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Cancer Gene Therapy (2021) 28:321–334
Chimeric Antigen Receptor-modified T cells targeting EphA2 for the
immunotherapy of paediatric bone tumours
Kenneth Hsu1 ● Shiloh Middlemiss1 ● Federica Saletta1 ● Stephen Gottschalk 2 ● Geoffrey B. McCowage3 ●
Belinda Kramer 1
Received: 5 May 2020 / Revised: 14 August 2020 / Accepted: 21 August 2020 / Published online: 1 September 2020
© The Author(s), under exclusive licence to Springer Nature America, Inc. 2020
Chimeric Antigen Receptor (CAR) T-cell therapy, as an approved treatment option for patients with B cell malignancies,
demonstrates that genetic modification of autologous immune cells is an effective anti-cancer regimen. Erythropoietinproducing Hepatocellular receptor tyrosine kinase class A2 (EphA2) is a tumour associated antigen expressed on a range of
sarcomas, including paediatric osteosarcoma (OS) and Ewing sarcoma (ES). We tested human EphA2 directed CAR T cells
for their capacity to target and kill human OS and ES tumour cells using in vitro and in vivo assays, demonstrating that
EphA2 CAR T cells have potent anti-tumour efficacy in vitro and can eliminate established OS and ES tumours in vivo in a
dose and delivery route dependent manner. Next, in an aggressive metastatic OS model we demonstrated that systemically
infused EphA2 CAR T cells can traffic to and eradicate tumour deposits in murine livers and lungs. These results support
further pre-clinical evaluation of EphA2 CAR T cells to inform the design of early phase clinical trial protocols to test the
feasibility and safety of this immune cell therapy in paediatric bone sarcoma patients.
Despite substantial improvements in the survival of children
diagnosed with cancer over recent decades, children with
recurrent or metastatic bone tumours such as osteosarcoma
(OS) or Ewing sarcoma (ES) face a poor prognosis. The
recent establishment of Chimeric Antigen Receptor (CAR)
based T-cell immunotherapy as an effective treatment for B
cell malignancies  demonstrates that gene modification of
immune cells to target tumour associated antigens (TAA) is
a powerful method of disease control. It is well documented
that T cells have the capacity to infiltrate bone tumours
[2–4], however, the significance of their presence has long
been a subject of conjecture: some , but not all studies 
report a relationship between T-cell presence within
tumours and treatment outcomes. Studies attempting to
generate autologous cytotoxic T cells from bone tumour
patients have also reported variable results, demonstrating
the difficulties in developing endogenous tumour infiltrating
lymphocytes (TIL) as a therapeutic option [5, 6]. Adoptive
transfer of gene modified, CAR bearing T cells offers an
alternative strategy that bypasses the need for identification
and expansion of endogenous TIL, with proven efficacy at
least in B cell malignancies .
By design, CAR T cells are functionally uncoupled from
requirements of MHC-mediated antigen presentation and
co-stimulatory receptor engagement, and are unaffected by
TCR-mediated T regulatory cell (Treg) suppression that
mitigates against their presence within the tumour microenvironment . Successful translation of CAR T-cell
therapies into effective treatments for bone tumours requires
identification of suitable TAAs, coupled with effective
delivery to the tumour. Currently there are a range of TAA’s
being investigated in both pre-clinical studies and in early
phase trials as putative targets for sarcomas. These include
Human Epidermal Growth Factor Receptor 2 (HER2) [7, 8],
* Belinda Kramer
1 Children’s Cancer Research Unit, Kid’s Research, The Children’s
Hospital at Westmead, Westmead, NSW 2145, Australia
2 Department of Bone Marrow Transplant and Cellular Therapy, St.
Jude Children’s Research Hospital, Memphis, TN, USA
3 Children’s Cancer Centre, The Children’s Hospital at Westmead,
Westmead, NSW 2145, Australia
Supplementary information The online version of this article (https://
doi.org/10.1038/s41417-020-00221-4) contains supplementary
material, which is available to authorised users.
the ganglioside antigen GD2 , type-I insulin-like growth
factor receptor (IGF1R), tyrosine kinase-like orphan
receptor 1 (ROR1) , Interleukin-13 Receptor α2 (IL-
13Rα2)  and a range of melanoma TAAs . Our
work focuses on the EphA2 protein, which normally acts as
a tyrosine kinase receptor for Ephrin signalling during
embryonic development , with post-development
expression largely confined to some epithelial cell layers
[14, 15]. EphA2 overexpression is widely reported across a
broad range of cancer types [16, 17], with functional significance for tumour formation and spread, prompting an
increasing interest in this protein as a therapeutic target in
cancer [18, 19]. In OS and ES, EphA2 overexpression has
been linked to oncogenic signalling , the promotion of
angiogenesis  and tumour aggressiveness . More
broadly, in tumours such as colorectal cancer , breast
cancer , pancreatic cancer , melanoma  and lung
cancer , EphA2 expression has been associated with
tumour proliferative and migratory capacity. EphA2 overexpression in tumours has been shown to have prognostic
significance in lung cancer [27, 28], renal cell carcinoma
, ovarian cancer  and brain tumours .
We hypothesise that EphA2 is a promising target in both
OS and ES due to high levels of expression in tumours,
contrasting with low-level expression in normal bone
[20, 32]. Moreover, EphA2 expression in malignant tissue
is reported to reveal targetable peptide epitopes that are
unavailable for binding in normal epithelial tissues .
EphA2 protein has been investigated as an immunotherapy
target in solid tumours using antibody-drug conjugates [34–
36], as a source of immunogenic peptides [37, 38] and as a
ligand of agonists to promote EphA2 degradation to
enhance T-cell activity against the tumour . In preclinical studies testing EphA2 CAR T-cell efficacy, antitumor cytotoxicity has been demonstrated against oesophageal squamous cell carcinoma cells in vitro , in both
in vitro and in vivo models of non-small-cell lung cancer
 and glioma and medulloblastoma, dependent on route
of delivery [42–44]. Also in glioma, where EphA2 is being
utilised in the development of tumour imaging diagnostics
, early phase clinical trials testing EphA2 CAR T cell
safety and efficacy are registered as completed
(NCT02575261) or on-going (NCT03423992). In this
study, we first determine the applicability of EphA2 CAR
T cells in targeting paediatric bone sarcomas, with consideration of the extent to which EphA2 expression may
lead to on-target but off-tumour effects in normal tissues.
