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www.impactjournals.com/oncotarget/ Oncotarget, October, Vol.4, No 10

Immunological targeting of tumor cells undergoing an epithelial- mesenchymal transition via a recombinant brachyury-yeast vaccine

Duane H. Hamilton1,*, Mary T. Litzinger1,*, Alessandra Jales1, Bruce Huang1, Romaine I. Fernando1, James W. Hodge1, Andressa Ardiani1, David Apelian2, -HϑUH\6FKORP1,*, and Claudia Palena1,* 1 Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 2 GlobeImmune Inc., Louisville, Colorado * These authors contributed equally to the work Correspondence to: Claudia Palena, email: [email protected] Keywords: epithelial-mesenchymal transition, brachyury, T-box , cancer vaccine, T-cell immunotherapy Received: August 16, 2013 Accepted: September 24, 2013 Published: September 26, 2013

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT: The embryonic T-box transcription factor brachyury is aberrantly expressed in a range of human tumors. Previous studies have demonstrated that brachyury is a driver of the epithelial-mesenchymal transition (EMT), a process associated with cancer progression. Brachyury expression in human tumor cells enhances tumor invasiveness in vitro and in vivo, and induces resistance to various conventional therapeutics including chemotherapy and radiation. These characteristics, and the selective expression of brachyury for a range of human tumor types vs. normal adult tissues, make brachyury an attractive tumor target. Due to its intracellular localization and the “undruggable” character of transcription factors, available options to target brachyury are currently limited. Here we report on the GHYHORSPHQWDQGFKDUDFWHUL]DWLRQRIDQLPPXQRORJLFDOSODWIRUPIRUWKHHϒFLHQW targeting of brachyury-positive tumors consisting of a heat-killed, recombinant Saccharomyces cerevisiae (yeast)–brachyury vector-based vaccine (designated as GI-6301) that expresses the full-length human brachyury . We demonstrate that human dendritic cells treated with recombinant yeast-brachyury can activate and H[SDQGEUDFK\XU\VSHFL¿F&'+ and CD8+ T cells in vitroWKDWLQWXUQFDQHϑHFWLYHO\ lyse human tumor cells expressing the brachyury protein. Vaccination of mice with UHFRPELQDQW\HDVWEUDFK\XU\LVDOVRVKRZQKHUHWRHOLFLWEUDFK\XU\VSHFL¿F&'+ and CD8+ T-cell responses, and to induce anti-tumor immunity in the absence of toxicity. Based on these results, a Phase I clinical trial of GI-6301 is currently ongoing in SDWLHQWVZLWKDGYDQFHGWXPRUVWRRXUNQRZOHGJHWKLVLVWKH¿UVWYDFFLQHSODWIRUP aimed at targeting a driver of tumor EMT that has successfully reached the clinical stage.

INTRODUCTION known as the epithelial-mesenchymal transition (EMT) [3, 4]. Although brachyury expression was initially reported in The T-box transcription factor T, also known as embryonic but not adult tissues [2], data mining of human EUDFK\XU\ZDVLQLWLDOO\LGHQWL¿HGDVDSURWHLQUHTXLUHG RNA libraries and RT-PCR analysis of multiple tissues for murine embryonic mesodermal development [1, 2], recently revealed brachyury to be expressed in a range of which involves the conversion of stationary epithelial cells human tumors and not in human adult normal tissues, with into migratory, invasive mesenchymal cells via a process the exception of testis and a subpopulation of B cells from www.impactjournals.com/oncotarget 1777 Oncotarget 2013; 4: some healthy donors [5]. Immunohistochemistry studies vaccination of tumor-bearing mice resulting in anti-tumor of human carcinoma biopsy specimens using a brachyury- activity. These and other studies have shown that yeast VSHFL¿FPRQRFORQDODQWLERG\ 0$E DOVRGHPRQVWUDWHG FRXOGHI¿FLHQWO\DFWLYDWHGHQGULWLFFHOOV '&V YLD7ROO the expression of brachyury in human lung and breast OLNHUHFHSWRUV 7/5V DQGFRQVHTXHQWO\LQGXFHWKHPWR carcinomas, both in primary tumors and metastases, produce high levels of type I cytokines, including IL-2, while limited levels of brachyury were seen in testis and 71)ĮDQG,)1Ȗ>@7KH³\HDVWFRPSRQHQW´RI some specimens of thyroid, and no other adult normal the recombinant yeast, therefore, is an integral part of tissues examined [6]. High levels of brachyury have also the vaccine platform in its ability to activate the innate previously been reported in human chordomas [7, 8]. immune system and might partly contribute to the anti- In addition to being essential for embryonic WXPRUHI¿FDF\RIDUHFRPELQDQW\HDVWFRQVWUXFW>@ development, recent studies have shown that EMT may In the studies reported here, we have constructed a also be critical to the progression of carcinomas, as recombinant Saccharomyces cerevisiae (yeast)–brachyury DFTXLVLWLRQRIPHVHQFK\PDOIHDWXUHVE\HSLWKHOLDOWXPRU vector-based vaccine (designated as GI-6301), consisting FHOOVPD\IDYRUWKHLUGLVVHPLQDWLRQDQGWKHDFTXLVLWLRQ of heat-killed Saccharomyces cerevisiae that expresses of resistance to various cancer therapies [9, 10]. Recent the full-length human brachyury protein. We report studies have characterized the transcription factor KHUHIRUWKH¿UVWWLPHWKDW D KXPDQ'&VWUHDWHGZLWK brachyury as a driver of EMT in human carcinomas recombinant yeast-brachyury can activate previously [11]. Overexpression of brachyury in human epithelial established human EUDFK\XU\VSHFL¿F 7FHOO OLQHV E  FDQFHUFHOOOLQHVOHGWRDPRUH¿EUREODVWRLGPRUSKRORJ\ UHFRPELQDQW \HDVWEUDFK\XU\±WUHDWHG '&V FDQ H[SDQG a switch from the expression of epithelial markers such as human EUDFK\XU\VSHFL¿F&'+ T cells from peripheral E-cadherin to mesenchymal markers such as N-cadherin, blood of healthy donors and cancer patients, and (c) ¿EURQHFWLQ DQG YLPHQWLQ DQG DQ LQFUHDVH LQ PLJUDWLRQ UHFRPELQDQW \HDVWEUDFK\XU\±WUHDWHG '&V FDQ H[SDQG and invasion in in vitro assays. Conversely, silencing of KXPDQEUDFK\XU\VSHFL¿F&'+ T cells. It is also shown brachyury in human tumor cell lines resulted in the loss here that vaccination of mice with recombinant yeast- of mesenchymal features, including loss of migration and EUDFK\XU\FDQHOLFLWEUDFK\XU\VSHFL¿F&'+DQG&'+ invasiveness in vitro, and markedly decreased their ability T-cell responses capable of reducing tumor burden in an to metastasize in xenograft models [11]. Previous studies experimental model of metastasis. This is accomplished have also demonstrated that high levels of brachyury in in the absence of any interference with wound healing, human carcinoma cells drive resistance to chemotherapy or any effect on pregnancy/birth rates and other general and radiation [12]. toxicology measurements. Based on these results, a Phase While brachyury is a transcription factor of nuclear I clinical trial of GI-6301 is currently ongoing in patients localization, recent studies have shown that human with advanced tumors [19]; to our knowledge, this is the carcinoma cells can present brachyury peptides in the ¿UVWYDFFLQHSODWIRUPDLPHGDWWDUJHWLQJDGULYHURIWXPRU context of major histocompatibility complex (MHC) EMT that has successfully reached the clinical stage. class I molecules on the tumor cell surface. This was GHPRQVWUDWHGE\WKHLGHQWL¿FDWLRQRIDEUDFK\XU\PHU RESULTS peptide that was used to expand human brachyury- VSHFL¿F&'+ T cells from the blood of cancer patients in vitro which, in turn, were able to lyse brachyury- Recombinant yeast-brachyury–treated human positive tumor cells in an MHC class I–restricted manner '&VDFWLYDWHEUDFK\XU\VSHFL¿FKXPDQ&'+ T [5]. In addition, it has recently been shown that patients cells UHFHLYLQJ D SURVWDWHVSHFL¿F DQWLJHQ 36$ ±GLUHFWHG vaccine in combination with anti-CTLA4 MAb, or a carcinoembryonic antigen (CEA)–directed vaccine, +XPDQ'&VFXOWXUHGIRUGD\VLQWKHSUHVHQFHRI GHYHORS EUDFK\XU\VSHFL¿F 7 FHOOV SRVWYDFFLQDWLRQ UHFRPELQDQWKXPDQ*0&6)DQG,/ZHUHLQFXEDWHG most likely via the mechanism of antigen cross- for 48 hours with either heat-killed control yeast or presentation [13]. These studies provided evidence of the KHDWNLOOHG UHFRPELQDQW \HDVWEUDFK\XU\ DW D '&WR immunogenicity of brachyury in humans and its potential yeast ratio of 1:10. Treatment with either construct to serve as a vaccine target. (control yeast or recombinant yeast-brachyury) resulted A previously characterized therapeutic vaccine LQ D  D VXEVWDQWLDO LQFUHDVH LQ WKH SHUFHQWDJH RI '&V platform [14-18] consists of heat-killed recombinant H[SUHVVLQJ &' &' DQG 0+&FODVV , PROHFXOHV Saccharomyces cerevisiae \HDVW  PRGL¿HG WR H[SUHVV E DQLQFUHDVHLQWKHÀXRUHVFHQFHLQWHQVLW\RI&'DQG tumor-associated antigen(s). For example, a recombinant MHC-class II molecules, and (c) enhanced production of yeast-CEA vaccine was previously used in vitro to ,/FRPSDUHGWRXQWUHDWHG'&V 6XSSOHPHQWDO7DEOH HI¿FLHQWO\DFWLYDWHPXULQHDQGKXPDQ7FHOOVWKDWZHUH 1). It was next examined whether recombinant yeast- lytic against CEA-expressing targets, and in vivo for EUDFK\XU\±WUHDWHGKXPDQ'&VFRXOGHI¿FLHQWO\VWLPXODWH www.impactjournals.com/oncotarget 1778 Oncotarget 2013; 4: 7DEOH  $FWLYDWLRQ RI KXPDQ EUDFK\XU\VSHFL¿F &'+ T cells by recombinant yeast-brachyury–treated DCs ,)1Ȗ 'RQRU 'RQRU 7FHOOVFRQWURO\HDVW±WUHDWHG'&V 250.8 (± 132.8) 95.2 (± 4.1)

