 minutes at 4°C. Fluorophore labeled CAR
T cells were determined by using a BD FACSCelesta flow cytometer. Data were analyzed
using FlowJo 7.6 software. Data are representative of three independent experiments. .
Analysis of CAR T-cell persistence in mouse peripheral blood lymphocytes
To examine the persistence of CAR T cells, whole mouse blood collected by retro-orbital
bleeding was analyzed as following: 50 μL of blood was incubated with the antibody against
CD3-PerCP/CD4-FITC/CD8-PE in the dark for 15 minutes at room temperature. After lysis
of red blood cells, cells were fixed with 0.45 ml 1× BD FACS Lysing Solution (BD
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Bioscience, Franklin Lakes, NJ) for 15 minutes at room temperature. The fixed cells were
then subjected to flow cytometric analysis by using FACSCelesta flow cytometer (BD
Bioscience, Franklin Lakes, NJ). Data from flow cytometry were further analyzed by using
FlowJo 7.6 software. Absolute cell numbers per microliter of blood were determined by using
TruCount tubes (BD Bioscience, Franklin Lakes, NJ) as described in the manufacturer’s
instruction.
Cytotoxicity assays in vitro
Each line of glioma cells were cocultured with target-selective CAR T cells or mock T
cells at effector:target ratio of 3:1, 1:1, and 1:3. After 18-hour culture, supernatant lactate
dehydrogenase (LDH) activity was determined by using the CytoTox 96®
Non-radioactive
Cytotoxicity Kit (Promega, Madison, WI) as previously described (27).
Cytokine release assay
CAR T cells or mock T cells were cocultured with glioma cells at a 3:1 ratio in a 96-well
culture plate. After 24-hour coculture, the release of IFNγ, TNFα, and IL2 cytokines from
activated CAR T cells or from mock T cells was determined by using an ELISA kit
(MultiSciences Biotechnology, Hangzhou, China) as described in the manufacturer’s
instructions.
In vivo efficacy studies
To generate subcutaneous xenograft models, 6- to 8-week old female NOD/SCID mice
were subcutaneously inoculated with 3 × 106 U251-EGFR or 2 × 10
6 U251-EGFRvIII cells at
the right flank. After the tumor volume increased to 75–120 mm3, the mice were randomly
divided into two groups (n = 6–10 per group) for receiving treatment regimen. The control
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group of mice received intravenous (i.v.) injection of mock T cells (activated T-cells
transduced with eGFP-CAR). The treatment group received i.v. injection of M27-28BBZ
CAR T cells (activated T cells transduced with EGFR-CAR). Twenty four hours prior to
CAR T cell–infusion, the mice were intraperitoneally (i.p.) injected with 200 mg/kg
cyclophosphamide to deplete host lymphocytes and to enhance the tumor treatment efficacy
of the administered T cells (28). Two doses of 1×107 M27-28BBZ CAR T cells were i.v.
injected via the tail vein in 200 μl of PBS on days 14 and 17. Tumors were measured by
using calipers, and tumor volumes were calculated by using the formula V = (length × width2)
/ 2. Animal body weights and tumor volume measurements were carried out twice weekly.
The mice were euthanized when their body weight loss was greater than 20%
of the initial weight, the mean tumor volume exceeded 2000 mm3, or the tumors became
ulcerated in the Mock control groups.
To generate the U251-luci-EGFR orthotopic model, 1 × 106 U251-luci-EGFR cells were
intracranially implanted into 6- to 8-week-old female NOD/SCID mice (n=8 mice per group).
To generate the U251-luci-EGFR/ U251-luci-EGFRvIII orthotopic model, the mixture of 7.5
× 105 U251-luci-EGFR and 2.5 × 10
5 U251-luci-EGFRvIII cells were intracranially
implanted into 6- to 8-week-old female NOD/SCID mice (n=7 mice per group). To generate
the U251-luci-EGFRvIII orthotopic model, 5 × 105 U251-luci-EGFRvIII cells were
intracranially implanted into 6- to 8-week-old female NOD/SCID mice (n=6 mice per group).
Implantation was performed by using a stereotactic surgical device with injection of tumor
cells at 1 mm right and at 1 mm anterior to the bregma and at 3 mm into the brain on day 0.
Seven days after surgery, the mice were randomly divided into two groups. On day 8, the
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mice were intravenously injected with a single dose of 1 × 107 CAR T cells in 200 μl of PBS
via the tail vein. Bioluminescent measurements were used as surrogates for tumor volume.
The transduction efficiency of CAR T cells used in the assays was ~50%. All animal
experiments were performed by following the protocol approved by the Shanghai Cancer
Institute Experimental Animal Care Commission.