We report on the use of a lentiviral (LV) vector to generate
EphA2 CAR T cells that specifically target and kill EphA2-
positive (EphA2+) OS and ES cell lines in vitro, and using
in vivo subcutaneous OS and ES mouse models demonstrate that EphA2 CAR T-cell administration to animals
with established tumours can result in complete tumour
regression, with extended survival in a dose and delivery
route-dependent manner. In addition, these CAR T cells can
traffic to, and eradicate metastatic deposits of human OS
cells in the livers and lungs of mice when delivered
Materials and methods
Immunohistochemistry analysis of EphA2
expression of tumour samples and normal tissues
EphA2 protein expression was analysed by immunohistochemical (IHC) staining of formalin-fixed paraffin embedded (FFPE) tissues derived from a cohort of paediatric
sarcoma biopsies, assembled in tissue microarray (TMA)
, from in house derived and commercially available
normal tissue TMAs (BioSB, Santa Barbara) and tissues
from the experimental in vivo models described below.
Staining was performed using a BOND-RX automatic
immunostainer (Leica Biosystem) according to standard
protocol. Briefly, samples were sectioned at 4 µm and slide
deparaffinization was performed with Bond Dewax solution
(AR9222, Bond) followed by rehydration and heatmediated epitope retrieval (AR9640, Bond). Slides were
incubated at room temperature for 1 h with EphA2 antibody
(6997, Cell Signaling) diluted in Primary Antibody Diluent
(AR9352, Bond). Staining was visualised using a Polymer
Refine Detection kit (DS9800, Bond) and nuclei were
counterstained with haematoxylin. The slides were scanned
using an Aperio CS2 virtual microscope (Leica Biosystem).
Tumours with ≥10% of cells showing EphA2 membrane
staining (or a combination of membrane and cytoplasmic
staining) of any intensity were considered EphA2-positive
(EphA2+). EphA2 staining intensity was also scored on a
semi-quantitative scale of 0 to 3 as follows: no staining (0),
weakly positive staining (1+), moderately positive staining
(2+) and strongly positive staining (3+).
A humanised EphA2 antibody that binds to the CAR
targeted epitope (#HPAB-0453-CN, Creative Bio-labs)
could not be used in this study due to in-compatibility with
the immunostainer system. Since antigen recognition can
vary between antibody reagents, analysis of paediatric sarcoma biopsies using this antibody would be of particular
relevance in identifying patients with CAR targetable
EphA2 expression in future clinical trials.
Lentiviral construct and vector production
A lentiviral transfer construct to drive expression of a 2nd
generation EphA2 CAR in gene modified T cells was
constructed using a codon-optimised and custom synthesised (GeneArt, Thermo Fisher Scientific) humanised
322 K. Hsu et al.
single-chain variable fragment (scFv) specific for EphA2,
4H5 , upstream of an IgG1 hinge region and followed
by 41BB and CD3ζ as endodomains [42, 43]. The CAR
sequence was inserted downstream of the human Elongation Factor-1α (huEF1-α) promoter within a selfinactivating (SIN) lentiviral backbone (kindly provided by
Dr S, Ginn, Children’s Medical Research Institute, NSW,
Australia). A control EphA2 construct (ΔEphA2 CAR, Δ)
retained the same promoter, scFV, hinge and transmembrane configuration, but with sequences for the cytoplasmic
signalling domains (41BB and CD3ζ) deleted to disable
induction of cytotoxic activity upon antigen engagement.
Two EphA2 CAR vectors, with their respective ΔEphA2
CAR controls, were used in the study, containing an additional transmembrane anchored, truncated protein sequence
downstream of a 2A sequence at the 3′ end of the CAR
cassette to facilitate antibody detection of transduced CAR
T cells. The first of these expressed truncated CD19 peptide
(tCD19)  and the second expressed a custom truncated
epidermal growth factor receptor (tEGFR) . Lentiviral
vector stocks were generated using PEI mediated transfection of 4 plasmids (transfer plasmid, 2 packaging plasmids
and VSV-G envelope plasmid) of 293T cells, and collection
of vector containing supernatant in LV Max Production
medium (Thermo Fisher Scientific). Purification and concentration of vector stocks were undertaken using the
KrosFlo KR2i tangential filtration instrument and mPES/
500 kD column (Repligen). Vector titre was determined
using quantitation of functional transduction of SUP-T1
cells and detection of extracellular tCD19 or tEGFR by
antibody labelling and flow cytometry, or by q-PCR
quantitation of lentiviral backbone sequences in genomic
DNA (gDNA) extracted from transduced HT1080
A panel of human sarcoma cell lines including OS, ES,
rhabdomyosarcoma and other soft tissue sarcomas were
used for experiments in vitro. SJSA, HOS-143B, HOS, 778,
SW872 and SW982 cell lines were kindly provided by A/
Prof Jia Lin Yang (Sarcoma and Nano-oncology Group,
Adult Cancer Program, University of New South Wales,
Sydney). All other cell lines, including the human cancer
cell lines (TF-1 and SUP-T1) and the neuroblastoma cell
line SHEP, were purchased from ATCC (Manassas, Virginia, USA) or DSMZ (Braunschweig, Germany). All cell
lines used throughout were subjected to Short Tandem
Repeat (STR) profiling by CellBank Australia (Westmead,
Australia) to confirm identity.