7FHOOVUHFRPELQDQW\HDVWEUDFK\XU\±WUHDWHG'&V 577.5 (± 33.2) 200.1 (± 7.2)

'&VJHQHUDWHGIURP3%0&VRI+/$$+KHDOWK\GRQRUVZHUHFXOWXUHGIRUGD\VLQWKHSUHVHQFHRI*0&6) DQG,/)RUWKHODVWKRXUVRIFXOWXUH'&VZHUHWUHDWHGZLWKFRQWURO\HDVWRUUHFRPELQDQW\HDVWEUDFK\XU\ '&\HDVWUDWLRRI '&VZHUHKDUYHVWHGDQGXVHGWRVWLPXODWHEUDFK\XU\VSHFL¿F&'+7FHOOV 7FHOOV'& UDWLRRI $IWHUKRXUVFXOWXUHVXSHUQDWDQWVZHUHDQDO\]HGE\(/,6$DVVD\IRU,)1ȖSURGXFWLRQ5HVXOWV SJPO UHSUHVHQWWKHPHDQRIUHSOLFDWHPHDVXUHPHQWV “6' DIWHUVXEWUDFWLRQRIEDFNJURXQGZLWK7FHOOVDORQH

A Donor 3 Donor 4 1.0 Control Tetramer Brachyury Tetramer 0.8

0.6

0.4

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% Tetramer+/ CD8+ T cells 0.0 Control Brachyury Control Brachyury Yeast Yeast Yeast Yeast B 3 Control Tetramer 0.10 % 2.57 % Brachyury Tetramer

2 Control Tetramer Brachyury TetramerBrachyury

1 CD8 CD8 % Tetramer+/ CD8+ T cells

0 Donor 5 Donor 6 Donor 7 Donor 8 Donor 9 Donor 10 Donor 11

C D 100 40 25 p< 0.001 10-1 20 30 10-2 15 20 10-3 10 10 10-4 % Specific Lysis % Specific Lysis 5

-5

Normalized Brachyury mRNA Normalized Brachyury 10 0 0 SW480 H460 SW480 H460 IgG anti-MHC class I SW480