Bioluminescence imaging
Bioluminescence imaging was performed by using the IVIS system (IVIS, Xenogen,
Alameda, CA). Briefly, tumor-bearing mice were intraperitoneally injected with D-Luciferin
(150 mg/kg) and imaged under isoflurane anesthesia after 10 minutes. The images were
quantified by using Living Image software (Caliper Life Sciences,Alameda, CA).
Immunohistochemical analysis
To assess the infiltration of human T cells into tumors, formalin-fixed and
paraffin-embedded tumor tissues were immunostained by using anti-CD3 (Thermo Fisher
Scientific, Waltham, MA). A normal rabbit IgG was used as an isotype-matched control. The
procedures were performed as previously described (27). Briefly, after deparaffinization and
rehydration, the sections were exposed to 3% H2O2 in methanol to eliminate endogenous
peroxidase activity and then blocked with bovine serum albumin (1%) for 30 min at room
temperature (RT). After blocking, the sections were incubated with primary rabbit
anti-human CD3 mAb overnight at 4°C. After PBS wash, the sections were incubated with
peroxidase-conjugated secondary antibodies (ChemMate™ DAKO EnVision™ Detection Kit,
Peroxidase/DAB, Rabbit/Mouse) for 45 min at RT. The sections were visualized by using a
diaminobenzidine staining kit (Dako Corporation, Carpinteria, CA) and then counterstained
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with hematoxylin, dehydrated, cleared, mounted, and photographed. The
DAB-immunostained sections were analyzed by bright-field microscopy using an Olympus
microscope (OLYMPUS IX71, Japan) equipped with an image analysis software.CD3+ cells
were quantified by measuring the number of stained T cells in each section by using the
Image-Pro Plus (Media Cybernetics, Rockville, MD) software. Sections from three mice in
each group were subjected to determination of T-cell infiltration for statistical analysis. The
mean count of the three areas was obtained and expressed as the absolute number of CD3+
cells per 0.95 mm2 (200× magnification).
Statistical analysis
All data were presented as the mean ± SE. Statistical significance was determined by using
a two-way or one-way ANOVA with Bonferroni post-test for multi-sample comparisons or
the unpaired two-tailed Student t test for comparisons between two samples. The overall
survival statistics were calculated by using the log-rank test. GraphPad Prism 5.0 was used
for statistical calculations. P< 0.05was considered statistically significant.
Results
Humanized scFv recognizes overexpressed EGFR and EGFRvIII in tumor cells
To avoid anaphylaxis caused by mouse scFv-derived CAR T cells (29), we generated a
panel of humanized antibody fragments derived from a mouse EGFR monoclonal antibody
7B3 that specifically bound EGFR287-302
(Supplementary Table S1 and Supplementary Fig.S1).
EGFR and EGFRvIII-transfected U87MG and U251, two cancer cell lines with endogenous
EGFR overexpression (A431 and CAL27), normal cells L-02 (Human hepatocyte cell) and
RWPE-1 (human prostate epithelial cell) as well as primary keratinocytes were used in the
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following biological assays. The expression of EGFR was confirmed by western blot
(Supplementary Fig.S2). After two rounds of affinity maturation and binding specificity
enrichment, M27 scFv clone was selected for further characterization of its binding
specificity. The scFv-derived from anti-EGFR antibody C225 (cetuximab) and mAb806 were
used as controls (Supplementary Fig.S3). Flow cytometric analysis showed that C225 scFv
bound to most of the cells except for U87MG cells (Fig. 1A). The binding curves of C225
scFv to U251 cells and to the three healthy keratinocytes were similar. 806 scFv showed
observable binding on EGFR- and EGFRvIII-overexpressing U87MG and U251cells and
three primary keratinocytes. The mean fluorescence intensity (MFI) of 806 scFv on
U87MG-EGFRvIII, U251-EGFRvIII, U87MG-EGFR, and U251-EGFR cells was 22.6±0.42,
58.1±0.85, 1.57±0.05, and 9.81±0.08, respectively. The MFI of 806 scFv on three primary
keratinocytes was 0.71±0.04, 0.57±0.02, and 0.60±0.03. Humanized M27 scFv showed
distinguishable binding on EGFRvIII-overexpressing cells and EGFR-overexpressing
U87MG and U251 cells. The MFI of M27 scFv on U87MG-EGFRvIII, U251-EGFRvIII,
U87MG-EGFR, and U251-EGFR cells was 12.2±0.31, 19.4±0.50, 0.39±0.01, and 0.78±0.02,
respectively. The EC50 of M27 and 806 scFv on U87 MG-EGFR cell was 3.9 μM and 2.4 μM
respectively, whereas the EC50 of M27 and 806 scFv on U87 MG-EGFRvIII cell was 99.5
nM and 66.4 nM respectively (Supplementary Fig.S4). These results indicated that M27 scFv
had a lower affinity to EGFR- or EGFRvIII-overexpressing cells than 806 scFv. However,
M27 scFv did not bind to any of the three primary keratinocytes tested. In addition, all
anti-EGFR scFv demonstrated binding to endogenous EGFR-overexpressing cell lines A431
and CAL27. M27 scFv, however, showed relatively less affinity than other two scFv in
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binding to A431 and CAL27 (Fig. 1A). The MFI of different scFv proteins on the cells tested
is shown (Fig. 1B). Additionally, we also evaluated the binding ability of M27 scFv to normal
cells L-02 (human hepatocyte cell) and RWPE-1 (human prostate epithelial cell), which
endogenously express EGFR. The results showed that M27 scFv also could not bind to these
two cells (Supplementary Fig.S5). These data indicate that the M27 scFv specifically bound
EGFR and EGFRvIII overexpressed on tumor cells but not EGFR expressed on normal cells.
