O iginal A icle
T Cell-Ac i a ing Mesenchymal S em Cells
as a Bio he apeu ic o HCC
A pad Szoo ,
1,2,3
Abishek Vaidya,
1,2,3
Mi eya Paulina Velasquez,
1,2,3
Zhuyong Mei,
1,2,3
Daniel L. Gal an,
1
Da id To es,
1
Ad ian Gee,
1,2,3
And as Heczey,
1,2,3
and S ephen Go schalk
1,2,3,4
1
Cen e o Cell and Gene The apy, Texas Child en’s Hospi al, Hous on Me hodis , Baylo College o Medicine, Hous on, TX 77030, USA;
2
Texas Child en’s Cance Cen e ,
Texas Child en’s Hospi al, Baylo College o Medicine, Hous on, TX 77030, USA;
3
Depa men o Pedia ics, Baylo College o Medicine, Hous on, Texas 77030, USA;
4
Depa men o Pa hology and Immunology, Baylo College o Medicine, Hous on, TX 77030, USA
The ou come o ad anced s age hepa ocellula ca cinoma
(HCC) emains poo , highligh ing he need o no el he apies.
Gene ically modified mesenchymal s em cells (MSCs) a e
ac i ely being explo ed as cance he apeu ics due o hei
inhe en abili y o mig a e o umo si es. We easoned ha
MSCs can be gene ically modified o edi ec T cells o Glypi-
can-3 (GPC3)
+
HCC, and gene ically modified hese wi h i al
ec o s encoding a GPC3/CD3 bispecific T cell engage
(GPC3-ENG), a bispeci c T cell engage specific o an i ele an
an igen (EGFR III), and/o cos imula o y molecules (CD80 and
41BBL). Cocul u e o GPC3
+
cells,GPC3-ENG MSCs, and T cells
esul ed in T cell ac i a ion, as judged by in e e on g(IFNg)
p oduc ion and killing o umo cells by T cells. Modifica ion
o GPC3-ENG MSCs wi h CD80 and 41BBL was equi ed o
an igen-dependen in e leukin-2 (IL-2) p oduc ion by T cells
and esul ed in as e umo cell killing by edi ec ed T cells.
In i o, GPC3-ENG MSCs ±cos imula o y molecules had an i-
umo ac i i y in he HUH7 HCC xenog a model, esul ing in a
su i al ad an age. In conclusion, MSCs gene ically modified o
exp ess GPC3-ENG ±cos imula o y molecules edi ec T cells o
GPC3
+
umo cells and ha e po en an i umo ac i i y. Thus,
u he p eclinical explo a ion o ou modified app oach o
GPC3- a ge ed immuno he apy o HCC is wa an ed.
INTRODUCTION
Hepa ocellula ca cinoma (HCC) is he hi d leading cause o cance
dea hs wo ldwide, wi h o e 500,000 people a ec ed. The majo i y o
pa ien s a e diagnosed wi h agg essi e ad anced disease, which has an
o e all 5-yea su i al a e o less han 15%.
1
Ac i a ing he immune
sys em o he apeu ic benefi holds he p omise o imp o e ou comes
o HCC because i does no ely on he cy o oxic mechanisms o con-
en ional he apies.
Glypican 3 (GPC3),
2
a glycophospha idylinosi iol-linked memb ane-
associa ed p o ein, is a p omising immuno he apeu ic a ge o HCC.
I plays an impo an ole in g ow h and dedi e en ia ion o HCC,
3,4
and is exp essed in 67%–90% o umo s, bu no in heal hy, adul
no mal issues.
2,5
The GPC3-specific monoclonal an ibody (mAb)
GC33 has been e alua ed in ea ly phase clinical s udies. In usion o
GC33 was sa e; howe e , only limi ed an i umo ac i i y was obse ed
ha co ela ed wi h he in ensi y o GPC3 exp ession.
6
One s a egy o
imp o e he an i umo ac i i y o GPC3- a ge ed immuno he apies is
o exp ess GPC3-specific chime ic an igen ecep o s (GPC3-CARs) o
T cell ecep o s on T cells. Indeed, GPC3-specific T cells had po en
an i umo ac i i y in p eclinical HCC models,
7–9
and clinical phase I
es ing in humans is in p og ess. Howe e , he b oade applica ion
o au ologous cell p oduc s, such as CAR T cells, may ul ima ely be
limi ed because hese cell p oduc s a e no eadily a ailable and
equi e a significan “on si e”in as uc u e o p oduce.
Allogeneic “o - he-shel ”cell p oduc s, including mesenchymal s em
cells (MSCs), ha e he po en ial o o e come hese limi a ions.
Human MSCs a oid allo ecogni ion and, due o hei inhe en abili y
o a fic o umo si es, a e ac i ely being explo ed o deli e cy o oxic
payloads o cance cells.
10–15
Fo example, o HCC, i has been
shown ha p oduc ion o he chemokines chemokine (C-C mo i )
ligand 2 (CCL2) and chemokine (C-X-C mo i ) ligand 8 (CXCL8)
by HCC p omo es MSC mig a ion o umo si es.
16
He e, we epo he gene a ion o MSCs ha a e gene ically modified
o exp ess bispecific T cell engage s ha consis o one single chain
a iable agmen (scF ) specific o GPC3 and a second scF specific
o CD3 (GPC3-ENG). MSCs exp essing GPC3-ENG (GPC3-ENG
MSCs) edi ec ed T cells o GPC3
+
umo cells, as judged by cy okine
p oduc ion and cy oly ic ac i i y. GPC3-specific T cell ac i a ion by
GPC3-ENG MSCs was u he enhanced by he p o ision o CD80
and 41BBL cos imula ion. In addi ion, GPC3-ENG MSCs induced
umo eg ession in an HCC xenog a mouse model, which was asso-
cia ed wi h a significan su i al ad an age.
RESULTS
GPC3-ENG MSCs Redi ec T Cells o GPC3
+
Tumo Cells
We gene ically modified human MSCs wi h VSVG-pseudo yped
len i i al ec o encoding GPC3-ENG and GFP (Figu e 1A). Mean
Recei ed 22 July 2017; accep ed 25 July 2017;
h p://dx.doi.o g/10.1016/j.om o.2017.07.002.
Co espondence: S ephen Go schalk, Cen e o Cell and Gene The apy, Baylo
College o Medicine, 1102 Ba es S ee , Sui e 1770, Hous on, TX 77030, USA.
E-mail: [email p o ec ed]
Molecula The apy: Oncoly ics Vol. 6 Sep embe 2017 ª2017 The Au ho s. 69
This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/).
ansduc ion e ficiency was 93.3% ( ange: 86.1%–97.8%; n = 6), as
judged by fluo escence-ac i a ed cell so ing (FACS) analysis (Figu es
1B and 1C). To quan i y GPC-ENG molecules in cell cul u e media,
we de eloped an ELISA using ecombinan GPC3-ENG p o ein as
a s anda d. Al hough indi idual GPC3-ENG MSCs sec e ed a mean
o 81 pg ( ange: 60.4–94.33) o GPC3-ENG p o ein pe cell in
24 h , no GPC3-ENG p o ein was de ec ed in he media o non- ans-
duced (NT) MSCs (Figu e 1D). Pheno ypic analysis o GPC3-ENG
MSCs e ealed no significan change in pheno ype o NT MSCs, as
judged by cell adhe ence, fib oblas mo phology, and exp ession o
MSC su ace ma ke s (CD90 R95%; CD105 R95%; CD45 %1%;
Figu e S1).
To demons a e ha GPC3-ENG MSCs edi ec T cells o umo cells
exp essing he a ge an igen, we used GPC3
+
(HUH7 and G401) and
GPC3