Cell lines were cultured and maintained in filter capped
tissue culture flasks (Corning, New York), and maintained
in Dulbecco’s Modified Eagle’s Medium (DMEM) or
RPMI 1640 (Life Technologies) supplemented with 10%
heat-inactivated foetal calf serum (FCS) (Life Technologies), and maintained at 37 °C with 5% CO2. Adherent cells
were maintained in exponential growth phase by trypsin
passaging (Trypsin-EDTA 0.05%, Life Technologies),
when required, every 3–4 days. Suspension cells (TF-1 and
SUP-T1) were maintained between 0.3–5 × 105 viable cells/
mL in RPMI 1640 supplemented with 2 ng/mL GM-CSF
(AF300–03, Lonza) and 1% L-glutamine (Life
CAR T-cell production
Human T cells were obtained and used under approved
Human Research Ethics Committee (HREC, Sydney Children’s Hospitals Network) approved protocols. T cells were
sourced from normal donor Buffy Coats (BC) obtained
under a Material Supply Agreement via the Australian Red
Cross Lifeblood. Peripheral blood mononuclear cells
(PBMC) were isolated by centrifugation using Lymphoprep
(7811, Stem Cell Technologies) in accordance with manufacturer’s instructions. T cells were enriched using the
EasySep human CD3 positive selection kit (Stem Cell
Technologies) and cultured in OpTimizer T-cell expansion
medium supplemented with immune cell SR (Thermo
Fisher Scientific) and Interleukin 7 (IL-7) and Interleukin-
15 (IL-15) at 10 and 5 ng/ml respectively (Miltenyi Biotec).
T cells were activated using T cell TransAct (Miltenyi
Biotech) for 1 day prior to transduction with EphA2 and Δ
CAR LV vectors, and further expanded for 8–10 days
before being used in vitro or in vivo assays. Cultured and
transduced T-cell suspensions were labelled for flow cytometric analysis with antibodies to CD3 in combination with
CD19 or EGFR and had an approximate CD4:CD8 ratio of
30:70, with more than 50% memory (central and effective)
T cells as assessed by flow cytometry using CD45RA and
CCR7 as markers.
Adherent tumour cell lines were harvested from flasks using
trypsin and washed with FACS Buffer (0.1% BSA%/0.1%
sodium azide in PBS) prior to antibody labelling for
detection of EphA2 surface expression with PE Mouse
IgG2b, k isotype control or PE anti-human EphA2
(#400313 and #356803, Australian Biosearch) for 30 min at
4 °C in the dark. The murine cell line NIH3T3 was included
as a negative control for EphA2 labelling on adherent cells,
and TF-1 cells (grown in suspension) were included as a
human cancer negative control, as this line does not express
EphA2, unlike the majority of adherent human cancer cell
lines surveyed (data not shown). TF-1 cells were taken
directly from culture and washed with FACS Buffer prior to
Chimeric Antigen Receptor-modified T cells targeting EphA2 for the immunotherapy of paediatric bone. . . 323
labelling as described above. After labelling, cell suspensions were washed in FACS buffer and resuspended for
analysis using a Guava® easyCyte Flow Cytometer (Merck
Millipore). EphA2 positive and negative control cell lines
used to determine the gating regions were SHEP and
NIH3T3 respectively. Forward and side scatter were used to
discriminate live and dead cells. InCyte Software for
Guava® easyCyte Systems (Merck Millipore) was used for
Human EphA2+ sarcoma cell lines and murine NIH3T3
cells (negative control) were co-cultured under different
effector/target cell (E:T) ratios with nontransduced T cells
(T-NT), control ΔCAR T cells (T-Δ) or EphA2- CAR
T cells (T-EphA2) for ~54 h. This time point was determined in preliminary experiments (impedance-based
tumour cell killing, below) as that at which cytotoxicity
could be detected against both OS and ES cell lines over a
range of E:T ratios. At the end of incubation, the plates were
washed three times to remove T cells, and remaining cells
were quantified using the CellTiter-Glo 2.0 assay (Promega)
according to the manufacturer’s instructions. Tumour cell
viability was calculated based on luminescence – (T cell and
tumour cell co-culture – T cell alone)/untreated cancer
cell) × 100%.
Impedance-based tumour cell killing assay
Continuous tumour cell killing was determined and monitored over time using the xCELLigence system (ACEA
Biosciences) as a means of determining kinetics of CAR Tcell response to co-cultivation with tumour cell lines. The
OS cell line 143B and ES cell line A673 were plated in 16-
well E-Plates at 3000 and 5000 cells per well cultured for
20–24 h. CAR-T cells were added at E:T ratios of 1:1, 1:5
and 1:25 with nontransduced T cells (T-NT) or control
ΔCAR T cells (T-Δ) at an E:T ratio of 1:1. EphA2- CAR
T cells (T-EphA2) and tumour cells were co-cultured for an
additional 80–120 h, and cell index values were collected
every 15 min. Cell index data were normalised to the time
point immediately prior to the addition of T cells.
Analysis of cytokine production
Various ratios of EphA2- CAR T cells (T-EphA2), alongside T-NT or control T-Δ cells, were co-cultured with cell
line tumour cells at different E:T ratios for 48 h. Supernatants were then removed and analysed by ELISA for
human Interferon-γ (IFN-γ, R&D systems) and human
Interleukin-2 (IL-2, R&D systems), in accordance to the
Osteosarcoma and Ewing sarcoma subcutaneous
All animal work was carried out under protocols approved
by the Children’s Hospital at Westmead (CHW) and
Children’s Medical Research Institute (CMRI) Animal
Ethics Committee. Mice were purchased from the Animal
Resources Centre (ARC), Perth, Australia, and randomised on arrival to control or treatment groups To
determine the growth kinetics of the OS cell line 143B
and ES cell line A673, 6–8-week-old female NOD scid
gamma (NSG) mice received initial (Day 0) subcutaneous
injections of 5 × 106 tumour cells in 100 µL of Matrigel®
Basement Matrix HC (#354248, BioStrategy) and Dulbecco’s Modified Eagle Medium (DMEM) (#1195–065,
Life Technologies) containing 1% penicillin/streptomycin
(#15140–122, Life Technologies) mixed at 1:1 ratio,
using 27 G × 13 mm insulin syringes (#TER00141, Terumo, Medshop). Tumour measurements (mm3) were taken
three times per week using electronic callipers until
tumour volume (Volume = (Length × Width × Height)/2)
reached an endpoint of 1000 mm3, at which point mice
were humanely euthanased.