)LJXUH([SDQVLRQRIEUDFK\XU\VSHFL¿F&'+7FHOOVLQUHVSRQVHWR\HDVWEUDFK\XU\௅WUHDWHG'&VA, Autologous T cells from 2 HLA-A2+KHDOWK\GRQRUVZHUHVWLPXODWHGZLWKLUUDGLDWHGFRQWURORUEUDFK\XU\\HDVW௅WUHDWHG'&V '&\HDVWUDWLRRI IRU ,96F\FOHV$WGD\RI,967FHOOVZHUHVWDLQHGZLWK&'),7&DQGD3(ODEHOHGEUDFK\XU\SHSWLGHWHWUDPHURUDFRQWURO&09 WHWUDPHU,QGLFDWHGLVWKHSHUFHQWDJHRIEUDFK\XU\ EODFNEDUV RUFRQWURO&09 JUD\EDUV WHWUDPHUSRVLWLYH&'+ T cells as determined E\ÀRZF\WRPHWU\B3HUFHQWDJHRIEUDFK\XU\RUFRQWURO&09WHWUDPHUSRVLWLYH&'+ T cells in PBMCs of 7 HLA-A2+ healthy donors VWLPXODWHGIRU,96F\FOHVZLWK\HDVWEUDFK\XU\௅WUHDWHG'&V,QVHUWVKRZVDUHSUHVHQWDWLYH)$&6SORWVWDLQLQJIRUGRQRUC, Brachyury mRNA expression normalized to GAPDH OHIWSDQHO DQGF\WRWR[LF7FHOOO\VLVRI6: +/$$+) and H460 (HLA-A2neg) cells with &'+7FHOOVSXUL¿HGDWWKHHQGRI,96IURPGRQRUD$VLPLODU&7/DVVD\ZDVFRQGXFWHGZLWK6:WXPRUFHOOVSUHYLRXVO\ LQFXEDWHGZLWK—JPORIFRQWURO,J*RUDQWL0+&FODVV,0$EIRUPLQEHIRUHWKHDGGLWLRQRIEUDFK\XU\VSHFL¿F&7/V7KH(7UDWLR ZDV%DUVUHSUHVHQWWKHPHDQRIWULSOLFDWHPHDVXUHPHQWVHUURUEDUVUHSUHVHQW6(0 www.impactjournals.com/oncotarget 1779 Oncotarget 2013; 4: HLA-A2+±UHVWULFWHG EUDFK\XU\ SHSWLGH±VSHFL¿F KXPDQ the human colon carcinoma HLA-A2+ 6: DQG WKH &'+ T cells in vitro '&V JHQHUDWHG IURP SHULSKHUDO lung HLA-A2neg H460 line, which are both positive for blood mononuclear cells (PBMCs) of two HLA-A2+ brachyury expression (Fig. 1C, left panel). As shown healthy donors (Table 1, donors 1 and 2) were treated LQ)LJXUH& ULJKWSDQHO &'+ T cells expanded with with control yeast or recombinant yeast-brachyury at a UHFRPELQDQW\HDVWEUDFK\XU\±WUHDWHG'&VSUHIHUHQWLDOO\ '&WR\HDVWUDWLRRI)RUW\HLJKWKRXUVDIWHUWUHDWPHQW lysed the HLA-A2+ 6: FHOOV WKXV LQGLFDWLQJ WKH '&V ZHUH LUUDGLDWHG DQG XVHG WR VWLPXODWH DOORJHQHLF MHC preference of the cytotoxic effect. MHC-restriction EUDFK\XU\VSHFL¿F&'+ T cells that were generated by was also investigated by performing a lysis assay with XVLQJDEUDFK\XU\VSHFL¿FPHUSHSWLGHDVSUHYLRXVO\ 6:FHOOVSUHWUHDWHGZLWKDFRQWURO,J*YVDQDQWL described [5]. As shown in Table 1 with both donors, 0+& FODVV , EORFNLQJ $E $V VKRZQ LQ )LJXUH ' UHFRPELQDQW \HDVWEUDFK\XU\±WUHDWHG '&V VWLPXODWHG EORFNDGHRI0+&FODVV,VLJQL¿FDQWO\GHFUHDVHGWKHO\VLV EUDFK\XU\VSHFL¿F 7 FHOOV WR SURGXFH KLJKHU OHYHOV RI RI6:FHOOVUHLQIRUFLQJWKHLGHDWKDWVWLPXODWLRQRI ,)1ȖWKDQFRQWURO\HDVW±WUHDWHG'&V DSSUR[LPDWHO\ 3%0&VZLWKUHFRPELQDQW\HDVWEUDFK\XU\±WUHDWHG'&V vs. 250 pg/ml for donor 1 and 200 vs. 95 pg/ml for donor LQGXFHV EUDFK\XU\VSHFL¿F &7/V WKDW DUH FDSDEOH RI  UHVSHFWLYHO\ 7KH ORZHU OHYHO RI ,)1Ȗ REVHUYHG LQ lysing brachyury-positive tumor cells in an MHC class I– UHVSRQVHWRFRQWURO\HDVW±WUHDWHG'&VZDVLQDJUHHPHQW restricted manner. with previous observations [14-16]; as stated above, To further evaluate the ability of recombinant yeast- FRQWURO HPSW\ \HDVWFDQVWLPXODWH'&VYLD7/5VDQGWKH EUDFK\XU\±WUHDWHG '&V WR DFWLYDWH EUDFK\XU\VSHFL¿F DFWLYDWHG'&VLQWXUQFDQDFWLYDWH7FHOOVLQDQRQVSHFL¿F &'+ T cells, T-cell lines were established in vitro using manner to secrete type I cytokines. UHFRPELQDQW\HDVWEUDFK\XU\±WUHDWHG'&VIRUWZR,96 F\FOHV IROORZHG E\ VWLPXODWLRQ ZLWK &'/DFWLYDWHG ([SDQVLRQ RI EUDFK\XU\VSHFL¿F &'+ T cells '&VSXOVHGZLWKWKH+/$$+ brachyury 9-mer peptide following in vitro stimulation with recombinant 7SIRUDQDGGLWLRQDO,96F\FOHDW7FHOOWR$3& yeast-brachyury–treated DCs DQWLJHQSUHVHQWLQJFHOOV UDWLR2QWKHVL[WKGD\RI,96 WKHSHUFHQWDJHRIEUDFK\XU\±VSHFL¿F&'+ T cells was determined by tetramer staining. As shown in Figure 2A, To investigate whether recombinant yeast- DPDUNHGH[SDQVLRQRIEUDFK\XU\VSHFL¿F&'+ T cells EUDFK\XU\±WUHDWHG '&V FRXOG JHQHUDWH DQG H[SDQG was observed in two out of three donors tested (donors DXWRORJRXV EUDFK\XU\VSHFL¿F &'+ T cells from 3 and 4). When compared for their cytotoxic activity PBMCs, autologous T cells from two HLA-A2+ healthy against tumor targets endogenously expressing brachyury donors (Fig. 1A, donors 3 and 4) were stimulated for two )LJ %  &'+ 7 FHOOV SXUL¿HG IURP WKH 3%0&V RI in vitroVWLPXDWLRQ ,96 F\FOHVZLWKFRQWURO\HDVW±RU GRQRUVDQGZHUHDEOHWRHI¿FLHQWO\O\VH6:FHOOV UHFRPELQDQW \HDVWEUDFK\XU\±WUHDWHG '&V DW D 7 FHOO (HLA-A2+ and brachyury-high), while lower lysis was WR'&UDWLRRI$WWKHHQGRI,967FHOOVZHUH observed against the MCF7 breast carcinoma cell line stained with a PE-labeled brachyury peptide tetramer or that is HLA-A2+ but expresses lower levels of the target a control CMV peptide tetramer. As shown in Figure 1A, EUDFK\XU\ )LJ& 7XPRUO\VLVPHGLDWHGE\SXUL¿HG WKHSHUFHQWDJHRIEUDFK\XU\WHWUDPHUSRVLWLYH&'+ T cells &'+ T cells was proportional to the percentage of was higher in cultures stimulated with recombinant yeast- EUDFK\XU\WHWUDPHU&'+ T cells observed for each donor. EUDFK\XU\±FRPSDUHGWRFRQWURO\HDVW±WUHDWHG'&V7KH 6LPLODU VWXGLHV ZHUH FRQGXFWHG WR GHWHUPLQH detection of some level of brachyury tetramer positive ZKHWKHUEUDFK\XU\VSHFL¿F7FHOOVFRXOGDOVREHH[SDQGHG FHOOVLQ7FHOOVVWLPXODWHGZLWKFRQWURO\HDVW±WUHDWHG'&V from PBMCs obtained from two patients. might be attributed, as indicated above, to the ability of PBMCs from patients 1 and 2 were stimulated for two FRQWURO\HDVWWRHIIHFWLYHO\DFWLYDWH'&VWRSURGXFHKLJK F\FOHV ZLWK UHFRPELQDQW \HDVWEUDFK\XU\±WUHDWHG '&V levels of type I cytokines which, in turn, could induce the ,96  )LJ '  IROORZHG E\ RQH F\FOH ZLWK &'/ QRQVSHFL¿FH[SDQVLRQRIVRPH&'+ T cells. WUHDWHG '&V SXOVHG ZLWK WKH 7S EUDFK\XU\ SHSWLGH By using PBMCs from seven additional HLA-A2+ ,96)LJ' $ERXWDQGRIEUDFK\XU\ healthy donors (Fig. 1B, donors 5-11; insert is donor 5) WHWUDPHU SRVLWLYH&'+ T cells were detected in T-cell ZHFRQ¿UPHGWKHVHREVHUYDWLRQVDPDUNHGH[SDQVLRQRI cultures from patients 1 and 2, respectively, at the end of EUDFK\XU\WHWUDPHUSRVLWLYH&'+ T cells was observed ,96&'+7FHOOVSXUL¿HGIURPWKHFXOWXUHRI3%0&V LQGRQRUVWHVWHGDWWKHHQGRI,967RH[DPLQHWKH from patient 1 were assayed for cytotoxic activity against F\WRO\WLF DFWLYLW\ RI WKH H[SDQGHG EUDFK\XU\VSHFL¿F WKHFRORQ6:DQGOXQJ+WXPRUFHOOOLQHVERWK &'+ T cells, a cytotoxic T lymphocyte (CTL) assay was HLA-A2+ and brachyury-positive. As shown, brachyury- performed with T cells expanded from PBMCs of donor VSHFL¿F&7/VVXFFHVVIXOO\O\VHGERWKWXPRUFHOOOLQHVin DIWHUWZRURXQGVRI,96ZLWK\HDVWEUDFK\XU\±WUHDWHG vitro (Fig. 2E). '&V&'+7FHOOVZHUHSXUL¿HGE\QHJDWLYHVHOHFWLRQ using magnetic beads and used as effectors against www.impactjournals.com/oncotarget 17 0 Oncotarget 2013; 4: Expansion of CD4+ EUDFK\XU\VSHFL¿F 7 FHOOV '&VWRH[SDQGEUDFK\XU\VSHFL¿F&'+ T-cell populations following stimulation with recombinant yeast- was also investigated. PBMCs from nine healthy donors brachyury–treated DCs were stimulated in the presence of control yeast– or UHFRPELQDQW\HDVWEUDFK\XU\±WUHDWHGLUUDGLDWHG'&V '& WR\HDVWUDWLRRI IRURQHRUWZR,96F\FOHV$WWKH The ability of recombinant yeast-brachyury–treated HQGRIHDFKF\FOH&'+7FHOOVZHUHSXUL¿HGE\QHJDWLYH