(A549) cell lines (Figu e S2). To show ha GPC3-ENG bind
GPC3
+
umo cells as well as T cells, we incuba ed cells wi h condi-
ioned media om GPC3-ENG exp essing MSCs o NT MSCs. Cells
we e hen washed and incuba ed wi h ecombinan GPC3-Fc ( o
T cells) o CD3e-Fc ( o umo cells) p o ein. Bound ecombinan
p o ein was de ec ed wi h an Fc an ibody. The Fc an ibody eadily
bound o T cells and umo cells ha had been ini ially incuba ed
wi h media GPC3-ENG-exp essing MSCs. In con as , no binding
was de ec ed o cells ha had been incuba ed wi h media om NT
MSCs, indica ing ha GPC3-ENGs eadily bind o T cells wi h hei
CD3-specific scF and o umo cells wi h hei GPC3-specific scF
(Figu e S3). Fo co-cul u e assays, we gene a ed NT MSCs, GPC3-
ENG MSCs, o MSCs gene ically modified wi h an ENG molecule
ecognizing an i ele an an igen (EGFR III-ENG MSCs). These
we e mixed wi h umo cells a MSC o umo cell a ios o 1:3 and
1:10 in he p esence o human T cells. A e 24 h , media was collec ed
and he p esence o in e e on g(IFNg) and in e leukin-2 (IL-2) was
de e mined by ELISA. GPC3-ENG MSCs induced T cells o p oduce
significan amoun s o IFNg(p < 0.05) only in he p esence o GPC3
+
umo cells in con as o EGFR III-ENG MSCs (Figu e 2A). How-
e e , GPC3-ENG MSCs did no induce T cells o p oduce IL-2 (Fig-
u e 2B). To assess he abili y o GPC3-MSCs o induce T cell killing o
umo cells, we cocul u ed MSCs in he p esence o T cells and umo
cells ha we e gene ically modified o exp ess enhanced GFP (eGFP)
Figu e 1. Gene a ion o GPC3-ENG MSCs
(A) Scheme o len i i al ec o encoding GPC3-ENG and
GFP. (B and C) Rep esen a i e FACS diag am and sum-
ma y da a (GPC3-ENG MSCs [n = 6] and NT MSCs [n = 6])
o GFP exp ession pos - ansduc ion. (D) De ec ion o
GPC3-ENG p o ein in media o GPC3-ENG and NT MSCs
a e 24 h o cul u e (n = 6, pe o med in duplica es).
fi efly luci e ase usion gene (eGFP. Luc).
Al hough GPC3-ENG MSCs induced T cell
killing o GPC3
+
a ge cells, no T cell killing
o GPC3

a ge cells was obse ed (p < 0.001;
Figu e 2C). Media, NT MSCs, o EGFR III-
ENG MSCs did no induce killing o any a ge
cells, confi ming specifici y. Thus, GPC3-ENG MSCs ac i a e human
T cells in an an igen-dependen manne and induce an igen-depen-
den umo cell killing.
GPC3-ENG MSCs Exp essing CD80 and 41BBL Induce T Cells o
P oduce IL-2 in an An igen-Dependen Manne
Because we did no obse e an igen-dependen IL-2 p oduc ion, we
nex gene a ed GPC3-ENG MSCs ha also exp essed cos imula o y
molecules (CD80, 41BBL, o CD80 and 41BBL) on hei cell su ace
(Figu es S4A and S4B). The e was no significan di e ence in e-
ga ds o GPC3-ENG p o ein p oduc ion be ween di e en MSC
popula ions (Figu e S4C). We pe o med co-cul u e assay as
desc ibed abo e wi h GPC3-ENG MSC.CD80, GPC3-ENG
MSC.41BBL, o GPC3-ENG MSC.CD80+41BBL a an MSC o u-
mo cell a io o 1:10 in he p esence o T cells. All MSC popula ions
induced significan p oduc ion o IFNgby T cells in he p esence o
GPC3
+
(HUH7 and G401) umo cells in compa ison o GPC3