Molecular Construction of CAR cassette and Generation of CAR T cells
We generated a series of recombinant lentiviral vectors encoding various CAR cassettes
that had an EGFR287-302
scFvantibody, an intracellular human T-cell costimulatory domain
derived from human CD28, CD137, and CD3ζ chains, and a linker domain of human CD8
hinge and CD28 transmembrane regions (M27-28BBZ and 806-28BBZ). We also constructed
a CAR cassette lacking the intracellular CD137 signaling domain (M27-28Z). The scheme of
recombinant lentiviral constructs is shown in Fig. 2A.
To determine the expression of the EGFR CAR on the genetically modified T-cell surface,
different CAR-transduced T cells were determined by flow cytometry using biotinylated
human-EGFR-F(ab’)2 fragment antibody and PE-conjugated streptavidin. The transduction
efficiency was ranged from 51.9% to 74.8%. As a control, mock T cells were transduced with
the lentiviral vector encoding GFP gene. The transduction efficiency of mock T cells was
determined by eGFP expression (Fig. 2B).
M27-derived CAR T cells lyse EGFR-and/or EGFRvIII-overexpressing tumor cells
To determine whether CAR T cells targeting EGFR/EGFRvIII could selectively recognize
and eliminate EGFR/EGFRvIII-positive human glioblastoma cells, cytotoxicity assays were
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performed by incubating the genetically modified CAR T cells with either control cells or
EGFR- or/and EGFRvIII-overexpressing glioblastoma cells. The results showed that both
M27-28Z and M27-28BBZ CAR T cells efficiently lysed the
EGFR/EGFRvIII-overexpressing glioblastoma cells but not untransfected U87MG and U251
cells (Fig. 2C and D). Mock T cells showed very weak cytotoxicity compared to M27-derived
CAR T cells (Fig. 2C). The M27-28BBZ CAR T cells showed higher cytotoxic activities than
M27-28Z CAR T cells in lysis of both EGFR and EGFRvIII-overexpressing U87MG or
U251 cells at 1:1 and 3:1 effector:target (E:T) ratio (P < 0.05, Fig. 2C and D). These data
suggested that M27-28BBZ CAR T cells would be a better candidate for further evaluation
and development.
