cancer immunology immunotherapy; Cll
`so
`no. 5 (May 2011)
`eral Collection
`
`
`
`Volume 60: Number5 - May 2011
`
`
`
`Soto BL, HankJA, Van De Voort TJ, SubramanianL, PolansAS,
`OriginalArticles
`Rakhmilevich AL,Yang RK, Seo S,Kim K, Reisfeld RA, Gillies SD,
`Sondel PM:
`TadmorT, ZhangY, Cho H-M,PodackER, Rosenblatt JD:
`The anti-tumoreffect of resveratrol alone or in combination
`The absence ofB lymphocytes reduces the numberand function
`with immunotherapyin a neuroblastoma model
`731
`ofT-regulatorycells and enhancesthe anti-tumorresponse
`YangS, Luca G,Liu F, JiY, Yu Z, Restifo NP Rosenberg SA, Morgan RA:
`ina murine tumor model
`609
`In vitro generated anti-tumorT lymphocytes exhibit distinct
`Kim S-K,Wu X, RagupathiG,Gathuru J, Koide F, Cheung N-K,
`subsets mimickingin vivo antigen-experienced cells
`739
`Panageas K,Livingston PO:
`Impact of minimal tumor burden on antibody response
`Erratum
`tovaccination
`621
`Vasievich EA, Chen W, HuangL:FeeSta
`Enantiospecific adjuvantactivity of cationic lipid DOTAPin cancer
`Yang S, GattinoniL, Liu F, Ji, Yu Z, Restifo NP Rosenberg SA,
`vaccine
`629
`Morgan RA:
`Battke C, Ruiss R,Welsch U,WimbergerP, LangS,Jochum $,Zeidler R:
`Erratum to: In vitro generated anti-tumorT lymphocytes exhibit
`Tumour exosomesinhibit binding oftumour-reactive antibodies
`distinct subsets mimicking in vivo antigen-experienced cells
`751
`to tumourcells and reduce ADCC
`639
`Battke C, KremmerE, Mysliwietz J, Gondi G, Dumitru C, BrandauS,
`Meeting Report
`Lang 5,Vullo D, SupuranC,Zeidler R: ee
`Generation andcharacterization of thefirst inhibitory antibody
`Ochoa MC, Hervas-StubbsS, Palazon A, Martinez-Forero |, BerraondoP,
`targeting tumour-associated carbonic anhydrase XII
`649
`Sarobe P MelcherA, Melero |:
`;
`;
`;
`International symposium on CTL and immunostimulation,
`Shirota H, Klinman DM:
`A
`CpG-conjugated apoptotic tumorcells elicit potent tumor-specific
`Pamplona(Spain), October 26th and 27th 2010
`753
`immunity
`659
`Amos SM, Pegram HJ, Westwood JA, John LB, Devaud C, Clarke C,
`Restifo NP Smyth MJ, Darcy PK, Kershaw MH:
`Adoptive immunotherapy combined with intratumoral TLR agonist
`delivery eradicates established melanoma in mice
`671
`Lee JH, Chen Y, ChanJL, Qian Y, Goydos JS:
`Molecular analysis of melanoma-induced sentinel lymph node
`immune dysfunction
`685
`Forthcomingarticles in print
`Wiegering V, Eyrich M, Rutkowski S, Wélfl M, Schlegel PG, Winkler B:
`TH1 predominance is associated with improved survivalin pediatric
`Published online and citable with the DOI(Digital Object
`medulloblastomapatients
`693
`:
`Identifier) can befoundatspringerlink.com
`Xu M,WangX,Cai¥,Zhang H,Yang H,Liu C,Zhang C:
`WY ~ oe i
`InstructionsforAuthors
`An engineered superantigen SEC2 exhibits promising antitumor
`be 5
`WYiy
`activity and low toxicity
`705
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`i
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`5
`Instructions for Authors are now available only on the journal's
`He Q,Li J, Yin W, Song Z,ZhangZ, YiT,Tang J,Wu D,Lu ¥, Wang Z,Liu D,
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`oa
`t
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`Zhang X,Hu Z, Gao J:
`i
`Low-dose paclitaxel enhancesthe anti-tumorefficacy of GM-CSF
`ieee 2
`surface-modified whole-tumor-cell vaccine in mouse model|hadnedinGeienemine
`of prostate cancer
`715
`Indexed in/abstracted by Current Contents and IndexMedicus
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`

`

`Cancer Immunol Immunother (2011) 60:739-749
`DOT 10.1007/s00262-01 1-0977-7
`
`
`
`
`This material may be protected by Copyrightlaw (Title 17 U.S. Code)
`
`In vitro generated anti-tumor T lymphocytes exhibit distinct
`subsets mimicking in vivo antigen-experienced cells
`
`Shicheng Yang - Gattinoni Luca - Fang Liu -
`Yun Ji» Zhiya Yu - Nicholas P. Restifo -
`Steven A. Rosenberg « Richard A. Morgan
`
`Received: 19 October 2010/ Accepted: 1] January 2011 /Published online: 9 February 2011
`© Springer-Verlag (outside the USA) 2011
`
`Abstract The T-lymphocyte pool can be subdivided into
`naive (Tn), effector memory (Tem), and central memory
`(Tcm) T cells.