AB C Donor 3 Donor 4 Donor 10 0 2.5 Control Tetramer 60 10 Brachyury Tetramer 50 2.0 10-1 40 1.5 10-2 30 1.0 10-3 20

% Specific Lysis 10-4 0.5 10 Normalized Brachyury mRNA Normalized Brachyury % Tetramer+/ CD8+ T cells 0.0 0 10-5 3 4 10

Donor MCF7 MCF7 MCF7 MCF7 SW480 SW480 SW480 SW480

D E Patient 1 Patient 2 Patient 1

1.0 1.0 Control Tetramer 40 Brachyury Tetramer 0.8 0.8 30

0.6 0.6 20 0.4 0.4

% Specific Lysis 10 0.2 0.2 % Tetramer+/ CD8+ T cells 0.0 0.0 0 IVS 2 IVS 3 IVS 2 IVS 3 SW480 H441 F 1200 200 HSA protein HSA protein Brachyury protein * Brachyury protein 1000 150 800

600 100 Ȗ SJPO Ȗ SJPO 400 IFN- IFN- 50 200

0 0 Control Brachyury Control Brachyury yeast yeast yeast yeast

)LJXUH*HQHUDWLRQRIEUDFK\XU\VSHFL¿F&'+ and CD4+7FHOOUHVSRQVHVXSRQVWLPXODWLRQZLWK\HDVWEUDFK\XU\௅ WUHDWHG'&VA, Autologous T cells from 3 HLA-A2+KHDOWK\GRQRUVZHUHVWLPXODWHGZLWKLUUDGLDWHGFRQWURO\HDVW௅RU\HDVWEUDFK\XU\௅ WUHDWHG'&V '&\HDVWUDWLRRI IRU,96DQGWKHQZLWKEUDFK\XU\SHSWLGHSXOVHG'&VIRUDQDGGLWLRQDO,96F\FOH ,96 6KRZQLV WKHSHUFHQWDJHRI&'+ T cells that co-stained with a brachyury tetramer (black bars) or a control CMV tetramer (gray bars). B, On day 6 RI,96&'+7FHOOVZHUHSXUL¿HGE\QHJDWLYHVHOHFWLRQDQGD&7/DVVD\ZDVSHUIRUPHGRYHUQLJKW (7UDWLRRIIRUGRQRUVDQG and 30:1 for donor 10). 111In-labeled HLA-A2+6: EUDFK\XU\KLJK DQG0&)WXPRUFHOOV EUDFK\XU\ORZ ZHUHXVHGDVWDUJHWVC, Brachyury mRNA expression in indicated tumor cells, normalized to GAPDH. D, Percentage of tetramer positive (brachyury vs. control) LQ&'+ T cells expanded from blood of 2 breast cancer patients stimulated in vitroZLWK\HDVWEUDFK\XU\௅WUHDWHG'&VIRU,96IROORZHG E\RQH,96F\FOHZLWKEUDFK\XU\SHSWLGHSXOVHG'&VE3XUL¿HG&'+ T cells from patient 1 were used in a cytotoxic assay with labeled HLA-A2+EUDFK\XU\SRVLWLYH6:DQG+FHOOV (7UDWLRRI F*HQHUDWLRQRIEUDFK\XU\VSHFL¿F&'+ T-cell responses upon VWLPXODWLRQZLWK\HDVWEUDFK\XU\௅WUHDWHG'&V$XWRORJRXV7FHOOVIURPKHDOWK\GRQRUVZHUHVWLPXODWHGIRU,96F\FOH OHIWSDQHOGRQRU  RU,96 ULJKWSDQHOGRQRU ZLWKLUUDGLDWHGFRQWURORU\HDVWEUDFK\XU\௅WUHDWHG'&V '&WR\HDVWUDWLRRI $WWKHHQGRI,96  GRQRU RU,96 GRQRU &'+7FHOOVZHUHSXUL¿HGE\QHJDWLYHVHOHFWLRQDQGUHVWLPXODWHGZLWKDXWRORJRXVLUUDGLDWHG3%0&V 7 FHOOVWR$3&UDWLRRI LQWKHSUHVHQFHRIFRQWUROSURWHLQ +6$ RUSXUL¿HG+LVEUDFK\XU\SURWHLQ —JPO IRUKRXUV6XSHUQDWDQWV ZHUHDQDO\]HGIRU,)1ȖSURGXFWLRQE\(/,6$UHVXOWVDUHVKRZQLQSJPO%DUVUHSUHVHQWPHDQRIGXSOLFDWHPHDVXUHPHQWVHUURUEDUV UHSUHVHQW6' p<0.05. www.impactjournals.com/oncotarget 17 1 Oncotarget 2013; 4: selection and restimulated with autologous irradiated EUDFK\XU\±EXWQRWFRQWURO\HDVW±WUHDWHG'&VSURGXFHG PBMCs (T cells-to-APC ratio of 1:3) in the presence of ,)1ȖLQUHVSRQVHWRWKHSXUL¿HGEUDFK\XU\SURWHLQEXW FRQWUROSURWHLQ KXPDQVHUXPDOEXPLQ+6$ RUSXUL¿HG QRW FRQWURO SURWHLQ ,Q WRWDO EUDFK\XU\VSHFL¿F &'+ His-brachyury protein. Cell culture supernatants were T-cell responses were generated from three of nine healthy DQDO\]HGIRU,)1ȖSURGXFWLRQE\(/,6$$VGHSLFWHG donors tested. Altogether these results indicated that LQ )LJXUH ) IRU WZR GLIIHUHQW GRQRUV &'+ T cells UHFRPELQDQW\HDVWEUDFK\XU\±WUHDWHG'&VDUHFDSDEOHRI SXUL¿HGIURPFXOWXUHVVWLPXODWHGZLWKUHFRPELQDQW\HDVW H[SDQGLQJERWKEUDFK\XU\VSHFL¿F&'+DQG&'+ T cells