To determine the selective cytotoxicity of M27-28BBZ CAR T cells in lysis of tumor cells,
CAR T cells were incubated with primary keratinocytes and EGFR/EGFRvIII-overexpressing
tumor cells, respectively. 806 scFv-derived 806-28BBZ CAR T cells were also included as a
control. The data showed that both 806- and M27-derived CAR T cells could efficiently lyse
EGFR- and EGFRvIII-overexpressingU87MG and U251 cells. The cytotoxicities of both
CAR T cells were similar in lysis of U251-EGFRvIII cells. 806-28BBZ CAR T cells showed
slightly higher cytotoxic effect on U87MG-EGFRvIII cells than on M27-28BBZ CAR T cells
(806-28BBZ vs M27-28BBZ at 1:3 E:T: 24.3%±2.0% vs 16.2%±0.9%; 806-28BBZ vs
M27-28BBZ at 1:1 E:T: 50.2%±3.7% vs 43.0%±2.5%; 806-28BBZ vs M27-28BBZ at 3:1
E:T: 68.9%±4.4% vs 57.8%±4.7%, Fig. 3A and B). Unlike M27-28BBZ, 806-28BBZ CAR T
cells demonstrated a cytotoxic effect on untransfected U251 cells at a 3:1 E:T ratio. At a 3:1
E:T ratio, both M27-28BBZ and 806-28BBZ CAR T cells could lyse almost all the
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U251-EGFR cells. At an E:T ratio of 1:1, M27 CAR T cells had a lower lysis capacity than
the 806 CAR T cells in EGFR-transfected U87MG and U251 cells(Fig. 3A and B). In
addition, both CAR T cells could effectively induce lysis of endogenous
EGFR-overexpressing cancer cell lines A431 and CAL27. At E:T ratio of 3:1, no significant
difference on the lysis capacity was observed between these two CAR T cells(Fig. 3C).The
M27-28BBZ CAR T cells had no measurable cytotoxic effect on all three primary
keratinocytes; however, the 806-28BBZ CAR T cells showed significantly higher cytotoxic
effect on all three keratinocyte lines at 3:1 E:T ratio (806-28BBZ vs M27-28BBZ on
keratinocyte-1: 31.9%±1.6% vs 16.9%±0.8%, P<0.001; 806-28BBZ vs M27-28BBZ on
keratinocyte-2: 38.8%±2.0% vs 11.7%±1.3%, P<0.001; 806-28BBZ vs M27-28BBZ on
keratinocyte-3: 54.1%±3.3% vs 20.6%±1.1%, P<0.001; Fig. 3D). Additionally, M27-28BBZ
CAR T cells had also no cytotoxic effect on other normal cells L-02 and RWPE-1
(Supplementary Fig. S6). This indicated that M27-28BBZ CAR T cells could selectively lyse
EGFR and/or EGFRvIII-overexpressing tumor cells but not EGFR-expressing normal cells.
M27-28BBZ CAR T cells produce cytokines in the presence of target cells
Cytokine secretion by CAR T cells in response to a target antigen indicates activation and
maintenance of an antigen-specific immune response. The secretion of TNFα, IL2, and IFNγ
from CAR T cells was determined to evaluate the activation of CAR T cells by
antigen-expressing tumor cells. Activated M27-28BBZ CAR T cells secreted significantly
more TNFα, IL2, and IFNγ than did mock T cells after incubation with
EGFR/EGFRvIII-overexpressing tumor cells (P<0.05, Fig. 3E). In addition, M27-28BBZ
CAR T cells produced greater concentrations of cytokines in the presence of
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EGFRvIII-overexpressing tumor cells than in the presence of EGFR-overexpressing tumor
cells. Secretion of these cytokines from CAR T cells was negligible after incubation with
untransfected U87MG and U251 cells (Fig. 3E). Cytokine secretion was not induced in
M27-28BBZ CAR T cells cocultured with three primary keratinocytes, further supporting
that M27-28BBZ CAR T cells would not cross react with keratinocytes.
M27-28BBZ CAR T cells suppress growth of EGFR/EGFRvIII-overexpressing tumors
To determine the therapeutic efficacy of genetically modified T cells, M27-28BBZ CAR T
cells and mock T cells were used to treat mice bearing U251-EGFR or U251-EGFRvIII
subcutaneous tumors. The results demonstrated thatM27-28BBZ CAR T cells could
significantly inhibit tumor growth in both xenografts. On day 42 following tumor cell
inoculation, significant reduction of tumor volume (P<0.001, Fig. 4A) and tumor weight
(P<0.0001, Fig. 4B) of the U251-EGFRvIII tumors was observed in the group treated
withM27-28BBZ CAR T cells when compared to the group of mock T cell treatment. On day
49 following tumor cell inoculation, the tumor volume and tumor weight of U251-EGFR
xenografts in the M27-28BBZ CART-cell group was also significantly lower than those in
the mock T-cell group (P< 0.05; Fig. 4C and D). These results indicated that M27-28BBZ
CAR T cells could efficiently suppress the growth of both U251- EGFR and U251-EGFRvIII
cells in vivo.
Efficacy of M27-28BBZ CAR T cells in orthotopic glioblastoma xenograft models
Given that the subcutaneous microenvironment might disturb the activity of CAR T cells,
we evaluated the antitumor activities of M27-28BBZ CAR T cells in an intracranial
glioblastoma xenograft that contained luciferase-transfected U251-EGFR and
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U251-EGFRvIII tumor cells. Previous studies have reported that the expression of EGFRvIII
in some cells is frequently associated with amplified expression of EGFR and that the
coexpression of both receptors within the tumor mass confers a worse prognosis in patients
with glioblastoma (12, 30). To model heterogeneous expression of EGFR and EGFRvIII in
glioblastoma, U251-luci-EGFR cells and U251-luci-EGFRvIII cells were mixed together at
3:1 ratio (EGFR-expressing cells grew much more slowly than EGFRvIII-expressing cells).