`In this study, we characterized in vitro
`short-term cultured anti-tumor human T lymphocytes
`generated by lentiviral
`transduction with an anti-tumor
`antigen TCR vector. Within 2 weeks of in vitro culture, the
`cultured T cells showed a Tem-like phenotype illustrated
`by a high percentage of CD62L and CD45ROcells. When
`the
`cells were
`sorted
`into
`populations
`that were
`CD45RO+/CD62L-(Tem), CD45RO+/CD62L+(Tem), or
`CD45RO’"/CD62L+(Tn) and co-cultured with antigen-
`matched tumor lines,
`the magnitude of cytokine release
`from these populations for IFNy (Tn < Tem < Tem) and
`IL-2 (In > Tcm > Tem) mimicked the types of immune
`cell responses observed in vivo. In comparing cell-medi-
`ated effector function, Tn were found to be deficient (rel-
`ative to Tem and Tem) in the ability to form conjugates
`with tumor cells and subsequent lytic activity. Moreover,
`analysis of the gene expression profiles of the in vitro
`cultured and sorted T-cell populations also demonstrated
`patterns consistent with their in vivo counterparts. When
`Tcm and Tem were tested for the ability to survive in vivo,
`Tem displayed significantly increased engraftment and
`persistence in NOD/SCID/ye‘~ mice. In general, a large
`percentage of in vitro generated anti-tumor T lymphocytes
`
`Electronic supplementary material The online versionof this
`article (doi: 10.1007/s00262-011-0977-7) contains supplementary
`material, which is available to authorized users.
`
`S. Yang - G. Luca - F. Liu- Y. Ji. Z. Yu -
`N. P. Restifo - S. A. Rosenberg » R. A. Morgan (J)
`Surgery Branch, Center for Cancer Research, National Cancer
`Institute, National Institutes of Health, 10 Center Drive,
`Building 10, CRC 3W-3864, Bethesda, MD 20892, USA
`e-mail: rmorgan@mail.nih.gov
`
`mimic a Tem-like phenotype (based on phenotype, effector
`function, and increased persistence in vivo), which sug-
`gests that these Tem-like cultured T cells may be optimal
`for adoptive immunotherapy.
`
`Keywords Gene therapy - Lentiviral vector - T-cell
`receptor - Central memory cells - Effector memory cells -
`Tumor immunity
`
`Introduction
`
`therapy (ACT) using tumor-infiltrating
`Adoptive cell
`lymphocytes (TIL) is a potent treatment for patients with
`metastatic melanoma and can mediate objective responses
`in 50-70% of patients [1-3]. However, not all patients have
`pre-existing tumor
`reactive T lymphocytes, and these
`tumor reactive lymphocytes are generally only found in
`melanoma patients, which limits the broad application of
`this approach. The transduction of peripheral blood lym-
`phocytes (PBL) with genes encoding anti-tumor TCRs
`using y-retroviruses readily renders autologous PBL from
`any patient into tumor killing T lymphocytes in vitro [4].