To determine CAR T-cell efficacy against established
tumours, NSG mice were first injected subcutaneously with
5 × 106 tumour cells on Day 0. Tumour measurements
(mm3) were taken thrice weekly, using electronic callipers
until the median tumour volume of the cohort reached
100–200 mm3 (measurable tumour) on Day 7 for both
models. At this point, mice were treated with either vehicle
(PBS), control T-Δ cells or T-EphA2 CAR T cells. In the
ES model, an additional group of animals received T-NT
control cells, cultured in parallel with the CAR T cell preparations. Vehicle or T cell suspensions were administered
either directly into the tumour (intratumoural, IT) via two
injections into the capsule surrounding the tumour, or
intravenously (IV) via the tail vein, in a total volume of
40 µL (IT) or 200 µL (IV) using 0.5 mL 27 G × 13 mm
insulin syringes. Mice were monitored for a maximum
holding time of 90 days or until experimental endpoints of
tumour volume ≥1000 mm3 or weight loss (g) ≥10%.
Experiment sample sizes were calculated to detect (80%
power) a minimum one third reduction in tumour size from
1000 ± 250 mm3 due to treatment, with a probability of
Type I error = 0.05.
Osteosarcoma metastatic model
6–8-week-old female NSG mice were injected via the
tail vein with a suspension of GFP expressing 143B
cells (2.5 × 105) at Day 0, a dose of tumour cells which
reliably produces >25 metastases in the lungs at Day 14.
Seven days after injection, mice were treated with either
324 K. Hsu et al.
vehicle (PBS), nontransduced T cells (T-NT), ΔCAR
T cells (T-Δ) or EphA2 CAR T cells (T-EphA2) which
were administered via the tail vein, 5 × 106 CAR T cells
(200 µL) using a 0.5 ml 27 G × 13 mm insulin syringes.
On Day 14–16 mice were humanely euthanased and the
lungs and liver of each mouse were harvested, and GFP
expressing metastatic nodules visible in these organs
were counted with the aid of a Nikon SMZ800 dissecting microscope and UV light (BioRad) by technical
staff blinded to treatment group. Organs were then fixed
in buffered neutral formalin 10% (Sigma Aldrich,
Australia) and embedded in paraffin. FFPE specimen
blocks were serially cut into 4-µm-thick sections and
stained with Haematoxylin and Eosin (H&E) using
standard histological methods. For human IHC antihuman CD3 staining of sections, slides were subjected
to low pH epitope retrieval treatment followed by 1 h
incubation with a 1:200 dilution of the anti-human CD3
(ab5690, Abcam), at room temperature, using the
BOND-RX automatic immunostainer protocol described above for EphA2 staining. For further quantitative
assessment of liver metastases, H&E stained slides were
scanned using an Aperio CS2 virtual microscope (Leica
Biosystems) and the Aperio GENIE Image Analysis
Tool (Leica Biosystems) was used to classify tumour
regions and determine the percentage of tumour compared with surrounding normal background tissue (data
not shown). For analysis of T cell infiltration into lung
and livers of mice, CD3+ T cell density of normal tissues, metastatic deposits and combined tissues,
respectively, were scored manually and calculated as an
average number of infiltrating CD3+ cells out of three
representative high-power fields. Experiment sample
sizes were calculated to detect (80% power) a reduction
in metastatic burden of 50% in treatment groups from a
mean of 25 ± 10 nodules per organ in control groups,
with a probability of Type I error = 0.05.
Graph-Pad Prism version 6.0 (GraphPad Software, Inc.)
was used for all statistical analyses. Log-rank (Mantel–Cox)
test was used for survival curve comparisons. Two-tailed
Student’s t tests and two-way ANOVA tests were used to
compare experimental groups where indicated in Figures
and Tables. The number of donors used for experiments, the
number of independent experiments performed and the
number of mice per group are given in Figure legends. P <
0.05 was considered to indicate a statistically significant
difference between groups.
Fig. 1 Expression of tumour-associated antigen target EphA2. a
IHC staining of diagnostic OS tumour sections showing weak/Intermediate (left) compared with strong (right) EphA2 staining (brown)
against the blue counterstain. b IHC staining of diagnostic ES tumour
sections showing weak/intermediate (left) compared with strong (right)
EphA2 staining (brown) in against the blue counterstain. Scale bars
(200 µm) are shown on the bottom left of each panel. c Flow cytometric analysis of EphA2 expression on adult and paediatric sarcoma
cell lines (X axis). Bars indicate mean ± SEM of the MFI (Y axis) for at
least three independent experiments. SHEP (neuroblastoma) and TF-1
(erythroleukemia) cell lines were used as positive (SHEP) and negative
(TF-1) controls, respectively, for antibody labelling.
Chimeric Antigen Receptor-modified T cells targeting EphA2 for the immunotherapy of paediatric bone. . . 325
EphA2 expression in patient tumour samples and
TMA slides containing biopsy cores from 31 OS and 39 ES
cases were stained and scored for EphA2 as described
above. In the overall patient cohort, 16/70 cases (23%) were
found to be EphA2-positive. The distribution of weak and
moderate/strong EphA2 expression was 10/70 (14.5%) and
6/70 cases (8.5%), respectively. OS cases showed EphA2
expression more commonly (14/31, 45%) than ES cases (2/
39, 5%). Of the 14 positive OS cases, 9/14 (64%) were
weakly positive, while 5/14 (36%) were moderately to
strongly EphA2-positive. The two positive ES cases were
one each of the weak and moderate/strong category.
