Selective Targeting of Glioblastoma with EGFRvIII/EGFR Bi … · 2018. 9. 8. · M27-derived CAR T...

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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:

[email protected])

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

Research. on February 13, 2021. © 2018 American Association for Cancercancerimmunolres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 10, 2018; DOI: 10.1158/2326-6066.CIR-18-0044

mailto:[email protected]

http://cancerimmunolres.aacrjournals.org/

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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.



https://en.wikipedia.org/wiki/Epidermal_growth_factor_receptor


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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




18

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




19

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




20

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




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




22

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




23

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




24

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30

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




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




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




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




34

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).




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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