The mixed cells were intracranially implanted into mice (day 0). After 6 days since
glioblastoma xenografts were established, the engraftment of U251-EGFR and
U251-EGFRvIII cells was confirmed by bioluminescence imaging. On day 7 following initial
tumor engraftment, the mice were intravenously injected with one dose of 1×107M27-28BBZ
CAR T cells or mock T cells. At day 28 after tumor inoculation (21 days after T-cell
injection), the tumors in the U251-EGFR xenograft model showed a significant reduction in
volume after treatment of M27-28BBZ CAR T cells compared to mock T cells (Fig. 5A and
B). The survival of mice in M27-28BBZ CAR T cell–treatment group was significantly
prolonged compared with that in the mock T cell–treatment group (P<0.05, Fig. 5C). In the
U251-EGFRvIII and U251-EGFR/EGFRvIII xenograft models, tumor growth in the
M27-28BBZ treatment group was suppressed compared to that in the mock group at day 21
after tumor inoculation (14 days after T-cell injection; Fig.5A and B). In both models, some
mice treated with M27-28BBZ CAR T cells survived for greater than 80 days
(U251-EGFR/EGFRvIII model: 2/6 mice, U251-EGFRvIII model: 1/6 mice). In M27-28BBZ
CAR T cell–treatment groups, median survival time was increased from 26 to 60 days in the
mice with U251-EGFRvIII xenografts and changed from 24 to 56 days in the mice with
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U251-EGFR/EGFRvIII xenografts compared with mice receiving mock T cells (P<0.05, Fig.
5C).
In vivo persistence of M27-28BBZ CAR T cells
To evaluate CAR T-cell persistence in vivo, tumor-bearing mice were euthanized at day 15
after intracranial tumor implantation (8 days after injection of CAR T cells). The persistence
of CAR T cells in the murine peripheral blood and the infiltration of CAR T cells into brain
tissues were determined in the orthotopic glioblastoma xenograft models. Flow cytometric
analysis showed a significant increase (P<0.05) of both CD4+ and CD8
+ T-cells, with a
predominance of CD8+ T-cells, in the M27-28BBZ group compared to the mock group in all
three models (Fig. 6A). More T cells in the EGFR/EGFRvIII heterogeneity model persisted
than in EGFRvIII and EGFR groups. The infiltration of human T cells was determined in the
tumor-bearing mouse brain tissues by immunostaining with antibody to human CD3 (Fig.
6B-D). Human CD3+ cells robustly infiltrated residual tumors after M27-28BBZ CAR T-cell
infusion. In contrast, very few T cells could be detected in tumor lesions after mock T-cell
treatment (Fig. 6B-D). More infiltrating T cells were found in EGFRvIII-overexpressing
tumor lesions than in EGFR-overexpressing tumors (U251-EGFRvIII vs U251-EGFR:
101.8±19.9 vs 62.1±20.7 CD3+ cells/mm
2, P<0.01, Fig. 6E). These data suggested that the
injected M27-28BBZ CAR T cells infiltrated antigen-expressing brain tumors in vivo and
were amplified and maintained.
Discussion
Mounting evidence indicates that intratumoral heterogeneity may be a cause of drug
resistance and tumor recurrence in cancer treatment (31). Glioblastoma is a highly
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heterogeneous tumor (32, 33) and one of the first cancers profiled in The Cancer Genome
Atlas (TCGA) project (34). Genetic alterations including mutations, rearrangements,
alternative splicing, and focal amplifications of EGFR have been found in 57% of
globlastomas (35). In addition, EGFR amplification and overexpression has been reported in
approximately 50-60% of glioblastomas (36, 37). EGFR variants including EGFRvIII and
de4 EGFR (38, 39) could increase proliferation and invasiveness of glioblastoma cells. One
study demonstrated that EGFRvIII is invariably accompanied by EGFR amplification,
although EGFR overexpression does not necessarily give rise to the EGFRvIII variant (23,
24). This partially explains why EGFRvIII-specific therapy was unable to completely
eliminate glioblastoma. Another study showed that EGFRvIII expression was lost in 80% of
relapsed tumors following vaccination with PEPvIII-KLH in Freund’s complete adjuvant,
suggesting that EGFRvIII-negative cell population predominated at the time of tumor
recurrence (40). Additionally, neither partial nor complete responses were observed in a total
of 10 glioblastoma patients treated with EGFRvIII-specific CAR T cells, although EGFRvIII
antigen completely disappeared in 2 patients after CAR T-cell infusion (18). This paradoxical
clinical readout could be ascribed to heterogeneity of EGFRvIII expression. To overcome the
heterogeneity of EGFRvIII, CAR-NK cells that could recognize both EGFR and EGFRvIII
were developed (41, 42). However, the antibodies used in the CARs such as C225, could bind
to normal EGFR, which is expressed in normal brain tissues (39, 43). Thus, these CAR-NK
cells may induce on-target, off-tumor toxicities in patients with glioblastoma even treated by
locally administration.