`Administration of anticMART-1 TCR-engineered PBL in
`patients with advanced metastatic melanomaresulted in a
`30% objective response rate [5, 6] and holds the promise
`for the treatment of other types of cancer. Genetic modi-
`fication of T cells in vitro with anti-tumor TCRs, followed
`by robust in vitro expansion, may avoid immunetolerance
`observed in vivo and can generate sufficient numbers of
`specific anti-tumor T cells for patient treatment.
`For TCR gene therapy, current protocols using y-retro-
`viral vectors require T cells to be actively dividing for
`efficient gene integration into cellular DNA. The need for
`fully dividing T cells limits the numberofcells that can be
`
`@) Springer
`Miltenyi Ex. 1027 Page 4
`
`Miltenyi Ex. 1027 Page 4
`
`

`

`740
`
`Cancer Immunol Immunother (2011) 60:739-749
`
`a t
`
`ransduced and often requires a second rapid expansion
`(REP) in order to generate enough cells for clinical appli-
`cation. However,
`this second expansion causes cells to
`becomefully differentiated and exhibit an effector memory
`phenotype that may impede in vivo persistence [7] and
`in vivo tumor killing efficacy [8]. Compared with y-retro-
`viral vectors,
`lentiviral vectors efficiently transduce non-
`dividing cells. The minimal
`requirement
`for
`lentiviral
`vector-mediated transduction of quiescent T cells is that T
`cells enter into the GI phase ofthe cell cycle. Following
`anti-CD3 activation, quiescent T cells easily move into the
`G1 phase within hours [9, 10]. The use of anti-CD3/CD28
`beads provided a simple activation method for lentiviral
`vector-mediated transduction and transgene expression
`{9, 11], and a clinical-scale transduction protocol has been
`reported to yield >1 x 10'° transduced T cells in a mini-
`mal culture period [9].
`In humans,
`the T-lymphocyte pool can be subdivided
`into naive antigen-inexperienced cells (Tn) and following
`antigen encounter into effector memory (Tem) and central
`memory (Tcm) T cells [12-15]. Tem cells are thought to
`contain a long-lived memory cell population displaying a
`capacity for self-renewal associated with high levels of
`phosphorylated transcription factors [16]. Recent studies
`suggestthat there are additional subsets of CD8* T-memory
`cells in mice [17] and in humans [18]. Central memory
`CD8*T cells and effector memory CD8* T cells have been
`identified in humans and animals and can be distinguished
`in part by the expression level of CCR7, CD62L [19] and
`the secretion of cytokines [15]. Animal studies suggestthat
`central memory T cells not only survive longer after ACT
`[7] but also confer superior anti-tumor reactivity compared
`with effector memory T cells and can leadto the eradication
`of large established tumors [20].
`The generation ofan active population of memory T cells
`is pivotal for effective vaccine and cell-based therapies to
`fight infectious diseases and cancer [21]. The phenotypic and
`functional characterization of immuneT cells after antigen
`challenge in vivo has been well studied [15, 22]. However,
`the immune-related characteristics of in vitro generated anti-
`tumor antigen TCR-engineered T cells remain to be eluci-
`dated. In this report, we systematically analyzed in vitro
`TCR gene-engineered T lymphocytes for their phenotype,
`function, and in vivo engraftment to determine the optimal
`type ofcell for use in adoptive cell therapy.
`
`Materials and methods
`
`Cell culture
`
`PBL used in this study were obtained from healthy
`donors
`or metastatic melanoma
`patients
`seeking
`
`4 Springer
`
`the Surgery Branch, National Cancer
`at
`treatments
`Institute, under approved clinical protocols. Briefly, PBL
`were collected by leukapheresis, and lymphocytes were
`separated
`by Ficoll/Hypaque
`cushion
`centrifugation,
`washed in HBSS, and resuspended at a concentration of
`1 x 10°%/ml
`in AIM-V medium (Invitrogen, Carlsbad,
`CA)
`supplemented with
`3001U/ml
`IL-2
`and
`5%
`heat-inactivated human AB serum (Valley Biomedical,
`Winchester, VA). Melanomacell lines included MART-
`1-positive HLA-A2* 526 and MART-|-positive HLA-A27
`938. 293T cells (ATCC, Manassas, VA) were cultured in
`DMEMsupplemented with 10% FCS, 100 U/ml penicillin/
`streptomycin, and 2 mM L-glutamine (Invitrogen). All cell
`lines were cultured at 37°C in a 5% CO, humidified
`incubator.