A

20000 Control yeast Brachyury yeast 15000 * *

* 10000 * *

5000 Brachyury-specific

CD4+ T-cell proliferation(cpm) 0 0.0 1.3 2.5 5.0 10.0 40.0 His-Brachyury protein [Pg/ml]

B

120 Control yeast Brachyury yeast 100

80 (pg/ml)

J 60 *

IFN- 40

20

0 C57/Bra C57/Bra peptide N Peptide A

C 25 HBSS )

3 Control yeast (4 YU) 20 Brachyury yeast (4 YU) 15

10

Area of wound Areawound of(mm 5

0 02468 10 Time (days) )LJXUH

A B 100

10-1 MC38 pCDNA pBr 10-2 p<0.001 49 Kd Brachyury 10-3 38 Kd ȕ-actin 10-4

10-5

10-6 Normalized Brachyury Normalized Brachyury mRNA 10-7 pCDNA pBrachyury MC38 C D 600 p<0.01 MC38 pcDNA 0.6 MC38-pCDNA MC38-pBr

400 0.4

MC38 pBrachyury * 200 0.2 Absorbance (570 nm)

Number ofinvasive cells * * 0 0 pCDNA pBrachyury 0255075 100 MC38 Culture time (hours)

E F 50 p<0.01 0.7 p=0.001

40 0.6 30

20 0.5 Lung Weight(g) 10

Number oflung metastases 0.4 0 0 pcDNA pBrachyury pcDNA pBr Control Brachyury yeast yeast MC38 MC38 )LJXUH$QWLWXPRUHIIHFWRI\HDVWEUDFK\XU\YDFFLQDWLRQLQWKH0&EUDFK\XU\PHWDVWDWLFWXPRUPRGHOBrachyury mRNA (A) and protein (B H[SUHVVLRQOHYHOVLQ0&FHOOVVWDEO\WUDQVIHFWHGZLWKHLWKHUDQHPSW\YHFWRU SF'1$ RUDYHFWRUHQFRGLQJ the full-length human brachyury (pBrachyury). C, In vitro(&0LQYDVLRQDVVD\IRU0&S&'1$DQG0&S%UDFK\XU\FHOOV 5HVXOWVRIRIH[SHULPHQWVDUHVKRZQLQWKHJUDSKDQGUHSUHVHQWDWLYHLPDJHVRIWKHERWWRPRIWKH¿OWHUVDUHDOVRVKRZQD, In vitro SUROLIHUDWLRQRI0&S&'1$DQG0&S%UDFK\XU\FHOOVDVDVVHVVHGE\WKH077DVVD\E2QHPLOOLRQWXPRUFHOOV 0&S&'1$ RU0&S%UDFK\XU\ ZHUHLQMHFWHGLQWUDYHQRXVO\YLDWDLOYHLQRQGD\LQWR&%/PLFH$WVDFUL¿FH GD\ OXQJVZHUHLQÀDWHG with India Ink and the number of lung tumor nodules enumerated. Representative images of lungs in each group are displayed. F, MC38- pBrachyury tumor cells (1 x 106FHOOV ZHUHLPSODQWHGLQ&%/PLFHYLDWDLOYHLQRQGD\0LFHZHUHVXEVHTXHQWO\YDFFLQDWHGRQGD\ ZLWKEUDFK\XU\\HDVWRUFRQWURO\HDVW <8[VLWHVZHHNO\ERRVWHUV $WVDFUL¿FHOXQJVZHUHLQÀDWHGZLWK,QGLDLQNDQGZHLJKHG,Q DOOJUDSKVHUURUEDUVLQGLFDWHWKHVWDQGDUGHUURURIWKHPHDQ 6(0 RIWULSOLFDWHPHDVXUHPHQWV www.impactjournals.com/oncotarget 17 4 Oncotarget 2013; 4: Table 2A: Recombinant yeast-brachyury vaccination does not induce toxicity +%66 Control yeast Brachyury yeast Body weight (normal ranges 18-26 g) 25.3 ± 1.7 24.5 ± 2.2 25.3 ± 2.1 &%& Q  Normal Normal Normal 6HUXPFKHPLVWU\ Q  Normal Normal Normal $XWRLPPXQHSDQHO Q  Normal Normal Normal +LVWRSDWKRORJ\ Q  Normal Normal Normal

*URXSVRIIHPDOH%DOEFPLFHZHUHYDFFLQDWHGZLWK+%66FRQWURO\HDVWRUUHFRPELQDQW\HDVWEUDFK\XU\ <8SHUVLWH at 4 sites) as described in the Materials and Methods section. One week after the last vaccination, animals were weighed, euthanized and tissues collected for evaluation.

