T Cell-Activating Mesenchymal Stem Cells as a Biotherapeutic for HCC

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 www.molecula he apy.o g 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). Molecula The apy: Oncoly ics 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 www.molecula he apy.o g 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 Molecula The apy: Oncoly ics 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 www.molecula he apy.o g 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). www.molecula he apy.o g Molecula The apy: Oncoly ics Vol. 6 Sep embe 2017 77