`
`Vector construction and lentivirus preparation
`
`lentiviral constructs utilized were derived from
`The
`pRRLSIN.cPPT.MSCV/GFP.wPRE harboring
`a green
`fluorescent protein (GFP) gene driven by the murine stem
`cell virus (MSCV) U3 promoter [23]. Woodchuck hepa-
`tilis virus response element (wPRE) was replaced with the
`truncated form oPRE, wherethe residual X protein andits
`promoter were deleted [24]. The oPRE sequence was
`synthesized and fused with Sal
`I and EcoR I restrictive
`enzyme sites (Epoch Biolabs, Missouri City, TX) and
`cloned into corresponding sites of pRRLSIN.cPPT.MSCV/
`GFP.wPRE to yield vector pLLV.GFP.oPRE. A lentivi-
`ral vector expressing the gpl100 TCR alpha and beta
`chains of DMF5 TCR targeting melanoma
`antigen
`MART-1 [25] was previously described [9, 26, 27]. All
`constructs are confirmed by restrictive enzyme digestion
`and sequencing.
`For lentivirus preparation, the day before transfection,
`20 x 10° 293 T cells were plated onto 150-mm? poly-p-
`Lysine-coated plates (BD Biosciences, San Jose, CA) using
`15 ml of culture medium. On the day of transfection, the
`medium wasreplaced with 15 ml fresh medium 3 h before
`transfection. Each plate received plasmid DNA 55 pg
`(transfer vector 22.5 ug, VSV-G 7.5 pg, pMDLg/pRRE
`15 ug and pRSV-Rev 10 pg) and 165 jl lipofectamine
`2000 (Invitrogen). Before transfection, 10 ml of culture
`medium was removed from the culture dishes and com-
`plexes of plasmid DNA and Lipofectamine 2000 were
`added evenly onto the medium. Six hours after transfec-
`tion, the plates were washed twice with PBS and 20 ml of
`fresh medium was added. The supernatant was collected
`48 h post-transfection, and cell debris was removed by
`centrifugation at 2,000xg for 10 min. Supernatant con-
`taining viruses was tittered using a p24 kit (ZeptoMetrix,
`Buffalo, NY) and was either used directly or stored at
`—80°C.
`
`Miltenyi Ex. 1027 Page 5
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`Miltenyi Ex. 1027 Page 5
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`The generation of anti-tumor T lymphocytes and FACS
`analysis/sorting
`
`Forcell preparation, briefly PBL were activated using anti-
`CD3/CD28 beads overnight, and next day, the cells were
`transduced with lentiviral vector harboring anti-tumor TCR
`by spinoculation as previously described in detail [9]. Six
`hours post-transduction,
`the cells were transferred to
`75-cm* flask and maintained below concentration of 10°/
`ml. Cell surface expression of CD3, CD4, CD8, CD27,
`CD127, CCR7, CD28, CD70, CD95, CD137, CD57,
`CD62L, and CD45RO was measured using fluorescein
`isothiocyanate (FITC)-, allophycocyanin (APC)-, phyco-
`erythrin (PE) PE-CY-7-, or APC-CY-7- conjugated anti-
`bodies (BD Biosciences, San Jose, CA). MART-1:27-35
`tetramer (PE or APC) was used (iTAg MHC Tetramer,
`Beckman Coulter, Fullerton, CA)
`to verify TCR gene
`transfer. Immunofluorescence staining was analyzed as the
`relative log fluorescence of live cells, determined using a
`FACscan flow cytometer (BD). A combination of forward
`angle light scatter and propidium iodide staining was used
`to gate out the dead cells, and | x 10° cells were analyzed.