Table 2B: Effect of recombinant yeast-brachyury vaccination on future pregnancies +%66 Control yeast Brachyury yeast 5DWHRISUHJQDQF\        Number of pups (range per mother) 54 (5-9) 52 (3-11) 51 (5-9) Condition of the newborns Normal Normal Normal Condition of the pups at weaning Normal Normal Normal Number of mice at endpoint 27 36 34

*URXSVRIIHPDOH&%/PLFHZHUHYDFFLQDWHGZLWK+%66FRQWURO\HDVWRUUHFRPELQDQW\HDVWEUDFK\XU\ <8 per site at 4 sites) weekly for a period of 4 weeks. Two weeks after the last vaccination, females in each group were bred (harem-breeding was performed). Rate of pregnancy, litter size, general condition of the pups, as well as development of the pups post-weaning were evaluated for each group. development, it was also investigated whether vaccination DQGH[SDQGEUDFK\XU\VSHFL¿FKXPDQ&'+DQG&'+ T with recombinant yeast-brachyury could have any effect cells in vitro from the blood of healthy donors and cancer RQ VXEVHTXHQW SUHJQDQFLHV RU WKH FRQGLWLRQ RI WKH patients, and elicits an effective immune response in vivo offspring. Female C57BL/6 mice were vaccinated with in vaccinated mice, which drives anti-tumor activity in the +%66RUFRQWURO\HDVWRUUHFRPELQDQW\HDVWEUDFK\XU\  absence of toxicity. YU per site at four sites) weekly for a period of 4 weeks. The approach described here could potentially be Two weeks after the last vaccination, females were bred applied towards the treatment of several types of cancer. and the rate of pregnancy, litter size, general condition of Expression of the brachyury protein has been previously the pups, as well as the development of the pups post- demonstrated with a polyclonal antibody in chordomas weaning were evaluated for each group. As shown in Table > @ ,Q VXEVHTXHQW VWXGLHV HPSOR\LQJ 573&5 > 2B, brachyury-yeast vaccination had no effect in any of 11, 21] or monoclonal antibodies in western blot or the parameters evaluated. immunohistochemistry [6], the expression of brachyury was also demonstrated in a range of human carcinomas, DISCUSSION including breast, lung, colorectal, pancreatic, and ovarian, and not in the majority of normal tissues evaluated. In The phenomenon of tumor EMT is now thought to many cases, brachyury was only expressed in a minority be critical to the progression of carcinomas by promoting of primary tumor cells while a higher proportion of WKHGLVVHPLQDWLRQRIFDQFHUFHOOVDQGWKHDFTXLVLWLRQRI brachyury-positive tumor cells and higher levels of mechanisms of resistance to various cancer therapies expression were observed in metastatic lymph nodes or >@7KLVVWXG\UHSRUWVIRUWKH¿UVWWLPHWKHGHYHORSPHQW distal metastases [6]. This is not surprising since the EMT and characterization of an immunological approach to process is thought to be dynamic and the transition from an potentially targeting tumor cells undergoing EMT via HSLWKHOLDOWRDPHVHQFK\PDOSKHQRW\SHFDQEHLQÀXHQFHG the use of a recombinant yeast vaccine that expresses by factors in the tumor microenvironment, such as IL-8 brachyury, a transcription factor able to drive EMT in DQG7*)ȕ>@,QDGGLWLRQWRFDUFLQRPDVEUDFK\XU\ human carcinomas [11, 12, 20]. We demonstrate here is also expressed in multiple myeloma and chronic that a recombinant yeast-brachyury vaccine can activate lymphocytic leukemia cells (unpublished data). Recent www.impactjournals.com/oncotarget 17 Oncotarget 2013; 4: data has also shown that brachyury is an independent poor control yeast can induce a strong innate immune response; prognostic indicator in [23], early colorectal WUHDWPHQW RI PXULQH RU KXPDQ '&V ZLWK FRQWURO \HDVW cancer [24], and breast cancer (unpublished data). HI¿FLHQWO\ PDWXUHG '&V DQG LQGXFHG VHFUHWLRQ RI W\SH Multiple studies have now demonstrated a I cytokines to similar levels than those elicited by TLR UHODWLRQVKLSEHWZHHQ(07DQGWXPRU³VWHPQHVV´>@ agonists. Thus it is confounding to consider the control %RWK³FDQFHUVWHPOLNH´FHOOVDQGWXPRUFHOOVXQGHUJRLQJ yeast vector as a true control for any immune response. EMT are characterized by drug resistance [10, 28], and Toxicology studies are important when dealing with are known to overexpress several transcription factors, a target involved in EMT as this process is also implicated LQFOXGLQJ 2&7 DQG 62; WKDW DUH LQYROYHG LQ WKH in tissue remodeling and wound healing. Here we have control of cell pluripotency [27, 29]. Thus, targeting of conducted toxicology studies in mice and reported no tumor cells undergoing EMT could also result in the toxicities associated with yeast-brachyury vaccination. elimination of cancer cells that exhibit tumor-initiating The murine toxicology studies, however, cannot substitute properties. Up to date, several transcription factors have for a carefully designed incremental dosing Phase I trial; EHHQ LGHQWL¿HG WKDW DUH DEOH WR GULYH WXPRU (07 RQH such trial has already been initiated employing GI-6301 distinction that has been reported between brachyury and in patients with metastatic cancer [19]. To our knowledge, RWKHUVLQFXGLQJ7:,6761$,/DQG6/8*LVWKHPRUH WKLVLVWKH¿UVWYDFFLQHSODWIRUPDLPHGDWWDUJHWLQJDGULYHU selective expression of brachyury in tumors vs. normal of tumor EMT that has successfully reached the clinical tissues [6, 21], which makes it an attractive target for stage. anti-tumor interventions. Transcription factors, however, DUHWKRXJKWWREH³XQGUXJJDEOH´E\GLUHFWFRQYHQWLRQDO MATERIALS AND METHODS small molecule inhibitor approaches due to their lack RIDVSHFL¿FJURRYHIRUWLJKWELQGLQJRIDQLQKLELWRU,Q addition, their intracellular localization would preclude Culture and maturation of DCs the use of monoclonal antibody targeting approaches. The use of a vaccine therapy that relies on T-cell mediated targeting of the transcription factor may circumvent these Peripheral blood used in this study was collected characteristics. Although transcription factors, such as from healthy donors and cancer patients after Institutional brachyury, would be expected to be found only in the Review Board