Examples of IHC membrane staining in OS and ES cases
are shown in Fig. 1a, b respectively. There was no significant association between EphA2 expression and disease
stage at clinical presentation, biopsy site (primary or
metastasis) or whether biopsies were obtained before or
after chemotherapy (Supplemental Table 1).
Across the two TMAs containing normal tissues, all
tissues tested were negative for EphA2 staining (Supplemental Fig. 1), including brain, skin, skeletal muscle, breast,
thyroid, liver, haematopoietic tissues (bone marrow, thymus, lymph node, spleen and tonsil), genitourinary system
tissues (adrenal gland, kidney, bladder, testes, prostate,
EF- 4H5 (EphA2)
T-NT cells T-Δ cells T-EphA2 cells
EGFR Alexa Fluor 488
T-NT cells T- Δ cells
Fig. 2 Vector schematics and LV CAR T-cell transduction efficiencies. a Schematic diagrams showing the EphA2 (T-EphA2) and
ΔCAR (T-Δ) constructs used to generate LV vectors for transduction
of T cells. For each A2 construct (containing either the tCD19 OR
tEGFR sequence downstream of the CAR sequence) an equivalent
control construct was used to generate Δ CAR T cells in all experiments. b Representative flow cytometry dotplots showing dual CD3
(FITC, Y axis) and tCD19 (PE, X axis) labelling of control nontransduced (T-NT) T cells, ΔCAR (T-Δ) or EphA2 CAR (T-EphA2)
T cells generated from the same donor. c Representative flow cytometry dotplots showing tEGFR (Alexa Fluor 488, X axis) against
Forward Scatter (Y axis) labelling of control nontransduced (T-NT)
T cells, ΔCAR (T-Δ) or EphA2 CAR (T-EphA2) T cells generated
from the same donor.
326 K. Hsu et al.
ovary and placenta), digestive system tissues (appendix,
stomach, colon, salivary gland and pancreas) and cardiovascular tissues (heart and lung).
EphA2 expression on sarcoma cell lines
All sarcoma cell lines tested by flow cytometry were positive for EphA2 protein expression when compared with a
positive control neuroblastoma cell line (SHEP) and the
negative control cell line TF-1 (Fig. 1c). OS Cell lines
143B, U2OS and MG63, ES lines A673 and ES8 and the
murine fibroblast cell line NIH3T3 (as a negative control)
were used for subsequent in vitro assays for EphA2 CAR T
cell (T-EphA2) efficacy. Cell lines 143B and A673 were
used in in vivo assays for EphA2 CAR T cell (T-EphA2)
Generation of EphA2 CAR T cells using LV vector
Gene transfer efficiencies for CD3/CD28 activated T cells
with either the EphA2CAR-tCD19 and EphA2CAR-tEGFR
LV (Fig. 2a) ranged between 35 and 81% (tCD19 vector)
and 5 and 62% (tEGFR vector). CAR T cells were further
expanded for up to 10 days prior to use in experiments,
typically with ~500-fold expansion. Control ΔCAR T cells
(T-Δ) transduced with the ΔEphA2 (Δ) CAR constructs
showed equivalent proportions of transduced cells. The
positive tCD19 (Fig. 2b) or tEGFR (Fig. 2c) transduced
CAR T cells was used to determine CAR T cell doses for
in vitro assay and in vivo delivery.
EphA2 CAR T-cell activation and cytotoxicity against
EphA2 expressing sarcoma cell lines in vitro
Forty-eight hour incubation of EphA2 CAR T cells with 3
OS and 2 ES cell grown in monolayer culture caused significant (P < 0.01) cytotoxicity at E:T ratios of between 1:1
and 1:27 (Fig. 3). No cytotoxic effect was seen after coculture with nontransduced T cells (T-NT) or ΔCAR T cells
(T-Δ), or after co-culture of EphA2 CAR T (T-EphA2) cells
with the EphA2 negative murine NIH3T3 cell line. This
potent effect was seen across four independent donors.
Quantitation of both IL-2 (Fig. 4a) and IFN- γ (Fig. 4b)
production by EphA2 CAR T cells, T-NT and T-Δ cells
over the same range of E:T ratios, following co-culture with
the same panel of tumour cell lines, showed significantly
higher levels (P < 0.05) of cytokine secretion by EphA2
CAR T cells over levels produced by control cells, indicative of antigen-specific tumour cell recognition by EphA2
CAR T cells. These differences peaked at E:T ratios of 1:1
and 1:3 with detectable cytokine secretion diminishing as
the number of CAR T cells relative to tumour cells declined.
In addition, the xCelligence real-time cell analysis assays
monitoring co-culture of T-EphA2 or control T cells with
either the OS cell line 143B or the ES cell line A673 over
the same time period similarly demonstrated that T-EphA2
Fig. 3 In vitro assessment of anti-tumour activity of CAR T cells
targeted against EphA2. Percentage (%) Tumour cell viability (Y
axes), quantitated using a CellTiter-Glo 2.0 assay after 48 h of coculture with IL-15/IL-17 expanded nontransduced (T-NT) T cells,
ΔCAR (T-Δ) or EphA2-targeted CAR (T-EphA2) T cells at the indicated E:T ratios (X axes) in the absence of exogenous cytokines. At
each point, percentage cell survival has been expressed relative to
untreated tumour cells (set as 100% viability). NE no effector cell. The
negative control cell line NIH3T3 (top left) does not express EphA2.
Data show the mean ± SEM from independent CAR T and control cell
preparations generated from PBMC of four different donors (biological replicates). For each cell line, mean values at each E:T ratio were
compared between T-EphA2 with either T-NT or T-Δ using a two-way
ANOVA (with Tukey’s multiple comparisons test) with statistically
significant differences indicated: *P < 0.05, **P < 0.01, ***P < 0.001,
****P < 0.0001.
Chimeric Antigen Receptor-modified T cells targeting EphA2 for the immunotherapy of paediatric bone. . . 327
cells, but not T-NT or T-Δ, were effective at killing tumour
cells at E:T ratios of 1:1, 1:5 and 1:25 (Supplemental Fig.