(A549) umo cells (p < 0.01; Figu e 3A). GPC3-ENG
MSC.CD80+41BBL no only induced he highes IFNgp oduc ion
by T cells (p < 0.05), bu also induced IL-2 p oduc ion in a
GPC3-dependen manne in con as o all o he MSC popula ions
(Figu e 3B). To u he in es iga e which a io o MSCs o umo
cells is needed o induce IFNgand IL-2 p oduc ion, we pe o med
a i a ion expe imen . Significan IFNgp oduc ion was obse ed
a an MSC o umo cell a io o 1:30 in con as o IL-2 p oduc ion
(Figu e S5). Specifici y o GPC3-ENG MSC exp essing cos imula o y
molecules was u he confi med in 24-h cy o oxici y assays, wi h
an igen-specific a ge cell killing o up o 60% (Figu e 3C). Killing
inc eased o g ea e han 99% in a 48-h cy o oxici y assay
(Figu e S6).
GPC3-ENG MSCs Induce An igen-Dependen T Cell P oli e a ion
We nex e alua ed whe he GPC3-ENG MSCs could induce an igen-
specific T cell p oli e a ion. GPC3-ENG MSCs ±cos imula o y
molecules, NT MSCs, o EGFR III-ENG MSCs we e cocul u ed
wi h umo cell lines a an MSC o umo cell a io o 1:10 in he p es-
ence o human T cells. A e 7 days, non-adhe en cells we e ha es ed
and he absolu e numbe s o CD3
+
T cells we e de e mined by FACS
analysis. All GPC3-ENG MSC popula ions induced T cell
Molecula The apy: Oncoly ics
70 Molecula The apy: Oncoly ics Vol. 6 Sep embe 2017
p oli e a ion in he p esence o GPC3
+
(HUH7 and G401) umo cells
in con as o media, NT MSCs, o EGFR III-ENG MSCs. In he
p esence o GPC3

umo s, no T cell p oli e a ion was obse ed,
confi ming specifici y (Figu e 4A). Pheno ypic analysis e ealed p e-
dominance o CD8
+
T cells (Figu e 4B), and expanded T cells had a
cen al memo y pheno ype (CCR7
+
CD45RA

;Figu es 4C and
4D). To confi m ha GPC3-ENGs by hemsel es do no induce
T cell p oli e a ion, T cells we e incuba ed wi h supe na an s o
GPC3-ENG o NT MSCs. Absolu e numbe s o T cells we e enume -
a ed a e 72 h , and no significan di e ence was obse ed be ween
bo h g oups (Figu e S7).
Figu e 2. GPC3-ENG MSCs Induce T Cells o
Recognize and Kill GPC3
+
Tumo Cells
(A and B) GPC3-ENG, EGFR III-ENG, o NT MSCs we e
cocul u ed wi h GPC3
+
(G401; HUH7) o GPC3

(A549)
cell lines a 1:10 (le panel) o 1:3 ( igh panel) MSC o
umo cell a ios in he p esence o human T cells (10:1
T cell o umo cell a io). A e 24 h , IFNg(A) o IL-2
(B) was de e mined by ELISA (n = 6, assay pe o med in
duplica es; GPC3-ENG e sus NT and CD19-ENG
MSCs: *p < 0.05, **p < 0.01, ***p < 0.001). (C) Luci e ase-
based cy o oxici y assays using GPC3-ENG, EGFR III,
o NT MSCs and GPC3
+
(G401.eGFP. Luc;
HUH7.eGFP. Luc) o GPC3

(A549.eGFP. Luc) cell
lines as a ge s a an MSC o umo cell a io o 1:10 in he
p esence o human T cells (10:1 T cell o umo cell a io;
n = 6; assay was pe o med in iplica es; box g aph;
whiske s: min, max; ***p < 0.001).
GPC3-ENG MSCs.CD80+41BBL Induce
Fas e T Cell Killing o GPC3
+
Tumo Cells
Ha ing es ablished ha GPC3-ENG
MSCs.CD80+41BBL induce mo e po en
T cell ac i a ion, as judged by IL-2 p oduc ion,
we wan ed o de e mine i his esul s in as e
umo cell killing by T cells. To s udy he
kine ics o umo cell killing, we used a spinning
disc con ocal mic oscopy assay, in which he
inco po a ion o p opidium iodide (PI) in o
dying cells is measu ed in eal ime. To quan i y
cell dea h, he a ea-co ec ed in ensi y o PI
fluo escence was calcula ed. GPC3-ENG
MSCs.CD80+41BBL induced as e T cell
killing o GPC3
+
umo cells in compa ison o
GPC3-ENG MSCs (p < 0.001). Nei he MSC
popula ion induced T cell killing o GPC3