approval and informed consent were nuclear compartment, short fragments of brachyury (9- obtained. PBMCs were isolated from peripheral blood 15 amino acids long) are processed and transported to the by centrifugation on a Ficoll density gradient. For the surface of the tumor cell in the context of MHC class I and SUHSDUDWLRQRI'&V3%0&VZHUHUHVXVSHQGHGLQ$,09 class II molecules to activate EUDFK\XU\VSHFL¿F&'+ and medium (Invitrogen, Carlsbad, CA) and allowed to adhere &'+7FHOOVUHVSHFWLYHO\$VVKRZQKHUH'&VWUHDWHG WR WKH VXUIDFH RI7ÀDVNV IRU  KRXUV DW ž& WKH with recombinant yeast-brachyury can indeed generate adherent cells were cultured for 7 days in AIM-V medium ERWKKXPDQ&'+DQG&'+7FHOOUHVSRQVHVVSHFL¿FIRU FRQWDLQLQJ UHFRPELQDQW KXPDQ *0&6)  QJPO  brachyury. The murine studies reported here also show and IL-4 (20 ng/ml) (Peprotech, Rocky Hill, NJ). For WKDWWKHUHFRPELQDQW\HDVWEUDFK\XU\FDQHOLFLWERWK&'+ PDWXUDWLRQRI'&VFRQWURO\HDVWRUDUHFRPELQDQW\HDVW DQG&'+ T-cell responses in vivo as well as anti-tumor EUDFK\XU\SUHSDUDWLRQDWDUDWLRRI\HDVWWR'&VRI activity. RUȝJPOUHFRPELQDQWKXPDQ&'OLJDQG &'/  A vaccine consisting of a recombinant yeast- SOXVȝJPOFURVVOLQNHUHQKDQFHU (Q]R/LIH6FLHQFHV brachyury has some potential favorable characteristics: Farmingdale, NY) were added to the cultures 48 hours recombinant yeast is relatively easy to generate, can be prior to use. SURSDJDWHGDQGVWRUHGDVDQ³RIIWKHVKHOI´GUXJDQGLV heat-killed and thus relatively safe [30, 31], as shown Human tumor cell lines E\DQH[FHOOHQWVDIHW\SUR¿OHLQSULRUFOLQLFDOVWXGLHVRI recombinant yeast vaccines directed against hepatitis C The human tumor cell lines used in this study, virus, mutated ras and CEA. In addition, the recombinant FRORQ6:EUHDVW0&)DQGOXQJ+DQG+ yeast-brachyury contains the full-length human brachyury were purchased from the American Type Culture + JHQHSURGXFWDQGWKXVFRXOGEHDEOHWRHOLFLWERWK&' &ROOHFWLRQ $7&&*DLWKHUVEXUJ0' DQGSURSDJDWHGDV + DQG&' T-cell responses regardless of the MHC allele(s) recommended by the ATCC. of the patient. While the recombinant yeast-brachyury YDFFLQH LV VKRZQ KHUH WR EH PRUH HI¿FDFLRXV WKDQ WKH Yeast vectors FRQWURO \HDVW LQ WKH LQGXFWLRQ RI EUDFK\XU\VSHFL¿F immune responses in vitro and in vivo and anti-tumor activity, the experiments reveal some degree of immune Two recombinant Saccharomyces cerevisiae effect with control yeast. Previous studies have shown that constructs were utilized in this study: a control empty www.impactjournals.com/oncotarget 17 Oncotarget 2013; 4: construct (designated as control yeast) and a construct Biotec, Auburn, CA) and re-stimulated (1 x 104&'+ T expressing the full-length human brachyury protein cells per well) with autologous irradiated PBMCs as APCs (designated as recombinant yeast-brachyury, GI-6301). &'WR$3&UDWLR  LQWKHSUHVHQFHRIHLWKHU+6$ The expression of brachyury in the recombinant yeast- (control protein) or His-brachyury protein (10 μg/ml) for brachyury vaccine, as detected with an anti-brachyury KRXUV&HOOFXOWXUHVXSHUQDWDQWVZHUHFROOHFWHG,)1Ȗ PXULQH0$ELVVKRZQLQ6XSSOHPHQWDO)LJXUH7KHVH ZDVPHDVXUHGE\(/,6$ vectors were engineered as previously described [32] DV SDUW RI D &RRSHUDWLYH 5HVHDUFK DQG 'HYHORSPHQW Flow cytometry $JUHHPHQW &5$'$ EHWZHHQWKH/DERUDWRU\RI7XPRU Immunology and Biology, NCI, and GlobeImmune, Inc. (Louisville, CO). 3(ODEHOHGDQWL&'DQWL&'DQWL&'DQWL HLA Class I, anti-HLA Class II, and appropriate isotype FRQWUROVZHUHSXUFKDVHGIURP%'%LRVFLHQFHV 6DQ'LHJR Peptides and CA). A PE-conjugated HLA-A2+/Tp2 brachyury peptide tetramer (NIH Tetramer Core Facility, Atlanta, GA) and 7KH KXPDQ EUDFK\XU\VSHFL¿F +/$$ ELQGLQJ a control HLA-A2+/CMV (NLVPMVATV) tetramer 9-mer peptide used in this study (designated as peptide (Beckman-Coulter, Brea, CA) were used. 7S ://3*767/  ZDV SUHYLRXVO\ GHVFULEHG >@ 7ZRSHSWLGHVGHULYHGIURPWKHVHTXHQFHRIWKHKXPDQ Detection of cytokines brachyury protein were used in experiments with C57BL/6 PLFHWKH&%UDSHSWLGH1 :6961*$97 DQGWKH &%UD SHSWLGH $ :6961*$9/  7R DVVHVV &'+ Culture supernatants were collected at indicated T-cell responses, a His-brachyury fusion protein was WLPHVDQGDQDO\]HGE\(/,6$IRUWKHSUHVHQFHRI,/ produced via a baculovirus expression system in insect %' %LRVFLHQFHV  RU ,)1Ȗ ,QYLWURJHQ  5HVXOWV ZHUH FHOOV+6$SURWHLQZDVXVHGDVFRQWURO 6LJPD6W/RXLV expressed in pg/ml. MO). Cytotoxic T-cell killing assay ([SDQVLRQRIEUDFK\XU\VSHFL¿F&'+ T cells in vitro 7DUJHWFHOOVZHUHODEHOHGZLWKȝ&LRI111Indium- ODEHOHGR[\TXLQROLQH $PHUVKDP+HDOWK6LOYHU6SULQJ 0' DQGSODWHG [3 cells per well) with effector cells %UDFK\XU\VSHFL¿F F\WRWR[LF 7 O\PSKRF\WHV at various effector-to-target (E:T) cell ratios. For MHC were generated from the blood of healthy donors or two class I blockade experiments, labeled target cells were different breast cancer patients post-vaccination with a LQFXEDWHGZLWKȝJPODQWLKXPDQ+/$$%&DQWLERG\ 08&&($75,&20EDVHG YDFFLQH %ULHÀ\ \HDVW 6HURWHF 5DOHLJK 1&  RU ,J* LVRW\SH FRQWURO %' WUHDWHG RU SHSWLGHSXOVHG LUUDGLDWHG  *\  '&V ZHUH Biosciences) for 30 min at 37°C, before the addition of used as antigen-presenting cells (APCs) with autologous HIIHFWRUFHOOV6XSHUQDWDQWVZHUHKDUYHVWHGDIWHUKRXUV PBMCs, used at a ratio of PBMC-to-APC of 10:1. and 111In release was measured by gamma counting. Cultures were maintained for 3 initial days in RPMI 1640 medium (Mediatech, Inc., Herndon, VA) supplemented ZLWKKXPDQ$%VHUXP *HPLQL%LR3URGXFWV:HVW Brachyury gene expression 6DFUDPHQWR&$ DQGIRUDGGLWLRQDOGD\VLQWKHVDPH medium supplemented with 20 U/ml of recombinant RNA from human and murine carcinoma cells human IL-2. After a 7-day culture period, designated as an ZDV SXUL¿HG XVLQJ WKH 4LDJHQ 51HDV\ NLW 9DOHQFLD in vitro VWLPXODWLRQ ,96 F\FOHFHOOVZHUHUHVWLPXODWHG CA). Real-time PCR was performed according to the as described above. PDQXIDFWXUHU¶V SURWRFRO XVLQJ  QJ F'1$ DQG WKH IROORZLQJ7DT0DQJHQHH[SUHVVLRQDVVD\VIURP$SSOLHG ([SDQVLRQRIEUDFK\XU\VSHFL¿F&'+ T cells in Biosystems: brachyury (Hs_00610080), human and vitro PXULQH*$3'+ (( 