2). This data is in line with previously reported potencies for
EphA2 CAR T cells generated using gamma-retroviral
constructs containing the same humanised EphA2 antibody
sequence [41, 42] but targeting EphA2+ glioma cell lines. It
demonstrates that constitutive expression of the EphA2
CAR molecule driven by the huEF1-α promoter can drive
the required response to EphA2 expressing sarcoma cell
lines to kill this tumour type at low E:T ratios, with effective
and specific activation upon antigen engagement.
EphA2 CAR T cell targeting of OS and ES cells in
established tumours in vivo
Two subcutaneous tumour cell models, one using the OS
cell line 143B and the second using the ES cell line A673,
were used to test the in vivo efficacy of Epha2 directed
CAR T cells in NSG mice (Fig. 5). In both models,
intratumoural (IT) injection of 5 × 106 EphA2 CAR
T cells per tumour (T-EphA2) resulted in tumour regression and eradication (Fig. 5a), whereas in mice receiving
treatment with ΔCAR T cells (T-Δ), nontransduced T cells
(T-NT) or vehicle (PBS), tumour growth continued in line
with previously established kinetics, resulting in euthanasia of tumour bearing mice at a median of 21 days after
tumour cell inoculation (Fig. 5a, b). Significant differences in size (P < 0.01) between regressing tumours in TEphA2 treated animals and growing tumours in control
treated mice were first evident at 8 and 9 days following
treatment in the OS and ES models respectively. Animals
receiving this dose of T-EphA2 showed extended survival
out to a mean of 74 days and 88 days in the OS and ES
models respectively (Fig. 5b). IT delivery of lower doses
of T-EphA2 cells (5 × 105 and 5 × 104 per tumour) in the
OS model did not have a significant effect on tumour
growth (data not shown) and these doses were omitted
from testing in the ES model.
IL – 2
Fig. 4 In vitro production of
IL-2 and IFN-γ by EphA2-
targeted CAR T cells.
Nontransduced (T-NT) T cells,
ΔCAR (T-Δ) or EphA2 CAR (TEphA2) T cells were cultured in
the absence of exogenous
cytokines in medium alone
(medium), negative control cells
(NIH3T3) or sarcoma cell lines
at indicated E:T ratios (X axes).
Supernatant collected after
48 hours was assayed for a IL-2
and b IFNγ production, and
quantitated by ELISA (Y axes).
Data show the mean ± SEM
from four independent T cell
donors (biological replicates).
For each cytokine and cell line,
results for two-way ANOVA
comparisons between T-EphA2
with either T-NT or T-Δ
conditions (with Tukey’s
multiple comparisons test) with
differences indicated: *P < 0.05,
**P < 0.01, ***P < 0.001 and
****P < 0.0001.
328 K. Hsu et al.
Tumour volume mm3 Tumour volume mm3
V T-∆ T-EphA2, 5 x 106 T-EphA2, 2 x 107 T-NT
a b c d
Days post inoculaon of tumour cells Days post inoculaon of tumour cells
Osteosarcoma Model Ewing Sarcoma Model
Fig. 5 Subcutaneous tumour growth and survival curves for OS
and ES tumour-bearing mice. a Tumour growth curves (volume,
mm3 over time) for subcutaneous OS tumours (left) and ES tumours
(right) in mice receiving EphA2 CAR T cells (T-EphA2) or control
treatments (PBS, T-Δ, NT-T) intra-tumourally on Day 7 (dotted line).
Mean ± SD for each group of animals is shown, n = 12 for the OS
model and n = 6 for the ES model. *-* indicates period over which
significant differences in mean tumour volume between T-EphA2 and
control treated tumours were seen (t-test with Holm-Sidak correction
for multiple comparisons). b Kaplan Meier survival curves for OS
(right) and ES (left) tumour bearing mice receiving EphA2 CAR
T cells (T-EphA2) or control treatments (PBS, T-Δ, NT-T) intratumourally. Statistical comparisons shown are between T-EphA2 and
control T-Δ treatments using the Log-Rank (Mantel–Cox) Test. c
Tumour growth curves (volume, mm3 over time) for subcutaneous OS
(right) and ES (left) tumours in mice receiving T-EphA2 CAR T or
control treatments (PBS, T-Δ or T-NT) intravenously on Day 7 (dotted
line). Mean + /- SD are shown for groups of mice, n = 6 mice per
group. *-* indicates period over which significant differences in mean
tumour volume between T-EphA2 and control treated tumours were
seen (t-test with Holm-Sidak correction for multiple comparisons). d
Kaplan–Meier survival curves for OS (right) and ES (left) tumourbearing mice receiving T-EphA2 or control treatments (PBS, T-Δ or TNT) intravenously. Statistical comparisons shown are between TEphA2 and control T-Δ treatments using the Log-Rank
Chimeric Antigen Receptor-modified T cells targeting EphA2 for the immunotherapy of paediatric bone. . . 329
By contrast with IT delivery, intravenous (IV) injection
of 5 × 106 EphA2 CAR T cells did not result in any significant tumour regression (Fig. 5c), nor extension of survival (Fig. 5d) in the OS model. In the ES model, this dose
led to a short (39 day) delay in tumour volume reaching the
experimental endpoint, but not tumour regression. However, IV administration of a higher dose of 2 × 107 cells/
mouse of T-EphA2 CAR T cells did result in tumour
regression and extended survival (Fig. 5c, d) in both OS and
ES models. Significant differences (P < 0.01) in tumour
volume between T-EphA2 and control treatments was first
seen in the ES model 15 days after treatment. In the OS
model, all tumours regressed to become undetectable,
however, due to the variability in tumour volume measured
throughout this process, no statistical differences were seen
between control and treated animals prior to control animals
being euthanased. As for experiments testing IT delivery, in
all animals receiving control treatments via IV delivery,
tumours grew according to previously established kinetics
resulting in euthanasia of animals at a median of 21 days
following tumour inoculation.