Severe toxicities including death have been reported in CAR T-cell immunotherapy due to
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21
on-target, off-tumor toxicities (44, 45). Thus, using cancer-selective antibodies as the
targeting moiety of the CARs may enhance the safety of this therapy. MAb 806, which binds
to the EGFR287-302
epitope, can bind to EGFR and to EGFRvIII overexpressed in cancer cells,
but not EGFR in normal cells. Unexpectedly, our study revealed that 806 scFv could bind to
primary keratinocytes, although with a much lower binding capacity than cetuximab. At
doses up to 24 mg/kg, ABT-806 (25) only induced mild dermatitis acneiform in 12 % (6/49)
of the patients and one grade 3 morbilliform rash. Even more promising is that ABT-414, at a
dose of 1.25 mg/kg, showed no skin toxicity and induced an objective response rate of 6.8%
in the 66 patients treated (26). In combination with radiation and temozolomide, ABT-414
was well tolerated and showed promising clinical benefit in patients with newly diagnosed
GBM (46). These studies support using antibodies targeting the EGFR287-302
epitope as an
immunotherapeutic strategy for patients with GBM. However, the study of ABT-414 had
ocular grade 3/4 adverse events (AEs) in 33% of patients overall, which may be ascribed to
the accumulation of the toxic MMAF. Another consideration with antibody-drug conjugates
is that they need internalization through receptor-mediated endocytosis. Its antitumor
activities depend on the binding avidity of the antibody and the endocytosis capacity of the
receptor. Unlike antibody-drug conjugates, CAR T cells do not possess the same
compound-associated toxicities and are capable of killing cancer cells independent of
receptor-mediated endocytosis. A single CAR T-cell may express thousands of CAR copies,
which may increase its binding avidity on target cells and maybe the reason why CAR T cells
comprising 806 scFv, a low-affinity EGFR binder, are capable of lysing normal keratinocytes
in vitro. In this study, we identified a humanized scFv M27 which did not bind to normal
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cells, but could bind to both EGFR and EGFRvIII in tumor cells at a relatively lower affinity
than 806. Like previous reports that EGFR antibodies could be tuned to low affinity to reduce
CAR T cell-associated toxicity against normal cells (47, 48), M27 CAR T cells showed no
toxicity against normal cells while retaining their tumor lysis capabilities. Additionally,
M27-28BBZ CAR T cells have relatively higher cytotoxicity against cancer cells with EGFR
or EGFR overexpression, compared with that of M27-28Z CAR T cells. These data indicated
that M27-28BBZ CAR T cells demonstrated effective antitumor activity with minimal
clinically relevant on-target, off-tumor toxicity.
In intracranial tumor xenograft models, M27-28BBZ CAR T cells efficiently suppressed
the growth of EGFR- and EGFRvIII-overexpressing tumors and increased survival of the
mice. Its antitumor activity against EGFRvIII tumors appeared more potent than those against
EGFR tumors. It might be explained by higher binding affinity of M27 scFv to EGFRvIII
than to EGFR. The xenografts in the EGFR/EGFRvIII mixture group were eradicated by
M27-28BBZ CAR T cells, and this may be ascribed to the enhanced amplification or
persistence of M27-28BBZ CAR T cells in the presence of EGFRvIII-positive tumor cells.
T-cell numbers in both blood and tumor tissues in the EGFR/EGFRvIII group were higher
than that in the EGFR group, and increased T-cell infiltration and persistence might facilitate
the elimination of tumor cells with EGFR overexpression.
Given their distinct tumor-specificity and antitumor activities, M27-28BBZ CAR T cells
represent a new bi-targeted antitumor agent for the treatment of patients with glioblastoma.
Additionally, as many other tumor types, including non-small cell lung cancer, breast cancer,
and head and neck squamous carcinoma, are characterized by EGFR amplification and/or
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EGFRvIII mutations (23, 49, 50), M27-28BBZ CAR T cells also have the potential for the
treatment of these cancer types as well.