Animals Control yeast or recombinant yeast-brachyury– WUHDWHG'&VZHUHLUUDGLDWHG *\ DQGXVHGDV$3&VWR stimulate autologous PBMCs in vitro at a PBMC-to-APC All mice were housed and maintained in ratio of 10:1. Cultures were maintained as above. At the PLFURLVRODWRU FDJHV XQGHU VSHFL¿F SDWKRJHQIUHH end of one to two in vitro ,96&'+ T cells were isolated conditions and in accordance with the Association for by negative selection with magnetic beads (Miltenyi Assessment and Accreditation of Laboratory Animal Care www.impactjournals.com/oncotarget 17 7 Oncotarget 2013; 4: (AAALAC) guidelines. All experimental studies were Toxicology study carried out under approval of the NIH Intramural Animal Care and Use Committee. *URXSV RI ¿YH IHPDOH %$/%F PLFH ZHUH vaccinated with 1 YU per site at four sites (4 YU total) of Murine CD4+DQG&'+ T-cell responses yeast control or yeast-brachyury weekly for 12 weeks, then HYHU\ZHHNVIRUDQDGGLWLRQDOZHHNVDQGVXEVHTXHQWO\ C57BL/6 mice were vaccinated weekly with either every month for 2 months. An additional group of 10 yeast control or recombinant yeast-brachyury vaccine at 1 FRQWURO DQLPDOV ZDV VLPLODUO\ LQMHFWHG ZLWK +%66 YU per site at four sites (4 YU total) for 4 weeks. Fourteen One week following the last injection, animals were days after the last vaccination, animals were euthanized, assessed for any potential toxicities in a blinded manner VSOHQRF\WHV FROOHFWHG DQG &'+ 7 FHOOV SXUL¿HG E\ at Charles River Pathology Associates (Wilmington, QHJDWLYH VHOHFWLRQ 0LOWHQ\L %LRWHF  &'+ T cells (1 MA) utilizing the following parameters: in-life body x 105) were plated with 5 x 105 irradiated syngeneic weight, histopathology (10 tissues), serum chemistry (7 spleen cells and indicated concentrations of either His- parameters), CBC (20 parameters), and autoimmunity (5 EUDFK\XU\ RU ȕJDODFWRVLGDVH ȕJDO  SURWHLQ $IWHU  parameters) as described previously [33]. GD\V RI LQFXEDWLRQ  ȝ&L RI >3H]-thymidine (Perkin- Elmer, Waltham, MA) was added per well; plates were Tumor cell transfection harvested after 16 hours and thymidine incorporation was measured using a 1450 Betaplate reader (Perkin-Elmer). Murine colon carcinoma MC38 cells were stably 3UROLIHUDWLRQZDVFRUUHFWHGIRUȕJDOFRXQWV)RU&'+ WUDQVIHFWHGZLWKDQHPSW\SF'1$72YHFWRU ,QYLWURJHQ T-cell responses, mice were vaccinated weekly with either GHVLJQDWHGDVSF'1$ RUDIXOOOHQJWKKXPDQEUDFK\XU\ vaccine at 0.025 YU per site at 4 sites (0.1 YU total) for encoding (pBrachyury) vector using a Nucleofector system 3 weeks, followed by 2 weeks rest, and splenocytes were according to the manufacturer’s recommendations (Lonza, FROOHFWHGDQGVWLPXODWHGIRUGD\VZLWKȝJPORI& :DONHUVYLOOH 0'  7UDQVIHFWHG FHOOV ZHUH VHOHFWHG LQ Bra peptide N or peptide A. T cells (1 x 105) were cultured media containing 100 μg/ml Zeocine (Invitrogen). with 5 x 105 irradiated syngeneic spleen cells along with ȝJPORIWKHEUDFK\XU\RUDFRQWURO+,9JDJSHSWLGH 649713$1, &XOWXUHVXSHUQDWDQWVZHUHFROOHFWHGDW Tumor cell migration, invasion and growth assays KRXUV,)1ȖZDVDVVHVVHGE\(/,6$5HVXOWVZHUH background-corrected for HIV peptide. Migration and invasion assays were performed as previously described [11]. RPMI 1640 supplemented with Effect of yeast-brachyury vaccination on wound )%6PHGLXPZDVDGGHGWRWKHORZHUFKDPEHUVDQG healing 1 x 105 cells in serum-free medium were added onto the XSSHUFKDPEHUVIROORZHGE\LQFXEDWLRQRYHUQLJKWDWÛ& Cell growth was evaluated using 3-4,5-dimethylthiozol-2, *URXSV RI ¿YH IHPDOH &%/ PLFH ZHUH GLSKHQ\OWHWUD]ROLXPEURPLGH 077  6LJPD$OGULFK vaccinated with 1 YU at four sites (4 YU total) of yeast 6W/RXLV02  control or recombinant yeast-brachyury weekly for 4 weeks. One week after the last injection animals were Experimental model of metastasis and antitumor anesthetized, shaved and wounded utilizing a 4 mm diameter AcuPunch skin biopsy punch (Acuderm Inc, vaccination Ft. Lauderdale, FL). Wounded areas were measured until closure. C57BL/6 mice were injected with 1 x 106 MC38- S&'1$ Q   RU 0&S%UDFK\XU\ Q   FHOOV Effect of yeast-brachyury vaccination on future intravenously into the tail vein. Forty days after tumor pregnancy LPSODQWDWLRQDQLPDOVZHUHHXWKDQL]HGOXQJVZHUHLQÀDWHG with India ink and tumor nodules were counted utilizing a dissecting microscope. For antitumor experiments, Groups of 10 female C57BL/6 mice were weekly mice were injected with 1 x 106 MC38-pBrachyury vaccinated with yeast control or yeast-brachyury at 1 YU cells intravenously into the tail vein; 4 days after tumor per site at four sites (4 YU total) for 4 weeks. An additional implantation, groups of mice were vaccinated with either group of 10 control animals were similarly injected with FRQWURO \HDVW Q   RU UHFRPELQDQW \HDVWEUDFK\XU\ +DQN¶V EDODQFHG VROXWLRQ +%66  2QH ZHHN DIWHU WKH Q  DWDGRVHRI<8DWIRXUVLWHV <8WRWDO DQG last injection, animals were placed for harem breeding. VXEVHTXHQWO\ZHHNO\IRUWKHGXUDWLRQRIWKHH[SHULPHQW The rate of pregnancy, litter size, as well as the general Mice were euthanized on day 36 post-tumor implantation; condition of the pups were evaluated. OXQJVZHUHLQÀDWHGZLWK,QGLD,QNYLVXDOO\LQVSHFWHGIRU www.impactjournals.com/oncotarget 17 Oncotarget 2013; 4: the presence of tumor nodules and weighed. opportunity for novel interventions against lung cancer. Clin Cancer Res. 2012; 18(14):3868-3879. Statistical analysis  9XMRYLF6+HQGHUVRQ63UHVQHDX12GHOO(-DFTXHV76 Tirabosco R, Boshoff C and Flanagan AM. Brachyury, a crucial regulator of notochordal development, is a novel 'DWD ZHUH DQDO\]HG XVLQJ *UDSK3DG 3ULVP biomarker for chordomas. J Pathol. 2006; 209(2):157-165. 9HUVLRQ*UDSK3DG 6RIWZDUH  'DWD SRLQWV LQ JUDSKV represent the mean of triplicate measurements ± standard