Mice with tumours cleared by EphA2 CAR T cell (TEphA2) administration were held for a maximum of
100 days post injection, and a proportion of these developed
histologically confirmed graft versus host disease (GVHD)
from approximately day 50. Signs of GVHD included
progressive weight loss, fur loss and eczema. This was
treated by administration of topical steroids and antibiotics
as directed by a veterinarian. Animals with GVHD were
euthanased when necessary according to ethical guidelines,
resulting in a decrease in surviving animal numbers after
50 days in the EphA2 CAR T treated group in the OS model
(Fig. 5b, d). The occurrence of GVHD was donor dependent
with all affected mice confined to experiments using T cells
derived from one (of two) donors.
Since animals that received control ΔCAR T cells or
nontransduced T cells were euthanased well before GVHD
was seen in EphA2 CAR T treated animals, it is difficult to
determine whether this side-effect of treatment was due to
non-specific EphA2 CAR T activity, directed through
antigen cross-reactivity, or due to more generalised human
vs mouse reactivity. We did note some positive non-specific
labelling for human EphA2 protein by IHC in mouse lung
and liver sections (Supplemental Fig. 1), in contrast to the
equivalent human tissues, indicating that EphA2 CAR T
cell cross-reactivity was a possibility. Staining for EphA2
expression using the humanised EphA2 antibody used for
generation of the EphA2 CAR in mouse tissues could elucidate the underlying mechanism for this side-effect and
assist in refining future pre-clinical studies.
EphA2 CAR T-cell efficacy against metastatic
osteosarcoma cells in vivo
GFP expressing 143B cells were used to establish disseminated metastatic disease in the livers and lungs of
recipient female NSG mice for in vivo testing of efficacy of
EphA2 directed CAR T cells (T-EphA2). In initial dose
finding studies, I.V. delivery of 2.5 × 105 GFP+ 143B cells
resulted in a substantial and reproducible metastatic burden
in both the lungs and livers of animals when assessed
14–16 days following injection (Supplemental Fig. 3). In
two experiments using independent donors, intravenous
delivery of 5 × 106 EphA2 CAR T cells (T-EphA2)/mouse
7 days after tumour cell inoculation resulted in either an
absence (donor 1T cells) of or a significant decrease (donor
2T cells) in the number of visible metastatic nodules
counted in both the lungs and livers of mice in comparison
to animals receiving ΔCAR T cells (T-Δ), nontransduced
T cells (T-NT) or vehicle (PBS; Fig. 6). Although it was of
No. metastatic lung deposits
No. metastatic liver deposits b
Fig. 6 EphA2 CAR T cells reduce metastatic osteosarcoma tumour
burden in mice. a Mean Day 14 macroscopic counts of GFP-positive
143B metastases on the external surfaces of lungs of mice receiving
EphA2 CAR T (T-EphA2) cells or control treatments (PBS, T-Δ or
NT-T) on Day 7. b Mean Day 14 macroscopic counts of GFP+ 143B
metastases on the external surfaces of livers of mice receiving EphA2
CAR T (T-EphA2) cells or control treatments (PBS, T-Δ or NT-T) on
Day 7. Error bars indicate ±SEM for two experiments using two
independent donors for T cells, n = 9 mice per group for donor 1 and
n = 4 mice per group for donor 2. Statistical comparisons shown
indicate significant differences (*P < 0.05, ****P < 0.0001, Student’s t
test) between mean values for treatment groups (T-Δ or T-EphA2) with
the NT-T control.
330 K. Hsu et al.
interest to test whether EphA2 CAR T-cell administration
could prolong survival in this model, assessment of metastatic burden for all treatment groups was routinely performed between 7 and 9 days post treatment (14–16 days
post tumour cell inoculation). The human 143B osteosarcoma cell line used in this model is highly efficient at
seeding metastatic deposits in both the lungs and livers of
recipient mice, and this end point was chosen to avoid
animals succumbing to high disease burden unexpectedly,
despite daily welfare monitoring.
T-cell infiltration and density in livers and lungs
Immunohistochemistry for human CD3 within sections of
liver and the lungs of mice-bearing metastases provided
direct evidence for the specificity of EphA2 CAR T-cell (TEphA2) targeting EphA2+ OS cells and their capacity to
traffic to disseminated tumour deposits in vivo. In both lung
and liver sections, T-cell density was highest in tissues close
to metastatic tumour deposits in mice receiving EphA2
CAR T cells when compared with those receiving either
nontransduced T cells (T-NT) or ΔCAR T cells (T-Δ; Fig.
7). In the lungs, EphA2 CAR T cells were preferentially
observed to be clustered around metastatic tumour deposits,
and depleted in number across areas of normal tissue (Fig.
7a, b). In the liver (Fig. 7c, d), CD3 + T cells were observed
both in association with, and not associated with metastatic
deposits in mice-receiving nontransduced (T-NT) and
ΔCAR T cells (T-Δ). By contrast, in mice receiving EphA2
CAR T cells (T-EphA2), CD3+ T cells were significantly
associated with metastatic tumours, at high density. In the
liver, the density of T cells in non-metastatic, normal
regions of the tissue was low. For one of two CAR T cell
PBS T – NT cells
T – A2 cells T- Δ cells
Normal Tissue Metastasis
Normal Tissue Metastasis
PBS T – NT cells
T- Δ cells T – A2 cells
Fig. 7 CAR T cell infiltration into lungs and livers of NSG mice. a
IHC staining for human CD3 in sections of lungs harvested at Day 14
from NSG mice treated with EphA2 CAR T (T-EphA2) cells or
control treatments, PBS, nontransduced T cells (T-NT) and ΔCAR
cells (T-Δ), on Day 7. Scale bar, 200 µm. Top row shows the extent of
T cell infiltration close to metastatic OS deposits in lungs of mice and
the bottom row shows T cell infiltration into surrounding normal tissue
for each treatment group. b IHC staining for human CD3 in sections of
livers harvested at Day 14 from NSG mice treated with EphA2 CAR T
(T-EphA2) cells or control treatments, PBS, nontransduced T cells (TNT) and ΔCAR cells (T-Δ), on Day 7. Scale bar, 5 µm. Top row shows
the extent of T cell infiltration in livers of receiving PBS (left) or
nontransduced (T-NT) control T cells (right), bottom row shows T cell
infiltration into lungs of mice receiving ΔCAR T cells (T-Δ) (left) or
EphA2 CAR T cells (T-EphA2) (right). Enlarged images taken from
were indicated in each section. c Quantitation of T-cell density in
normal, non-metastatic lung tissue compared with metastatic deposits.