A549 cells, confi ming specifici y (Figu es 5A
and 5B; Mo ies S1 and S2).
GPC3-ENG MSCs Ha e An i umo Ac i i y
In Vi o
We finally compa ed he an i umo ac i i y o
GPC3-ENG MSCs in i o. In he fi s se o
expe imen s, HUH7.eGFP. Luc cells we e
coinjec ed subcu aneously (s.c.) a a a io o 10:1 wi h GPC3-ENG
MSCs, GPC3-ENG MSC.CD80, GPC3-ENG MSC.41BBL, o GPC3-
ENG MSC.CD80+41BBL. On day 7, mice ecei ed an in a enous
(i. .) dose o 1 10
7
T cells. HUH7.eGFP. Luc coinjec ed wi h
EGFR 3-ENG MSC o EGFR III-ENG MSC.CD80+41BBL se ed
as con ols. A e T cell in usion, he e was a significan dec ease in
umo g ow h, as judged by bioluminescence imaging, in mice ha
ecei ed GPC3-ENG MSCs ±cos imula o y molecules in compa ison
o con ol mice (Figu es 6A and 6B). This esul ed in a significan
su i al ad an age o GPC3-ENG MSCs ±cos imula o y molecule
ea ed mice (p < 0.01; Figu e 6C). Howe e , p o ision o
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Molecula The apy: Oncoly ics Vol. 6 Sep embe 2017 71
cos imula ion did no imp o e su i al in compa ison o unmodified
GPC3-ENG MSCs. Nex , we de e mined he an i umo ac i i y o
GPC3-ENG MSCs in es ablished umo s. Mice we e injec ed s.c.
wi h HUH7.eGFP. Luc cells. On day 7, mice ecei ed a pe i umo al
injec ion o GPC3-ENG MSC.CD80+41BBL o EGFR III-ENG
MSC.CD80+41BBL, ollowed by an i. . dose o T cells on day 10. Con-
ols included umo -bea ing mice ha only ecei ed T cells o GPC3-
ENG MSC.CD80+41BBL. Only GPC3-ENG MSC.CD80+41BBL plus
T cells induced a eg ession o es ablished umo s, as judged by biolu-
minescence imaging (Figu es 7A and 7B). This esul ed in a signifi-
can inc ease in su i al in compa ison o mice ha only ecei ed
T cells (Figu e 7C).
DISCUSSION
In his s udy, we desc ibe he gene a ion o GPC3-ENG MSCs and
demons a e ha hese cells edi ec T cells o GPC3
+
HCC (Fig-
u e S8). Modi ying GPC3-ENG MSCs o exp ess CD80 and 41BBL
induced obus IL-2 p oduc ion, T cell p oli e a ion, and as e
T cell killing o umo cells in compa ison o unmodified GPC3-
ENG MSCs. In i o GPC3-ENG MSCs ±cos imula o y molecules
had po en an i- umo ac i i y in an HCC xenog a model.
Se e al chemo- and i adia ion-based he apies ha e been explo ed o
imp o e ou comes o pa ien s wi h ad anced s age HCC in he las
decade.
17
Mos o hem ha e limi ed an i- umo ac i i y, bes exem-
plified by so a enib, he only Food and D ug Adminis a ion
(FDA)-app o ed d ug o he ea men o ad anced s age HCC,
which imp o es o e all su i al by only 2 mon hs.
18
Among di e en
o ms o cell he apies, he in usion o au ologous, cy okine-ac i a ed
kille (CIK) cells has been explo ed in nume ous clinical s udies o
HCC.
19
Al hough CIK cells p olong p og ession ee and o e all su -
i al in he adju an se ing, hei an i umo ac i i y as he apy has
been limi ed.
20
In addi ion, CIK cells ha e o be p epa ed o indi id-
ual pa ien s, po en ially limi ing hei b oade clinical use. The same
limi a ions also apply o HCC-specific cell p oduc s, including GPC3-
CAR T cells.
Bispecific an ibodies, T cell ENG o BiTEs, ha ecognize a umo
associa ed an igen (TAA) and CD3 a e a ac i e o - he-shel p od-
uc s o edi ec esiden T cells o umo cells.
21
Ta ge ing CD19
+
hema ological malignancies wi h CD19-specific BiTEs has been
e y success ul in humans, leading o hei FDA app o al in 2014.
22
In con as , in usion o T cell ENG o BiTEs o solid umo s has
been less e ec i e. Lack o e ficacy is mos likely due o se e al ac o s,
including limi ed issue dis ibu ion and sho hal -li e o he in used
ecombinan p o ein.
23
Exp essing T cell ENGs o BiTEs a umo
Figu e 3. GPC3-ENG MSCs Exp essing CD80 and 41BBL Induce T Cells o
P oduce IL-2
(A and B) GPC3-ENG, GPC3-ENG.CD80, GPC3-ENG.41BBL, o GPC3-
ENG.CD80+41BBL MSCs we e cocul u ed wi h GPC3
+
(G401; HUH7) o GPC3

(A549) cell lines in 1:10 MSC o umo cell a ios in he p esence o human T cells
(10:1 T cell o umo cell a io). A e 24 h , IFNg(A) o IL-2 (B) was de e mined by
ELISA (n = 6, assay pe o med in duplica es; GPC3-ENG.CD80+41BBL e sus
GPC3-ENG MSCs: *p < 0.05, **p < 0.01, ***p < 0.001). (C) Luci e ase-based
cy o oxici y assays using GPC3-ENG, GPC3-ENG.CD80, GPC3-ENG.41BBL, o
GPC3-ENG.CD80+41BBL MSCs and GPC3
+
(G401. Luc; HUH7. Luc) o GPC3

(A549. Luc) cell lines as a ge s a a MSC: umo a io o 1:10 in he p esence o
human T cells (10:1 T cell o umo cell a io; n = 6; pe o med in iplica es; box
g aph; whiske s: min, max; ***p < 0.001).
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72 Molecula The apy: Oncoly ics Vol. 6 Sep embe 2017
si es should po en ially o e come his limi a ion.
24
To accomplish
his, we and o he s ha e exp essed T cell ENGs o diabodies in au ol-
ogous T cells (ENG T cells) ha mig a e o umo si es pos in u-
sion.
24–28
Al hough ENG T cells ha e po en an i umo ac i i y in
p eclinical models, hese cells s ill ha e o be p epa ed o indi idual
pa ien s.
29
To o e come his limi a ion, we he e now ha e explo ed
he use o o - he-shel MSCs o deli e GPC3-ENGs o HCC.
MSCs we e eadily ansduced by len i i al ec o s encoding ENG
molecules and GFP o e o i al ec o s encoding cos imula o y
molecules, as judged by FACS analysis. Sec e ion o ENG molecules
was confi med by ELISA. Al hough GPC3-ENG MSCs edi ec ed
Figu e 4. GPC3-ENG MSCs Induce An igen-
Dependen T Cell P oli e a ion
(A–D)GPC3-ENG,GPC3-ENG.CD80,GPC3-ENG.41BBL,
o GPC3-ENG.CD80+41BBL MSCs we e cocul u ed wi h
GPC3
+
(G401; HUH7) o GPC3