Acknowledgments
This work was supported by funding from the Supporting Programs of Shanghai Science
and Technology Innovation Action Plan (No.18431902900),Shanghai Municipal
Commission of Health and Family Planning (No. 201740124), the National Natural Science
Foundation of China (No.81672724 and No. 81472573), the Grant from the State Key
Laboratory of Oncogenes and Related Genes (No.91-17-17)and the seed fund of Renji
Hospital (No.RJZZ17-021).
Authors' Contributions
Conception and design: Zonghai Li
Development of methodology: Hua Jiang, Huiping Gao,Juan Kong, and Bo Song
Acquisition of data (provided animals, acquired and managed patients,provided facilities,
etc.): Bizhi Shi, Peng Wang andHuamao Wang
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational
analysis): Hua Jiang, Huiping Gaoand Bo Song
Writing, review, and/or revision of the manuscript: Zonghai Li and Hua Jiang
Administrative, technical, or material support (i.e., reporting or organizingdata, constructing
databases): Bo Songand Huamao Wang
Study supervision: Zonghai Li
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Figure Legends
Fig.1. FACS analysis demonstrated the binding of scFv-M27-Fc, scFv-806-Fc and
scFv-C225-Fc to the indicated target cells. To determine the binding of scFv-Fc proteins to
untransfected or transfected U87MG, U251 glioblastoma cells and three primary
keratinocytes, 1×106 cells were isolated and incubated with 5 μg/ml of scFv-M27-Fc,
scFv-806-Fc and scFv-C225-Fc antibodies for 45 minutes at 4°C. After wash with FACS
buffer, the cells were stained with an FITC-conjugated goat ant human IgG (H+L) for 45
minutes at 4°C. A, Fluorescence was assessed by using a BD FACSCelesta flow cytometer. B,
The mean fluorescence intensity (MFI) of scFv proteins binding to cells was determined by
flow cytometric analysis.
Fig.2. Construction and characterization of EGFR/EGFRvIII bi-targeted chimeric
antigen receptor (CAR) T cells. A, Schematic representation of CARs. M27-28Z CAR
consists of a scFv, a human CD8α hinge and a transmembrane (TM) region derived from
human CD28, and an intracellular signaling domain from human CD28 and human CD3ζ.
M27-28BBZ or 806-28BBZ CAR comprises a scFv, a human CD8α hinge, a CD28
transmembrane domain, and an intracellular signaling domain derived from human CD28,
CD137, and CD3ζ. B, Expression of EGFR-specific CARs on the lentivirus-transduced
human T cells were analyzed using flow cytometry. Transduction efficiency is shown. C and
D, In vitro cytotoxic activities of EGFR/EGFRvIII-targeted CAR T cells (M27-28Z or
M27-28BBZ). Primary human T cells transduced with the indicated lentiviral vectors were
incubated with the untransfected and transfected glioblastoma cells at the various
effector:target ratios for 18 hours. Cell lysis was determined using a standard nonradioactive
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31
cytotoxic assay. Data are representative of three independent experiments. Each data point
reflects the mean ± SEM of triplicates.
Fig.3. In vitro cytotoxic activity of and cytokine production by M27-28BBZ T cells in the
presence of tumor cells. Primary human T cells transduced with the indicated lentiviral
vectors were incubated with cell lines at different effector:target ratios for 18 hours. Cell lysis
was determined using a standard nonradioactive cytotoxic assay. Data are representative of
three independent experiments and shown as the mean ± SEM of triplicates. A, The cytotoxic
activities of M27-28BBZ and 806-28BBZ to U87MG, U87MG-EGFR and
U87MG-EGFRvIII cells. B, The cytotoxic assay of M27-28BBZ and 806-28BBZ using U251,
U251-EGFR and U251-EGFRvIII cells. C,The specific lysis of M27-28BBZ and 806-28BBZ
of primary keratinocytes from three different human skin specimens. D, The specific lysis of
M27-28BBZ and 806-28BBZ to A431 and CAL27 cells. Data are representative of three
independent experiments and shown as the mean ± SEM of triplicates (*, P<0.05; **, P<
0.01; ***, P< 0.001; two-tail Student t test). E, Human T cells transduced with lentiviral
vectors were cocultured with indicated cell lines at a 3:1 effector:target ratio. Culture
supernatants harvested at 24 hours after coculture were assayed for TNFα, IL2 and IFNγ by
ELISA. Data are representative of three independent experiments and presented as the mean
± SEM of triplicates.