`All FACS data were analyzed using FlowJo 8.1.1 software
`(Tree Star Inc., Ashland, OR). For cell sorting, 14-day
`cultured cells were washed twice with PBS and stained
`with anti-human CD8- or CD3-PE-Cy-7, CD45RO-FITC,
`CD62L-APC (BD Biosciences), and propidium iodide (PI)
`(50 pg/ml). Before sorting,
`the stained cells were passed
`through 40-j1m nylon cell strainer (BD), The cells were
`either gated on CD8 or gated on CD3 subsets depending on
`the specific experiment and using differentiation markers
`CD45RO and CD62L to yield CD62L-/CD45RO+ (Tem),
`CD62L+/CD45RO+ (Tem),
`and CD62L+/CD45RO—
`(Tn) populations. The
`sorting was performed on a
`FACSAria I cytometer (BD) equipped with 2 laser system
`(488-nm blue laser and 633-nm red laser); the cells were
`passed through 70-j1m nozzle under pressure of 70 psi. The
`cytometer was driven by BD FACSDiva version 6.1.3
`software.
`
`Illumina gene expression array
`
`Total RNA from sorted Tem, Tem, and Tn CD8 subpop-
`ulations was extracted, and 200 ng of total RNA was used
`to generate biotin-labeled cDNA using Il]umina TotalPrep
`RNA amplification kit
`(Ambion, Austin, TX). Biotin-
`labeled cDNA (750 ng) was hybridized to Sentrix Bead-
`Chip Array for Gene Expression (Human Ref-12 V2,
`Hlumina, San Diego, CA) and incubated at 58°C for
`16-20 h in an Illumina hybridization oven with rocker
`speed at 5. Beadchips were washed and stained according
`to Illumina’s protocol. Arrays were scanned by Illumina
`chip scanner, and images were analyzed by Bead Studio
`
`(Illumina, San Diego, CA). Data were exported and pro-
`cessed using Genespring GX 11
`(Agilent, Santa Clara,
`CA). Differentially regulated genes between different
`subpopulations were identified by statistical significance
`P < 0.05 and fold change >2., Significantly regulated genes
`were subjected to further analysis using Ingenuity Pathway
`software (Ingenuity, Redwood City, CA).
`
`Effector—-target conjugate formation assay
`
`The assay for effector—target cell conjugate formation has
`been previously reported [28]. Briefly, melanoma line 526
`was labeled with CFSE at a concentration of 1 4M using
`CFSE cell proliferation kit
`(Invitrogen) according to
`product manual;
`1 x 10’ melanoma 526 cells were
`labeled, and the cells were cultured for 48 h prior to use.
`Lentiviral TCR-vector-transduced PBL (14 days in culture)
`were sorted as described earlier. The co-culture was initi-
`ated by combining 3 x 10° each oftarget and effectorcell
`in 14-ml polypropylene round-bottom tubes (BD) by cen-
`trifugation at 137xg for 10 s. At the end ofincubation, the
`cells were stained with CD3 PerCP, CD8 APC, and
`MART-1 PE tetramer to analyze for T cell:tumor cell
`conjugates by FACS. FACS data were analyzed using
`FlowJo software. The percentage of effector—target con-
`jugate formation was determined using double positive
`cells divided by CFSE-labeled tumor cells:
`the statistic
`analysis was based on mean + SD from triplicate samples.
`
`Measurement of lymphocyte reactivity to antigen
`
`Transduced PBLeffector cells (1 x 10°) were co-cultured
`with melanoma lines (1 x 10°)
`in a final volume of
`0.2 ml in each well of a round-bottom 96-well plate. Cell
`culture supernatants were harvested and assayed 16h
`later for IFNy and IL2 by ELISA kit (Pierce Endogen,
`Rockford, IL). The culture supernatants were diluted to
`be in the linear range of the assay. Results represent
`mean + SD of triplicate cultures. The ability of trans-
`duced PBL to lyse melanoma tumor lines was evaluated
`using a Ser assay as described [29]. Briefly, 10° tumor
`cells were labeled for 1 h at 37°C with 100 wCi of *'Cr
`(GE Healthcare, Piscataway, NJ). Labeled target cells
`Q.% 10°) were co-cultured with effector cells at the ratios
`indicated for 4 h at 37°C in 0.15 ml of medium. Harvested
`supernatants were counted using a MicorBeta TriLux
`instrument
`(Perkin Elmer, Waltham, MA). Total and
`spontaneous *'Cr release was determined by incubating
`2 x 10° labeled target cells in either 2% SDS or medium
`alone for
`the aforementioned conditions,
`respectively.