(A549) cell lines a a 1:10
MSC o umo cell a io in he p esence o human T cells
(10:1 T cell o umo cell a io). A e 7 days, absolu enumbe
o CD3
+
cells (A), CD8/CD4 T cell a io (B), and p esence
o nai e (CD45A
+
,CCR7
+
), cen al memo y (CM;
CD45A

,CCR7
+
), e ec o memo y (EM; CD45A

,CCR7

),
o e ec o memo y RA (EMRA; CD45A
+
,CCR7

) T cells (C
and D) was de e mined by FACS analysis (n = 4; pe o med
in duplica es; GPC3-ENG.CD80+41BBL e sus GPC3-
ENG MSCs: *p < 0.05, **p < 0.01).
T cells o GPC3
+
umo cells (HUH7 and
G401), as judged by IFNgp oduc ion, no IL-2
p oduc ion was obse ed. This finding was
consis en wi h ou p e ious s udy, in which
IL-2 p oduc ion o CD19-ENG T cells was
dependen on he p esence o co-s imula o y
molecules on he cell su ace o umo cells.
26
Because HUH7 and G401 do no exp ess co-s i-
mula o y molecules, such as CD80 and 41BBL
(da a no shown), we exp essed CD80 and/o
41BBL on he cell su ace o GPC3-ENG
MSCs. Exp ession o CD80 and 41BBL was
equi ed o induce IL-2 p oduc ion by T cells
in he p esence o GPC3
+
umo s. Thus, u u e
s udies a e needed o unde s and he equi e-
men o CD80 and 41BBL cos imula ion in
ou sys em because, in gene al, cos imula ion
h ough CD80 alone is su ficien .
30
P o ision
o cos imula ion has been shown o imp o e
he killing capaci y o T cells edi ec ed wi h
ENG molecules o umo cells.
31
Al hough
G401 and HUH7 cells induced a simila
amoun o IFNgand IL-2 p oduc ion, HUH7
induced g ea e T cell p oli e a ion han G401
cells; in addi ion, he e we e sligh di e ences
in he pheno ype o expanded T cells. Cy okine
p oduc ion is one c i ical ac o ha de e mines
T cell p oli e a ion and pheno ype. Howe e , o he ac o s also play a
ole, including he absence o p esence o inhibi o y molecules
exp essed (e.g., PD-L1) o sec e ed (e.g., ans o ming g ow h ac o
b[TGF-b]) by umo cells. Fu he s udies a e needed o deciphe
di e ences be ween G401 and HUH7 cells.
He e, we show ha cos imula ion also imp o es he killing kine ics o
edi ec ed T cells, highligh ing ano he ad an age o p o iding cos i-
mula ion. Howe e , a e 24 and 48 h , no significan killing di e -
ence in condi ions ±cos imula ion was obse ed. This mos likely
is explained by assay di e ences (fluo escence mic oscopy e sus
luci e ase based). In addi ion, a e 48 h , g ea e han 99% o a ge
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Molecula The apy: Oncoly ics Vol. 6 Sep embe 2017 73

cells we e killed o all GPC3-ENG MSC condi ions es ed. In ou
s udy, we exp essed CD80 and 41BBL on he cell su ace o MSCs.
I migh be ad an ageous o gene ically modi y MSCs o sec e e usion
molecules ha consis o a umo -specific scF and he ex acellula
domain cos imula o y ligands
32
so ha T cells ecei e cos imula ion
di ec ly a he umo cells. Fu u e s udies a e planned o explo e
his app oach.
In i o, coinjec ion o umo cells and GPC3-ENG MSCs ollowed by
T cell injec ion on day 7 esul ed in obus an i umo ac i i y o
GPC3-ENG MSCs. Howe e , umo s e en ually p og essed. To un-
de s and he mechanisms o umo p og ession, we de e mined he
pe sis ence o eGFP. Luc-exp essing MSCs in i o. MSCs pe sis ed
o less han 28 days (Figu e S9), highligh ing he need o adminis e
mul iple doses o MSCs. Al hough, in his “p oo o concep ”s udy,
A
B
Figu e 5. Time-Lapse Con ocal Mic oscopic
Imaging o GPC3-ENG MSCs Induced Killing o
GPC3
+
Tumo Cells
(A) Rep esen a i e images (a 0 h and 12 h ) o eal-
ime de ec ion o cy oly ic ac i i y o T cells in he
p esence o GPC3
+
(HUH7) o GPC3