Fig.4. In vivo antitumor activities of M27-28BBZ CAR T cells on established
subcutaneous EGFR- or EGFRvIII-overexpressing glioblastoma xenografts. A,
NOD/SCID mice were subcutaneously inoculated with 2 × 106 U251-EGFRvIII cells on day
0. Two doses of 1×107 M27-28BBZ CAR T cells or mock T cells were intravenously
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32
administered on days 12 and 15. Data are presented as the mean tumor volume ± SEM.
Statistically significant differences between M27-28BBZ CAR T cells and mock T cells are
marked with asterisks [***, P<0.001; two-way analysis of variance (ANOVA) with
Bonferroni post-test]. B, On day 42 after tumor cell inoculation, the mice were euthanized.
Tumor weight was measured. Endpoint was defined by the tumors reaching a mean volume
of 2000 mm3, more than 20% body weight loss in mice, tumor ulceration, or inability of the
mice to ambulate. Efficacy was evaluated by measuring the reduction of the mean tumor
weight relative to mock T cells (***, P<0.001; two-tailed Student’s t test). C, NOD/SCID
mice were subcutaneously inoculated with 3 × 106 U251-EGFR cells. When the tumor
volume was approximately 100–120 mm3, mice bearing established subcutaneous tumors
were treated with two doses of 1×107 M27-28BBZ CAR T cells or mock T cells on days 14
and 17. Data are presented as the mean tumor volume ± SEM [*, P<0.05; two-way ANOVA
with Bonferroni post-test]. D, On day 49 after tumor cell inoculation, the mice were
euthanized. Tumor weight was measured. Efficacy was evaluated by measuring the reduction
of the mean tumor weight relative to Mock control (*, P<0.05; two-tailed Student’s t test).
Fig.5. Antitumor activities of M27-28BBZ T cells on established intracranial
glioblastoma xenografts in vivo. For orthotopic model of glioblastoma, 1 × 106
U251-luci-EGFR cells were implanted intracranially into NOD/SCID mice (n=8 mice per
group). For U251-luci-EGFR/ U251-luci-EGFRvIII models, 7.5 × 105 U251-luci-EGFR and
2.5 × 105 U251-luci-EGFRvIII cells were implanted intracranially into mice (n=7 mice per
group). For U251-luci-EGFRvIII models, 5 × 105 U251-luci-EGFRvIII cells were implanted
intracranially into mice on day 0 (n=6 mice per group). On day 7, mice were injected
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33
intravenously with 1 × 107 M27 CAR T cells or mock T cells. A, Representative IVIS images
depicting 6–7 of the mice from each of the groups treated with the indicated CAR T cells.
Mice were imaged weekly. Endpoint was defined by mice inability to ambulate. B, Tumor
burden was quantified as total flux in units of photons/second. Bars indicate means ± SE [*,
P<0.05; **, P< 0.01; (**); ***, P< 0.001; two-way analysis of variance (ANOVA) with
Bonferroni post-test]. C, Survival was plotted using a Kaplan-Meier curve; statistically
significant differences between the experimental groups were determined using log-rank
Mantel-Cox test (*, P<0.05).
Fig.6. The persistence of human T-cells from mice treated with the indicated genetically
modified T cells in orthotopic glioblastoma xenografts models. A, The quantification of
CD4+ and CD8
+ T cells in murine peripheral blood of intracranial tumor-bearing mice on day
15. Fifty microliters of murine blood was drawn at 8 days after indicated T-cell infusion to
determine T-cell counts. T cells were analyzed by flow cytometry using
anti-CD3-PerCP/anti-CD4-FITC/anti-CD8-PE and were enumerated using the count beads.
Data indicate means ± SEM number of T cells per microliter of blood as measured by flow
cytometry. Statistically significant differences were calculated by two-tailed Student’s t test
(*, P<0.05). B-D, The representative immunostaining images of CD3+ T-cell infiltration in
tumor-bearing mice brains. The brain tissues were harvested from intracranial tumor-bearing
mice on day 15. Formalin-fixed, paraffin-embedded mice brain tissue sections were
consecutively cut and stained for human CD3 (brown). [The images were taken with the
microscope (BX41, Olympus, PA) and camera (DP70) under ×200 magnifications. Scale bar
is 100μm]. E, the quantification of T-cell infiltration in brain tissues of intracranial
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tumor-bearing mice treated with M27-28BBZ and mock T cells. Data are expressed as mean
± SEM (*, P<0.05; **, P< 0.01; (**); ***, P< 0.001; two-tailed Student t test).
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Published OnlineFirst September 10, 2018.Cancer Immunol Res Hua Jiang, Huiping Gao, Juan Kong, et al. Bi-targeted Chimeric Antigen Receptor T CellSelective Targeting of Glioblastoma with EGFRvIII/EGFR
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