`Each data point was determined as a mean of quadrupli-
`cate wells. The percentage of specific lysis was calculated
`as indicated in figure legend.
`
`As
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`Cancer Immunol Immunother (201 1) 60:739-749
`
`Engraftment of Tem and Tem in NOD/SCID/)c/~
`mouse model
`
`Sorted CD3 Tem and Tem subpopulations were counted
`and suspended in PBS, and 1.7 x 10° cells were injected
`intravenously into NOD/SCID/yc-/~ mice without sup-
`plement of IL2. Thirty-seven days later, mice were killed
`and T lymphocytes were isolated from spleen, lymph nodes
`(LN), and lung. T cells from lymph node (LN) spleen were
`lysed with ACK lysing buffer (BioWhittaker) followed by
`two PBS washes. T cells from lung were minced andtissue
`debris was removed. Lymphocyte separation medium (MP
`Biomedicals) was used to purify T cells by centrifugation
`
`at 2,000 g for 20 min. After collecting lymphocytes, and
`washing twice, the cells were quantitated and subject to
`FACSanalysis.
`
`Results
`
`Phenotype and activity of in vitro generated anti-tumor
`T lymphocytes
`
`Using overnight activation with anti-CD3/CD28 beads
`followed
`by
`lentiviral
`vector—-mediated
`transduction
`(Fig.
`la),
`engineered cells
`from three donors were
`
`t
`
`Day
`
`Q osh
`FACS
`
`14
`0
`1
`
`Tem|Tcm
`
`Day O
`Day 14
`
`
`
`| Donor 1
`
`
`
`
`
`Donor 2
`
`
`
`
`anti-CD3/CD28 beads
`
`Gate on CD8
`
`CD45RO
`
`Tn
`
`CD62L
`
`
`
`
`
`Donor 4
`
`CD70
`
`
`
`Fig. 1 The in vitro cultured anti-tumor T lymphocytes exhibit
`distinct populations defined by differentiated markers CD45RO and
`CD62L. a Schematic illustration of in vitro generated anti-tumor T
`lymphocytes. PBMC were activated by anti-CD3/CD28 beads on day
`0, and on day 1 post-stimulation,
`the cells were transduced with
`lentiviral vector harboring MART-| antigen TCR and maintained in
`culture for
`14 days. b FACS analysis of
`in vitro cultured T
`lymphocytes defined by CD45RO and CD62L.Thein vitro cultured
`T lymphocytes were gated on CD8 T cells and analyzed using
`differentiated markers CD45RO and CD62L. The phenotype of the
`
`cultured T cells from 3 donors at day 0 and day 14 is shown in dot
`plots. C. The in vitro cultured anti-tumor T lymphocytes exhibit
`distinct populations defined by differentiated markers CD45RO and
`CD62L.Cells were gated on CD8, analyzed for CD45RO and CD62L
`expression followed by the individual markers as indicated. The
`expression of individual markers CD27, CCR7, etc. in each quadrant
`onleft was overlaid on the right. The histograms from red, blue, and
`green indicate cells from Tem, Tem, and Tn, respectively. Tem,
`effector memory T cells; Tem, central memory T cells; Tn, naive T
`cells. PBMC derived from donor 3 (color figure online)
`
`¥) Springer
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`Cancer Immuno! Immunother (2011) 60:739-749
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`expanded and harvested at day 14, and the phenotype of
`CD8* T cells was analyzed using differentiation markers
`CD45RO and CD62L. As shownin Fig. 1b, the Tem cells
`were the least abundant at day 0, while this phenotype was
`the most plentiful at day 14 following transduction and in
`vitro culture. The phenotyp