(A549) cell lines
and GPC3-ENG.CD80+41BBL MSCs (g een; 1:10
MSC o umo cell a io; 10:1 T cell o umo cell a io).
Cell dea h was isualized by inco po a ion o PI ( ed).
(B) Tumo cell killing a a ious ime poin s was
de e mined as an a ea o co ec ed mean pixel in-
ensi y (PI channel; in ensi y a ea 10
6
pixel). The
solid lines in he g aph ep esen he “bes - i ” linea
eg ession analysis (HUH7 cocul u e wi h GPC3-ENG
[blue] o GPC-ENG.CD80+41BBL MSCs [ ed]; A549
cells cocul u e wi h GPC3-ENG [g een] o GPC-
ENG.CD80+41BBL MSCs [yellow]. The do s show in-
di idual expe imen al alues (n = 5; expe imen s = 2;
***p < 0.001).
we did no explo e mul iple dosing schedules
o MSCs, his is he goal o u u e s udies. P o-
ision o cos imula ion did no imp o e he
an i- umo ac i i y o GPC3-ENG MSCs;
indeed, exp ession o 41BBL dec eased he an i-
umo ac i i y o GPC3-ENG MSCs. Se e al
ac o s ha e o be conside ed, including MSC
and T cell dose, and expe imen s a e in p og ess
o add ess his. In addi ion, mechanis ic s udies
in ega ds o he ole o 41BBL in ou sys em a e
needed. This should include s udying he T cell
infil a e wi hin umo s.
In ou second in i o model, we ocused on
he pe i umo al injec ion o MSCs, because,
as epo ed by o he s,
33
sys emic i. . adminis-
a ion o human MSCs esul ed in he ap-
ping o MSCs in he lung o mice (Figu e S10).
Al hough we injec ed MSCs pe i umo al, he e
could be an “abscopal e ec ” o dis an umo
si es. We plan o explo e his in u u e s udies, in which mice will
bea a second flank umo ha is no ea ed. In a-a e ial admin-
is a ion has been shown o significan ly imp o e he sys emic
dis ibu ion o human MSCs in xenog a models
34
and could be
explo ed in u u e s udies. Howe e , a p esen , i is unclea i he
obse ed biodis ibu ion o human MSCs in mice closely mimics
hei biodis ibu ion in humans.
35
Mo eo e , he p e e ed ou e
o GPC3-ENG MSC adminis a ion o HCC pa ien s will mos
likely depend on he clinical scena io. Fo example, al hough i. .
adminis a ion would be ideal o he he apy o lung disease, he
commones si e o HCC me as ases,
36
in a-a e ial adminis a ion
would be he ou e o choice o locally ad anced s age disease,
akin o ans-a e ial chemo emboliza ion (TACE), which is
commonly used o deli e chemo he apeu ic agen s di ec ly in o
locally ad anced HCC.
37
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74 Molecula The apy: Oncoly ics Vol. 6 Sep embe 2017
Ou s udy significan ly ex ends s udies by o he s. Comp e e al.
38
gene ically modified MSCs o exp ess a CEA bispecific diabody, a bis-
pecific mAb o ma ha equi es he exp ession o wo genes. CEA
bispecific diabody-sec e ing MSCs ac i a ed T cells, as e idenced by
an igen-specific T cell p oli e a ion. In i o, implan ed, MSC-seeded
sca olds suppo ed diabody p oduc ion and had an i umo ac i i y in
a colon cance xenog a model. Alipe a e al.
39
gene ically modified
he immo alized MSC line SCP-1 o exp ess CD133-specific T cell
ENG and 41BBL. CD133-ENG MSCs edi ec ed T cells o CD133
+
acu e myeloid leukemia (AML) cells, and 41BBL exp ession enhanced
cy okine p oduc ion and T cell p oli e a ion in i o. Ex i o ea -
men o AML cells wi h CD133-ENG MSC.41BBL and T cells
p e en ed eng a men o AML cells in NSG mice.
39
Like he wo
p e ious s udies, ou s udy is limi ed by elying on a xenog a model.
Al hough hese do no ecapi ula e he immunosupp essi e mic oen-
i onmen c ea ed by umo s, including HCC,
40
hey a e ideal o
s udy he in e ac ions o human umo cells, human MSCs, and
human T cells, which is c i ical o u u e clinical de elopmen .
Figu e 6. GPC3-ENG MSCs Redi ec T Cells In Vi o
o Kill HCC
An i umo ac i i y o GPC3-ENG MSCs in an HCC
xenog a model. Mice we e s.c. coinjec ed wi h 5 10
6
HUH7.eGFP. Luc cells and 5 10
5
GPC3-ENG (n = 5),
GPC3-ENG.CD80 (n = 4), GPC3-ENG.41BBL (n = 5),
GPC3-ENG.CD80+41BBL (n = 5), EGFR III-ENG (n = 5),
o EGFR III-ENG.CD80+41BBL (n = 5) MSCs on hei le
lowe lank. On day 7, mice ecei ed an i. . dose o
110
7
T cells. Tumo g ow h was ollowed by biolumi-
nescence imaging. (A) Rep esen a i e images o animals
( ed a ow: T cell injec ion). (B) Quan i a i e biolumines-
cence imaging esul s (solid lines: indi idual mice; adi-
ance = pho ons/s/cm
2
/s ). (C) Kaplan-Meie su i al
cu e (con ol e sus ea men g oups; *p < 0.05,
**p < 0.01, ***p < 0.001).
In summa y, ou s udy demons a es ha MSCs
gene ically modified o exp ess GPC3-ENG ±
cos imula o y molecules edi ec T cells o
GPC3
+
umo cells and ha e po en an i umo
ac i i y. Thus, GPC3-ENG MSCs may be a
p omising “o - he-shel cell he apy al e na-
i e” o cu en GPC3- a ge ed immuno he apy
app oaches ha ei he ely on he passi e in u-
sion o an ibodies o he adop i e ans e o
au ologous CAR T cells.
MATERIALS AND METHODS
Cell Lines and Cul u e Condi ions
G401 ( habdoid umo , ATCC) and HUH7
(hepa ocellula ca cinoma, a gi om D .
Xiao-Tong Song, Baylo College o Medicine)
we e used as GPC3
+
a ge s. The iden i y o
HUH7 was confi med by he Cha ac e ized
Cell Line Co e Facili y a MD Ande son Cance