d Quantitation of T-cell density in normal, non-metastatic liver tissue
compared with metastatic deposits. For both c and d, means are shown
for counts (CD3 positive T cells over 3 high-power fields, Y axis)
performed on tissue harvested from six mice from each group. Error
bars show ±SD. Statistical comparisons shown indicate significant
differences (**P < 001, ***P < 0.001, Student’s t test) between mean
values for treatment groups as indicated by the bars, ns no significant
Chimeric Antigen Receptor-modified T cells targeting EphA2 for the immunotherapy of paediatric bone. . . 331
donors used in this study, for which residual metastases
were counted at Day 14, staining of liver and lung sections
for EphA2 expression indicated that a proportion of residual
metastatic cells were negative, or below the level of
detection for IHC EphA2 staining (Supplemental Fig. 4).
However, EphA2+ residual tumour cells were also
observed, indicating that for this donor, with apparently
lower potency CAR T cells, an increased dose, or an
alternative dosing schedule may have been necessary to
completely eradicate established EphA2+ metastatic
While CAR T-cell therapy has been a highly effective
immune therapy for leukaemia, the treatment remains to be
translated effectively into the solid tumour context. Our work
demonstrates that EphA2 directed CAR T cells can effectively
kill EphA2 expressing OS and ES tumour cells in preestablished, localised xenografts in immune deficient mice,
associated with prolonged survival. In addition, EphA2 CAR
T cells caused a significant reduction or elimination of tumour
burden in a mouse model of disseminated OS metastases. In
the subcutaneous models of localised tumours, the effect was
dose and route of delivery dependent, with localised delivery
requiring a lower dose than systemic delivery, pointing to a
need to carefully consider the route of delivery to these
tumour types in patients. In the metastatic OS model, however, EphA2 CAR T-cells delivered systemically were capable of migrating into and disseminating throughout murine
lung and liver to effectively target metastatic tumour deposits.
Although nontransduced (T-NT) and Δ CAR (T-Δ) T cells
were also observed within these tissues, no reduction in the
numbers of metastatic nodules was observed in animals
receiving these control T-cell preparations. Our metastatic
model outcomes also highlighted variation in EphA2 CAR Tcell effect in preparations derived from different donors,
providing evidence that donor-specific factors will materially
affect CAR T-cell efficacy when this methodology is translated into the clinic. Compounding this variability, which may
relate to both intrinsic differences between donors, or extrinsic
factors such as PBMC harvesting and/or CAR T-cell manufacturing culture and transduction conditions will be the heterogeneity of EphA2 antigen expression within tumours. In
our metastatic OS model, EphA2 negative tumour cell
deposits were evident among those remaining after EphA2
CAR treatment, supporting predictions from the field that
antigen down-regulation or absence within tumours will likely
require administration of multivalent CARs or combinatorial
approaches using CARs to alternative TAAs.
As with other TAAs expressed in solid tumours, this study
identified variability between patients in relation to EphA2
expression (weak to strong) in addition to patients where no
EphA2 expression was observed. Although almost half of the
OS patient samples in our cohort expressed EphA2, only a
minor percentage of ES tumours did so. In contrast with other
TAAs being targeted for solid tumours that show some level
of normal tissue distribution , importantly, our data shows
that EphA2 protein expression is absent across a broad range
of normal tissues. The results of recently completed and ongoing early phase clinical trials targeting EphA2 expressing
gliomas will provide safety data regarding EphA2 as a
clinically applicable TAA for sarcoma. Our tEGFR EphA2
CAR construct is designed to be targetable by Cetuximab
(Erbitux) , an inhibitor of epidermal growth factor
receptor (EGFR), to provide a mechanism by which offtumour, but on-target toxic effects can be mitigated.
Recurrent bone sarcomas of childhood are low survival
malignancies, and new therapies for these patients are needed
to bring about the improvements in survival seen in other
types of childhood cancers over the past decades. Our study
provides pre-clinical evidence that EphA2 is a valuable target
for the development of CAR T-cell therapies for sarcomas,
adding another antigen to a growing list that will be needed to
comprehensively cover this heterogeneous group of solid
tumours. Alongside our demonstration of specificity and a
capacity for EphA2 CAR T cells to find their target in tissues
harbouring metastatic sarcoma tumour cells, we could find no
evidence for antigen expression that would predict for significant off tumour effects. In agreement with studies in brain
tumour models, our data shows superior efficacy when the
CAR T cells are delivered locally to the tumour, underlining
the need for consideration of delivery route for the design of
early phase clinical trial protocols for safety testing. Further
work focussing on combinatorial testing of EphA2 CAR
T cells with CAR T cells targeting alternative antigens, with
immune checkpoint blockade and with current conventional
chemotherapy agents will elucidate a way forward in
designing an effective CAR T-cell therapy for osteosarcoma
and Ewing sarcoma.
Acknowledgements The authors gratefully acknowledge the provision
of tumour TMA’s from the Sydney Children’s Tumour Bank Network
Funding This work was funded by project grants received from The
Kid’s Cancer Project (TKCP), Cancer Institute NSW through The
Kid’s Cancer Alliance (KCA) and The Australian and New Zealand
Sarcoma Association (ANZSA).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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