Cen e . A549 (lung ca cinoma, ATCC) cells we e used as nega i e
con ols. 293T cells (ATCC) we e used o packaging i al ec o s.
HUH7, A549, and 293T cell lines we e g own in DMEM (The mo
Scien ific), G401 in alpha-MEM (The mo Scien ific) media supple-
men ed wi h 10%–20% e al bo ine se um (FBS) (The mo Scien ific),
and 2 mmol/L Glu aMAX-I (In i ogen). The gene a ion o
eGFP. Luc-exp essing HUH7, G401 and A549 cells has been p e i-
ously desc ibed.
8
Human MSCs om heal hy dono s we e ob ained
unde a Baylo College o Medicine ins i u ional e iew boa d
(IRB)-app o ed p o ocol a e in o med consen was ob ained in
acco dance o he Decla a ion o Helsinki. MSCs we e cul u ed in
aMEM (Lonza) supplemen ed wi h 20% FBS (The mo Scien ific)
and 2 mmol/L Glu aMAX-I (In i ogen; comple e aMEM).
Cons uc ion o Vi al Vec o s Encoding T Cell ENG and
Cos imula o y Molecules
To gene a e a GPC3-specific T cell ENG molecule, a mini gene encod-
ing he immunoglobulin (Ig) hea y-chain leade pep ide and he
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Molecula The apy: Oncoly ics Vol. 6 Sep embe 2017 75
GPC3-specific scF (GC33) was syn hesized by The mo Fishe Scien-
ific.
41
This mini gene was subcloned in o an SFG e o i al ec o
con aining a sho se ine-glycine linke and a CD3-specific scFV
de i ed om OKT3.
24
The GPC3-ENG was hen subcloned in o
pCDH-CMV-MCS-EF1-GFP (Sys em Biosciences).The len i i al ec-
o encoding he EGFR III-specific T cell ENG was gene a ed in a
simila ashion using he EGFR III-specific scF 139.
42
Len i i al pa -
icles we e gene a ed in 293T cells using packaging plasmids (pPACK
Len i ec o Packaging Sys em, Sys em Biosciences) acco ding o he
manu ac u e ’s ins uc ions. MSCV e o i al ec o s encoding
CD80, 41BBL, o 41BBL and CD80 we e gene a ed by subcloning
Figu e 7. GPC3-ENG MSCs Ha e An i- umo Ac i i y
in Es ablished Tumo s
Mice we e s.c. injec ed wi h 1 10
6
HUH7.eGFP. Luc
cells on hei le lowe lank (n = 10). On day 7, mice
ecei ed a pe i umo al injec ion o 1 10
6
GPC3-
ENG.MSC.CD80+41BBL. On day 10, i e mice ecei ed
an i. . dose o 1 10
7
T cells. Tumo -bea ing mice
injec ed wi h EGFR III-ENG MSC.CD80+41BBL (n = 5) o
T cells (n = 5) se ed as con ols. Tumo g ow h was
ollowed by bioluminescence imaging. (A) Rep esen a i e
images o animals (g een a ow: MSC injec ion; ed a ow:
T cell injec ion). (B) Quan i a i e bioluminescence imaging
esul s (solid lines: indi idual mice; adiance = pho ons/s/
cm
2
/s ). (C) Kaplan-Meie su i al cu e (con ol e sus
ea men g oups; *p < 0.05).
CD80 om pORF.CD80 (In i oGen) and/o
41BBL om pORF.41BBL (In i oGen) in o
MSCV-I-GFP(M) (p o ided by he la e Elio Va-
nin,No hwes e nUni e si y Feinbe gSchoolo
Medicine). VSV-G-pseudo yped e o i al pa -
icles we e gene a ed as p e iously desc ibed.
43
Gene a ion o Gene ically Modi ied MSCs
MSCs we e g own o 60%–70% confluency in
T75 flasks and ansduced wi h i al ec o s a
an MOI o 10 and 4 mg/mL polyb ene (Sigma-
Ald ich). A e ansduc ion, MSC we e
expanded o 3–5 days p io o use.
Flow Cy ome ic Analysis
20,000 cells pe sample we e analyzed by a
FACSCalibu ins umen (BD Biosciences) us-
ing Cell Ques So wa e (BD Biosciences) and
a BD Can o II ins umen (BD Biosciences) us-
ing FACSDi a so wa e (BD Biosciences) and
analyzed using Kaluza Analysis 1.3 (Beckman
Coul e ) and FlowJo 10 (FlowJo LLC). Fo
pheno yping MSCs, he ollowing MAbs we e
used: CD105-PE (Mil enyi Bio ec), CD45-
Pe CP and CD90-APC (BD Biosciences),
41BBL-PE (BD Biosciences), and CD80-Pe CP
(eBioscience). Fo pheno yping T cells, he
ollowing MAbs we e used: CD3-APC, CD4-PE, CD8-APC, CCR7-
FITC, and CD45RA-APC (all BD Biosciences). GPC3 exp ession o
umo cell lines was de ec ed wi h YP7 mAb, which was p o ided
by Mi chell Ho (NIH).
44
Iso ype con ols included IgG1-PE, IgG1-
Pe CP, and IgG1-APC (bo h om BD Biosciences). Absolu e T cell
numbe s we e calcula ed using Coun B igh Absolu e Coun ing
Beads (The mo Fishe Scien ific).
ELISA
NT and GPC3-ENG ±cos imula o y molecules MSCs we e pla ed in
a 12-well pla e a a concen a ion o 5 10
4
/well in 2 mL o comple e
Molecula The apy: Oncoly ics
76 Molecula The apy: Oncoly ics Vol. 6 Sep embe 2017
aMEM and cul u ed o 24 h . To de e mine he ENG concen a ion
in condi ioned media, we fi s coa ed 96-well non- issue cul u e
ea ed pla es wi h human ecombinan GPC3 p o ein (R&D Sys-
ems) a 10 ng/well. Media om ENG-MSCs was pla ed and allowed
o incuba e o 1 h a oom empe a u e (RT). Goa an i-mouse
F(ab’) (Jackson ImmunoResea ch) was added and incuba ed a RT
o 1 h . The pla e was washed, and seconda y an i-goa ho se adish
pe oxidase (HRP) an ibody (Jackson ImmunoResea ch) was added.
A e 1 h incuba ion a RT, he pla e was washed and de eloping
agen was added ( e ame hylbenzidine [TMB] subs a e, Sigma-
Ald ich). Abso bance was ead a 450 nm. A s anda d cu e was
gene a ed using ecombinan GPC3-ENG p o ein (cus om syn hesis;
The mo Fishe Scien ific).
Cocul u e Assays
GPC3-ENG MSCs ±cos imula o y molecules and EGFR III-ENG
MSCs we e pla ed a a 1:3 o 1:10 a io wi h GPC3
+
(HUH7 and
G401) o GPC3

(A549) a ge cells. T cells, isola ed om pe iphe al
blood mononuclea cells (PBMCs) by CD3 selec ion (Mil enyi), we e
added a a 10:1 e ec o o a ge a io. Cocul u e supe na an was
collec ed a e 48 h and used o cy okine analysis. IFNgand IL-2
concen a ions we e de e mined using ELISA ki s (R&D Sys ems),
acco ding o he manu ac u e ’s ins uc ions.
Cy o oxici y Assay
Cy o oxic ac i i y o T cells agains a ge s was de e mined by a
luci e ase-based cy o oxici y assay. HUH7, G401, and A549 cells
exp essing eGFP/ Luc we e pla ed in 96-well fla bo om pla es a a
concen a ion o 1 10
4
cells/well in iplica es. GPC3-ENG
MSCs ±cos imula o y molecules and EGFR III-ENG MSCs we e
pla ed a a 1:10 MSC o umo cell a io, and T cells, isola ed om
PBMCs by CD3 selec ion (Mil enyi), we e added a a 10:1 e ec o
o a ge a io. Wells wi hou MSCs se ed as con ols. A e 24 h ,
luci e ase ac i i y was de e mined using a luci e ase assay ki acco d-
ing o he manu ac u e ’s ins uc ions (P omega) and an Infini e
M200 luminome e (Tecan T ading AG).
Time-Lapse Con ocal Mic oscopic Imaging and Image Analysis
MSC-induced a ge cell killing was quan i a i ely analyzed by a spin-
ning disk con ocal mic oscope (Zeiss) equipped wi h a CSU-X1A
5000 spinning disk uni (Yokogowa Elec ic Co po a ion) mul i-lase
module wi h wa eleng hs o 458 nM, 488 nM, and 514 nM and an
Axio Obse e Z1 mo o ized in e ed mic oscope equipped wi h a
p ecision mo o ized XY s age (Ca l Zeiss Mic oImaging). Tempe a-
u e was main ained a 37C and 5% CO
2
using an en i onmen al
con ol chambe . Zen 2012 so wa e (Zeiss) was used o acqui e
images in ime-lapse mode using a Zeiss Plan-Neoflua 20 0.4
NA objec i e on an O ca R2 CCD came a and o analyze a e age
GFP and PI in ensi y. A quan i a i e digi al image p ocessing pipe-
line, c ea ed in ImageJ (W.S. Rasband, NIH [1997–2014]), was used
o calcula e specific PI inco po a ion o HUH7 and A549 cells. Fi s ,
backg ound in ensi ies o PI channel we e sub ac ed; hen, aw
images we e h esholded based on hei in ensi y his og am. The
same h eshold was used on all analyzed images. PI inco po a ion
was de e mined as a mul iplica ion o he mean pixel in ensi y and
h esholded a ea.
In Vi o Expe imen s
Animal expe imen s we e pe o med on a p o ocol app o ed by he
Baylo College o Medicine Ins i u ional Animal Ca e and Use Com-
mi ee in acco dance wi h he Ame ican Associa ion o Labo a o y
Animal Science. Co-injec ion o umo cells and MSCs: 8- o
10-week-old NSG mice (NOD.Cg-P kdcscid/Il2 g m1Wjl/SzJ; JAX
Mice) we e s.c. injec ed wi h 5 10
6
HUH7.eGFP. Luc cells and
510
5
gene-modified MSCs on hei le lowe flank. On day 7,
mice we e injec ed i. . wi h 1 10
7
T cells. Sequen ial injec ion o u-
mo cells and MSCs: 1 10
6
HUH7.eGFP. Luc cells we e injec ed
s.c. on day 0, and 1 10
6
gene-modified MSCs we e injec ed pe i u-
mo al on day 7. On day 7, mice we e injec ed i. . wi h 1 10
7
T cells.
To gene a e su ficien T cells o in i o expe imen s, T cells we e ac i-
a ed om PBMCs wi h OKT3 (CRL-8001, ATCC) and CD28 (BD
Biosciences) an ibodies and expanded wi h ecombinan human
IL-7 and IL-15 (IL-7, 10 ng/mL; IL-15, 5 ng/mL; P oleukin; Chi on)
o 7–10 days. Mice we e imaged using he IVIS sys em (IVIS, Xeno-
gen), as p e iously desc ibed,
45
and eu hanized a p edefined
endpoin s o when hey me eu hanasia c i e ia in acco dance wi h
he Cen e o Compa a i e Medicine a Baylo College o Medicine.
S a is ical Analysis
G aphPad P ism 5 so wa e (G aphPad) was used o s a is ical
analysis. Da a we e p esen ed as mean ±SD o SEM. Fo compa ison
be ween wo g oups, a wo- ailed es was used. Fo compa isons o
h ee o mo e g oups, one-way ANOVA wi h Bon e oni’s pos - es
was used. Fo compa ison o PI inco po a ion, linea eg ession
analysis was pe o med. Fo he mouse expe imen s, su i al, de e -
mined om he ime o umo cell injec ion, was analyzed by he
Kaplan-Meie me hod and log- ank es .
SUPPLEMENTAL INFORMATION
Supplemen al In o ma ion includes en figu es and wo mo ies and
can be ound wi h his a icle online a h p://dx.doi.o g/10.1016/j.
om o.2017.07.002.
AUTHOR CONTRIBUTIONS
A.S. and S.G. designed he s udy. A.S., A.V., M.P.V., D.L.G., D.T., and
A.H. pe o med expe imen s. Z.M. and A.G. p o ided eagen s. All
au ho s con ibu ed o da a analysis and manusc ip p epa a ion.
CONFLICTS OF INTEREST
M.P.V., A.H., and S.G. ha e pa en applica ions in he fields o T cell
and/o gene he apy o cance .
ACKNOWLEDGMENTS
The au ho s would like o hank D . Malcolm B enne (Cen e o
Cell and Gene The apy, Baylo College o Medicine, Hous on, TX)
o help ul discussions and ad ice. This wo k was suppo ed by
NIH g an s P01CA094237 and 2 P30 CA125123-09. A.S. was sup-
po ed by he Hunga ian Talen P og am (NTP-NFTÖ-16-0912).
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Molecula The apy: Oncoly ics Vol. 6 Sep embe 2017 77